bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid appearance of connexin 26-positive gap junctions in centrilobular hepatocytes without induction of mRNA and protein synthesis in isolated perfused liver of female rat
Authors: Kojima Takashi , Sawada Norimasa , Oyamada Masahito , Chiba Hideki , Isomura Hiroshi , Mori Michio 
Published: April 2021

ABSTRACT
In the adult rat liver, the gap junction protein connexin 32 (Cx32) is evenly distributed in hepatocytes within the liver lobules, while connexin 26 (Cx26) is preferentially localized in hepatocytes in periportal zones. We report here that Cx26-positive gap junctions rapidly appear in the centrilobular hepatocytes of adult female rat livers during a 30 minute perfusion of the liver through the hepatic portal vein with a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and oxygen transport FC-43 fluid at a physiological flow rate without any changes in the distribution of Cx32. The change in the localization of Cx26 was closely related to that of E-cadherin, and there was no significant increase in the amounts of Cx26 protein and mRNA. The appearance of Cx26 in the centrilobular hepatocytes was inhibited by treatment with cytoskeleton disruptors such as colchicine and cytochalasin B, and intra-cytoplasmic transport inhibitors such as brefeldin A. The liver perfusion induced the appearance of Cx26 in the centrilobular hepatocytes only in female rats. Estrogen treatment of ovariectomized rats caused the appearance of both Cx26 and E-cadherin in centrilobular hepatocytes not only in the perfused liver but also in the non-perfused liver. Our results indicate that in the rat liver: (a) the localization of Cx26 can be modulated by a post-translational mechanism; (b) E-cadherin may play an important role in the formation of gap junctions composed of Cx26; and (c) the formation of gap junctions is regulated by female steroid hormones.
Expression of mouse LSP1/S37 isoforms S37 is expressed in embryonic mesenchymal cells
Authors: Misener V. L. , Hui C.-c. , Malapitan I. A. , Ittel M.-E. , Joyner A. L. , Jongstra J. 
Published: April 2021

ABSTRACT
Mouse LSP1 is a 330 amino acid intracellular F-actin binding protein expressed in lymphocytes and macrophages but not in non-hematopoietic tissues. A 328 amino acid LSP1-related protein, designated S37, is expressed in murine bone marrow stromal cells, in fibro-blasts, and in a myocyte cell line. The two proteins differ only at their N termini, the first 23 amino acid residues of LSP1 being replaced by 21 different residues in S37. The presence of different amino termini suggests that the LSP1 and S37 proteins are encoded by transcripts arising through alternative exon splicing. Here we report the genomic organization of the Lsp1 gene and show that the distinct N termini of LSP1 and S37 are encoded by two alternatively used exons, each containing a translational start codon. We also demonstrate that alternative 3′ acceptor sites are used in the splicing of exon 5. This results in LSP1 and S37 transcripts that either do or do not contain 18 bp encoding the 6 amino acids HLIRHQ of the acidic domain. Therefore, the Lsp1 gene encodes four protein isoforms: full-length LSP1 and S37 proteins, designated LSP1-I and S37-I and the same proteins without the HLIRHQ sequence, designated LSP1-II and S37-II. By in situ hybridization analysis we show that the S37 isoforms are expressed in mesenchymal tissue, but not in adjacent epithelial tissue, of several developing organs during mouse embryogenesis. This, together with our finding that S37 is an F-actin binding protein, suggests that S37 is a cytoskeletal protein of mesenchymal cells, which may play a role in mesenchyme-induced epithelial differentiation during organogenesis.
Nuclear calmodulin/62 kDa calmodulin-binding protein complexes in interphasic and mitotic cells
Authors: Portolés Manuel , Faura Magdalena , Renau-Piqueras Jaime , José Iborra Francisco , Saez Rosana , Guerri Consuelo , Serratosa Joan , Rius Eulalia , Bachs Oriol 
Published: April 2021

ABSTRACT
We report here that a 62 kDa calmodulin-binding protein (p62), recently identified in the nucleus of rat hepatocytes, neurons and glial cells, consists of four polypeptides showing pI values between 5.9 and 6.1. By using a DNA- binding overlay assay we found that the two most basic of the p62 polypeptides bind both single- and double-stranded DNA. The intranuclear distribution of calmodulin and p62 was analysed in hepatocytes and astrocyte precursor cells, and in proliferating and differentiated astrocytes in primary cultures by immunogold-labeling methods. In non-dividing cells nuclear calmodulin was mostly localized in heterochromatin although it was also present in euchro- matin and nucleoli. A similar pattern was observed for p62, with the difference that it was not located in nucleoli. p62/calmodulin complexes, mainly located over hete- rochromatin domains were also observed in interphasic cells. These complexes remained associated with the nuclear matrix after in situ sequential extraction with nucleases and high-salt containing buffers. In dividing cells, both calmodulin and p62 were found distributed over all the mitotic chromosomes but the p62/calmodulin aggregates were disrupted. These results suggest a role for calmodulin and p62 in the condensation of the chromatin.
Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells
Authors: äntti Jussi J , änen Sirkka Ker , Toikkanen Jaana , Kuismanen Esa , Ehnholm Christian , Söderlund Hans , Olkkonen Vesa M. 
Published: April 2021

