bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ectopic skeletal muscles derived from myoblasts implanted under the skin
Authors: Irintchev A. , Rosenblatt J. D. , Cullen M. J. , Zweyer M. , Wernig A. 
Published: April 2021

ABSTRACT
We investigated the potential of cultured myoblasts to generate skeletal muscle in an ectopic site. Myoblasts from a clonal cell line or from expanded primary cultures were injected under the skin of the lumbar region of adult syngenic Balb/c mice. One to 7 weeks after injection, distinct muscles, of greater mass in mice injected with clonal myoblasts (6-78 mg, n=37) than in mice injected with primary myoblasts (1-7 mg, n=26), had formed between the subcutaneous panniculus carnosus muscle and the trunk muscles of host animals. These ectopic muscles exhibited spontaneous and/or electrically-evoked contractions after the second week and, when stimulated directly in vitro, isometric contractile properties similar to those of normal muscles. Histological, electron microscopical and tissue culture examination of these muscles revealed their largely mature morphology and phenotype. The fibres, most of which were branched, were contiguous, aligned and capillarised, exhibited normal sarcormeric protein banding patterns, and expressed muscle-specific proteins, including desmin, dystrophin, and isoforms of developmental and adult myosin heavy chain. Enveloping each fibre was a basal lamina, beneath which lay quiescent satellite cells, which could be stimulated to produce new muscle in culture.
Presence of endplates (revealed by α-bungarotoxin and neurofilament staining), and the eventual loss of expression of neural cell adhesion molecule and extrasynaptic acetylcholine receptors, indicated that some fibres were innervated. That these muscle fibres were of implanted-cell origin was supported by the finding of Y-chromosome and a lack of dystrophin in ectopic muscles formed after subcutaneous injection of, respectively, male myoblasts into female mice and dystrophin-deficient (mdx) myoblasts into normal C57Bl/10 muscle. Our results demonstrate that an organised, functional muscle can be generated de novo from a disorganised mass of myoblasts implanted in an extramuscular subcutaneous site, whereby the host contributes significantly in providing support tissues and innervation. Our observations are also consistent with the idea that myogenic cells behave like tissue-specific stem cells, generating new muscle precursor (satellite) cells as well as mature muscle. Subcutaneous implantation of myoblasts may have a range of useful applications, from the study of myogenesis to the delivery of gene products.
Aryl-hydrocarbon receptor is an inhibitory regulator of lipid synthesis and of commitment to adipogenesis
Authors: Alexander David L. , Ganem Leonardo G. , Fernandez-Salguero Pedro , Gonzalez Frank , Jefcoate Colin R. 
Published: April 2021

ABSTRACT
The aryl-hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In mouse embryo fibroblasts, TCDD activates expression of multiple genes, including CYP1B1, the predominant cytochrome P450 expressed in these cells. Here, we analyze constitutive functions of the AhR in primary mouse embryo fibroblasts (MEFs) and spontaneously immortalized MEF cell lines derived from wild-type (WT) C57BL/6 mice and also from congenic mice with a targeted disruption of the AhR gene (AhR−/−). After multiple passages, primary MEFs exhibit spontaneous differentiation, growth cessation and senescence. Eventually, colonies of immortalized MEFs arise to provide clonal lines. The senescent phase occurs much earlier for AhR−/− MEFs, while immortalization is substantially delayed. Comparison of AhR−/− and WT MEFs also indicates that constitutive AhR activity is required for basal expression of CYP1B1 and suppresses lipogenesis in subconfluent cultures. Primary WT and AhR−/− MEFs and the corresponding lines undergo adipogenesis when treated at confluence with the appropriate hormonal inducers. Addition of TCDD before or concurrent with hormonal induction suppressed PPARγ mRNA and adipogenesis, as measured by lipid accumulation, glycerol phosphate dehydrogenase activity and stearoyl CoA desaturase type 1 mRNA expression. This effect of TCDD treatment was absent in AhR−/− MEFs, establishing the role of AhR in hormone-induced adipogenesis. Such hormonal activation of confluent MEFs and preadipocytes results in a limited proliferative expansion followed by irreversible growth arrest. TCDD-treated MEFs undergo the mitotic expansion but fail to exit the cell cycle. In AhR−/− MEFs, there is no such effect of TCDD. These findings implicate the AhR as a constitutive inhibitor of triglyceride synthesis, and as an early regulator of adipocyte differentiation. AhR interference with cell-cycle arrest in differentiation may be linked to the increased rate of senescence.
The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network
Authors: Gavet Olivier , Ozon Sylvie , Manceau Valérie , Lawler Sean , Curmi Patrick , Sobel André 
Published: April 2021

ABSTRACT
Stathmin is a small regulatory phosphoprotein integrating diverse intracellular signaling pathways. It is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3 and its two splice variants RB3’ and RB3’’. Stathmin itself was shown to interact in vitro with tubulin in a phosphorylation-dependent manner, sequestering free tubulin and hence promoting microtubule depolymerization. We investigated the intracellular distribution and tubulin depolymerizing activity in vivo of all known members of the stathmin family. Whereas stathmin is not associated with interphase microtubules in HeLa cells, a fraction of it is concentrated at the mitotic spindle. We generated antisera specific for stathmin phosphoforms, which allowed us to visualize the regulation of phosphorylation-dephosphorylation during the successive stages of mitosis, and the partial localization of stathmin phosphorylated on serine 16 at the mitotic spindle. Results from overexpression experiments of wild-type and novel phosphorylation site mutants of stathmin further suggest that it induces depolymerization of interphase and mitotic microtubules in its unphosphorylated state but is inactivated by phosphorylation in mitosis. Phosphorylation of mutants 16A25A and 38A63A on sites 38 and 63 or 16 and 25, respectively, was sufficient for the formation of a functional spindle, whereas mutant 16A25A38A63E retained a microtubule depolymerizing activity. Transient expression of each of the neural phosphoproteins of the stathmin family showed that they are at least partially associated to the Golgi apparatus and not to other major membrane compartments, probably through their different NH2-terminal domains, as described for SCG10. Most importantly, like stathmin and SCG10, overexpressed SCLIP, RB3 and RB3’’ were able to depolymerize interphase microtubules. Altogether, our results demonstrate in vivo the functional conservation of the stathmin domain within each protein of the stathmin family, with a microtubule destabilizing activity most likely essential for their specific biological function(s).
Articulins and epiplasmins: two distinct classes of cytoskeletal proteins of the membrane skeleton in protists
Authors: Huttenlauch Irm , Peck Robert K. , Stick Reimer 
Published: April 2021

ABSTRACT
The cortex of ciliates, dinoflagellates and euglenoids comprises a unique structure called the epiplasm, implicated in pattern-forming processes of the cell cortex and in maintaining cell shape. Despite significant variation in the structural organization of their epiplasm and cortex, a novel type of cytoskeletal protein named articulin is the principal constituent of the epiplasm in the euglenoid Euglena and the ciliate Pseudomicrothorax. For another ciliate, Paramecium, epiplasmins, a group of polypeptides with common biochemical properties, are the major constituents of the epiplasm. Using molecular tools and affinity purification we have selected polyclonal antibodies and identified epitopes of monoclonal antibodies that identify epitopes characteristic of articulins and epiplasmins. With these antibodies we have analysed the occurrence of the two types of cytoskeletal proteins in a dinoflagellate, a euglenoid and several ciliates. Our results indicate that both articulins and epiplasmins are present in these organisms, suggesting that both contribute to the organization of the membrane skeleton in protists. Articulins and epiplasmins represent two distinct classes of cytoskeletal proteins, since different polypeptides were labeled by articulin core domain-specific or epiplasmin epitope-specific antibodies in each organism studied. In one case, a polypeptide in Pseudomicrothorax was identified that reacts with both articulin core domain-specific and with anti-epiplasmin monoclonal antibodies; however, the epiplasmin monoclonal antibody epitope was mapped to the C terminus of the polypeptide, well outside the central VPV-repeat core domain that contains the articulin monoclonal antibody epitope and that is the hallmark of the articulins.
Isolation of the human genes encoding the Pyst1 and Pyst2 phosphatases: characterisation of Pyst2 as a cytosolic dual-specificity MAP kinase phosphatase and its catalytic activation by both MAP and SAP kinases
Authors: Dowd Stephen , Sneddon Alan A. , Keyse Stephen M. 
Published: April 2021

ABSTRACT
We have isolated the human genes encoding the Pyst1 (MKP-3) and Pyst2 (MKP-X) MAP kinase phosphatases. Both genes consist of three exons interrupted by two introns and lack an intron which is conserved in all the other members of this gene family characterised to date. This reinforces the conclusion that Pyst1 and Pyst2 are members of a distinct and structurally homologous subfamily of dual-specificity (Thr/Tyr) MAP kinase phosphatases. We find that Pyst2 mRNA is constitutively expressed in a wide variety of human cell lines including those derived from ovarian, bladder and breast cancers. While there is no evidence for inducible expression of Pyst2 mRNA in human skin fibroblasts in response to cellular stress, Pyst2 mRNA levels are moderately increased in response to serum stimulation. Pyst2 protein is predominantly cytosolic when expressed in COS-1 cells. In common with Pyst1, Pyst2 shows substrate selectivity for the classical p42 (ERK2) isoform of MAP kinase both in vitro and in vivo, displaying much reduced activity towards stress activated MAP kinase isoforms such as JNK-1 and p38/RK. Pyst2 binds p42 MAP kinase in vivo and both MAP kinase binding and substrate selectivity correlate with the ability of different recombinant MAP and SAP kinases to cause catalytic activation of the Pyst2 phosphatase in vitro.
Expression and localization of hepatocyte domain-specific plasma membrane proteins in hepatoma × fibroblast hybrids and in hepatoma dedifferentiated variants
Authors: Bender Virginie , Büschlen Sylvie , Cassio Doris 
Published: April 2021

ABSTRACT
We have studied two aspects of the plasma membrane of hepatocytes, highly differentiated epithelial cells that exhibit a particular and complex polarity. Using a genetic approach, we have distinguished between the expression/regulation of proteins specific for all three hepatocyte membrane domains and their organization into discrete domains. For this analysis we used a panel of previously isolated cell clones, derived from the differentiated rat hepatoma line H4IIEC3, and that present different expression patterns for liver-specific genes. This panel was composed of (1) differentiated clones, (2) chromosomally reduced hepatoma-fibroblast hybrids characterized by a pleiotropic extinction/reexpression of liver-specific genes and (3) dedifferentiated variant and revertant clones. The expression of 16 hepatocyte membrane polarity markers was studied by western blotting and immunolocalization. Even though cells of differentiated clones express all of these polarity markers, they are not polarized, and are therefore suitable for studying the regulation of plasma membrane protein expression, and for identifying gene products implicated in the establishment of membrane polarity. In hepatoma-fibroblast hybrids the expression of four markers, three apical (dipeptidylpeptidase IV, alkaline phosphodiesterase B10 and polymeric IgA receptor) and one lateral (E-cadherin), is down-regulated in extinguished clones and restored in reexpressing subclones, as previously reported for liver-specific functions. The dipeptidylpeptidase IV mRNA was undetectable or strongly reduced in extinguished hybrids, but expressed at a robust level in some of the reexpressing clones. Concerning the dedifferentiated variants, each has its own pattern of membrane marker expression (loss of expression of three to six markers), that differs from that of extinguished hybrids. Revertant cells express all of the membrane markers examined. Among all of these hepatoma derivatives, only cells of reexpressing hybrids are polarized, and form bile canaliculi-like structures, with spherical and even, for one clone, long tubular and branched forms. All apical markers examined are confined in these canalicular structures, whereas the other markers are excluded from them, and present on the rest of the membrane (basolateral markers) or at the cell-cell contacts (lateral markers). Cells of reexpressing hybrids also express simple epithelial polarity. Thus the expression of only a few hepatocyte-domain-specific plasma membrane proteins is subject to down-regulation, as is the case for liver-specific genes so far studied, and the expression of polarity markers and the formation of poles are dissociable events.
Vinculin but not α-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium
Authors: Perez-Moreno M. , Avila A. , Islas S. , Sanchez S. , González-Mariscal L. 
Published: April 2021

ABSTRACT
The establishment of the junctional complex in epithelial cells requires the presence of extracellular calcium, and is controlled by a network of reactions involving G-proteins, phospholipase C and protein kinase C. Since potential candidates for phosphorylation are the tight junction associated proteins ZO1, ZO2 and ZO3, in a previous work we specifically explored these molecules but found no alteration in their phosphorylation pattern. To continue the search for the target of protein kinase C, in the present work we have studied the subcellular distribution and phosphorylation of vinculin and α-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, α-actinin remains unchanged. The increased phosphorylation of vinculin is due to changes in phosphoserine and phosphothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calcium significantly increases the vinculin phosphorylation level; (2) H7 and calphostin C (both protein kinase C inhibitors) completely abolish this increase during a calcium switch; (3) inhibition of phosphorylation during a calcium switch blocks the subcellular redistribution of vinculin and α-actinin. These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junctional complex.
The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in fission yeast
Authors: Norbury Randa Craig and Chris 
Published: April 2021

ABSTRACT
Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this ‘S-M’ checkpoint control is fundamental to the maintenance of genomic integrity. Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the ‘cut’ phenotype. In an attempt to identify conserved molecules involved in the S-M checkpoint we have screened a regulatable murine cDNA library in S. pombe and have identified cDNAs that induce the cut phenotype in cells arrested in S phase by hydroxyurea. One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbed mitotic spindle functions, although spindle pole duplication was apparently normal. Both aspects of the phenotype induced by this cDNA product, which we term Sha1 (for spindle and hydroxyurea checkpoint abnormal), were suppressed by simultaneous overexpression of calmodulin. Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp). These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes.
Distinct signal transduction pathways are utilized during the tube formation and survival phases of in vitro angiogenesis
Authors: Mahooti Neta Ilan, Sepi , Madri Joseph A. 
Published: April 2021

ABSTRACT
Angiogenesis, the formation of new blood vessels from preexisting ones, occurs during development, wound healing and cancer and involves stages that orchestrate a network of cooperative interactions. Peptide growth factors and extracellular matrix (ECM) components are two major groups of angiogenesis mediators. Among the different ECM proteins, collagens have been well-associated with in vivo angiogenesis. Using human umbilical vein endothelial cells (HUVEC) grown in 3-D collagen gels we show that: (1) HUVEC do not survive well in 3-D collagen gels due to rapid induction of apoptosis. (2) VEGF, a potent in vivo angiogenic factor, fails to induce tube formation. (3) PMA was effective in inducing tube formation and survival in HUVEC dispersed in 3-D collagen gels, activating MAP kinase, phosphoinositide 3-OH kinase (PI-3-kinase) and Akt/PKB (protein kinase B) pathways. (4) VEGF was effective in preventing PMA-induced tube-like structure regression after PMA-withdrawal by (5) activating the mitogen activated protein kinase (MAPK), rather than the Akt/PKB, signaling pathway.
Furrow-associated microtubule arrays are required for the cohesion of zebrafish blastomeres following cytokinesis
Authors: Jesuthasan Suresh 
Published: April 2021

ABSTRACT
During the first few cleavages of the zebrafish embryo, daughter blastomeres are loosely associated immediately after furrow ingression, but then gradually cohere. Cohesion appears to be cadherin-dependent, as cadherin and β-catenin are found at the membrane between cohering blastomeres, and blastomeres fail to cohere in calcium-free medium. Cadherin and β-catenin are not initially found on the blastomere surface, but are deposited specifically at the furrow surface. An array of parallel microtubules is present on either side of the furrow tip during ingression, as seen by confocal microscopy of α-tubulin labelled embryos. Transient incubation of embryos in 1 µg/ml nocodazole at the start of furrowing, which causes a loss of the furrow array, inhibits the localization of β-catenin to the furrow surface but does not prevent furrow ingression. During ingression, intracellular membranes are transported to the furrow, as shown by labelling with DiD or DiOC6(3). Concentration of these membranes near the furrow surface is microtubule-dependent.
These findings suggest that microtubules are required for cohesion of blastomeres because they mediate trafficking of intracellular membranes to the furrow surface, where they are exocytosed and allow cohesion via cadherins.
The kar3p and kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers
Authors: Huyett Abigail , Kahana Jason , Silver Pamela , Zeng Xuemei , Saunders William S. 
Published: April 2021

ABSTRACT
Microtubules provide the substrate for intracellular trafficking by association with molecular motors of the kinesin and dynein superfamilies. Motor proteins are generally thought to function as force generating units for transport of various cargoes along the microtubule polymer. Recent work suggests additional roles for motor proteins in changing the structure of the microtubule network itself. We report here that in the budding yeast Saccharomyces cerevisiae microtubule motors have antagonistic effects on microtubule numbers and lengths. As shown previously, loss of the Kar3p motor stimulates cytoplasmic microtubule growth while loss of Kip2p leads to a sharp reduction in cytoplasmic microtubule numbers. Loss of both the Kip2p and Kar3p motors together in the same cell produces an intermediate phenotype, suggesting that these two motors act in opposition to control cytoplasmic microtubule density. A Kip2p-GFP fusion from single gene expression is most concentrated at the spindle poles, as shown previously for an epitope tagged Kar3p-HA, suggesting both of these motors act from the minus ends of the microtubules to influence microtubule numbers.
The axonemal microtubules of the chlamydomonas flagellum differ in tubulin isoform content
Authors: Johnson Karl A. 
Published: April 2021

ABSTRACT
Little is known of the molecular basis for the diversity of microtubule structure and function found within the eukaryotic flagellum. Antibodies that discriminate between tyrosinated alpha tubulin and post-translationally detyrosinated alpha tubulin were used to localize these complementary tubulin isoforms in flagella of the single-celled green alga Chlamydomonas reinhardtii. Immunofluorescence analysis of intact axonemes detected both isoforms along most of the lengths of flagella; however, each had a short distal zone rich in tyrosinated tubulin. Localizations on splayed axonemes revealed that the microtubules of the central-pair apparatus were rich in tyrosinated tubulin, while outer doublets contained a mixture of both isoforms. Immunoelectron analysis of individual outer doublets revealed that while tyrosinated tubulin was present in both A and B tubules, detyrosinated tubulin was largely confined to the wall of the B hemi-tubules. The absence of detyrosinated tubulin from the A tubules of the outer doublets and the microtubules of the central pair, both of which extend past the B hemi-tubules of the outer doublets in the flagellar tip, explained the appearance of a tyrosinated tubulin-rich distal zone on intact axonemes. Localizations performed on cells regenerating flagella revealed that flagellar assembly used tyrosinated tubulin; detyrosination of the B tubule occurred during later stages of regeneration, well after microtubule polymerization. The developmental timing of detyrosination, which occurs over a period during which the regrowing flagella begin to beat more effectively, suggests that post-translational modification of the B tubule surface may enhance dynein/B tubule interactions that power flagellar beating.