ABSTRACT
Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20°C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37°C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.
Nuclear import of the myogenic factor MyoD requires cAMP-dependent protein kinase activity but not the direct phosphorylation of MyoD
Authors: Vandromme Marie , Carnac Gilles , Gauthier-Rouvière Cécile , Fesquet Didier , Lamb Ned , Fernandez Anne 
Published: April 2021

ABSTRACT
MyoD is a nuclear phosphoprotein that belongs to the family of myogenic regulatory factors and acts in the tran-scriptional activation of muscle-specific genes. We have investigated the role of cAMP-dependent protein kinase (A-kinase) in modulating the nuclear locale of MyoD. Purified MyoD protein microinjected into the cytoplasm of rat embryo fibroblasts is rapidly translocated into the nucleus. Inhibition of A-kinase activity through injection of the specific inhibitory peptide PKI prevents this nuclear localisation. This inhibition of nuclear location is specifi-cally reversed by injection of purified A-kinase catalytic subunit, showing the requirement for A-kinase in the nuclear import of MyoD. Site-directed mutagenesis of all the putative sites for A-kinase-dependent phosphorylation on MyoD, substituting serine or threonine residues for the non-phosphorylatable amino acid alanine, had no effect on nuclear import of mutated MyoD. These data exclude the possibility that the effect of A-kinase on the nuclear translocation of MyoD is mediated by direct phosphoryla-tion of MyoD and imply that A-kinase operates through phosphorylation of components involved in the nuclear transport of MyoD.
On the molecular mechanism of the circadian clock: The 41,000 Mr clock protein of Chlorella was identified as 3-phosphoglycerate kinase
Authors: Walla Ortwin J. , Groot Egon J. de , Schweiger Manfred 
Published: April 2021

ABSTRACT
A 41,000 Mr polypeptide of Chlorella exhibits a circadian rhythm in its synthesis and possesses characteristic features of a putative essential clock protein as was proposed by the coupled translation-membrane model. Purification of this polypeptide and a microsequencing analysis yielded a N-terminal sequence of 35 amino acids that showed no homology to known sequences that were thought to be involved in circadian rhythm such as the per gene of Drosophila and the frq gene of Neurospora. However, strong homology was observed to 3-phospho- glycerate kinase (PGK) of different organisms. The highest homology (83%) of this Chlorella sequence was found with the PGK of wheat chloroplast.
PGK activity and the 41,000 Mr polypeptide co-purified through differential centrifugation and gel filtration. These data, and comparison with the physical properties of other known PGK molecules, support the conclusion that the 41,000 Mr polypeptide of Chlorella, a candidate for a putative essential clock protein, is 3-phosphoglycerate kinase.
Isolation and developmental expression of the amphibian homolog of the fibroblast growth factor receptor 3
Authors: Shi De-Li , Fromentoux Valérie , Launay Catherine , Umbhauer Muriel , Boucaut Jean-Claude 
Published: April 2021

ABSTRACT
Recent observations suggest that fibroblast growth factors (FGFs) and their receptors are involved in the control of embryogenesis. Several FGF receptor genes have been identified so far and their expression is differentially regulated. As part of a continuing effort to analyse the differential expression of FGF receptors and their potential role during amphibian development, we have isolated a Pleurodeles homolog of FGF receptor 3 (FGFR-3), which we designated PFR-3 because of its highest homology to human FGFR-3 (75% overall identity). PFR-3 is a maternally derived mRNA. While a low level of expression persists during the cleavage and gastrula stages, a significant increase in the mRNA was observed at the end of the gastrula stage. RNase protection analysis on dissected tissues showed that PFR-3 mRNA was mainly localized to the ectoderm at the early gastrula stage and then shifted to the embryonic neural tissues, whereas adult brain had decreased levels of PFR-3 mRNA expression. Consistent with the loss of FGF receptors during skeletal muscle terminal differentiation, PFR-3 as well as other FGF receptor mRNAs were undetectable in the adult skeletal muscle. However, highest levels of PFR-3 mRNA expression were found in the testis. In situ hybridization revealed strong expression in the germinal epithelium of the embryonic brain (especially the diencephalon and rhombencephalon) and neural tube, in the lens and the cranial ganglia. The epithelium of the developing gut, like the pharynx and esophagus, also prominently expressed PFR-3 mRNA. Other sites of expression were found in the liver and in the mesenchymal condensation sites of branchial arches. These observations further suggest that individual FGF receptor genes are differentially regulated during amphibian embryogenesis, and that PFR-3 may play a role in mesoderm induction and in other FGF-mediated developmental processes.
Expression and cellular localization of glucose transporters (GLUT1, GLUT3, GLUT4) during differentiation of myogenic cells isolated from rat fœtuses
Authors: Guillet-Deniau Isabelle , Leturque Armelle , Girard Jean 
Published: April 2021

ABSTRACT
Skeletal muscle regeneration is mediated by the proliferation of myoblasts from stem cells located beneath the basal lamina of myofibres, the muscle satellite cells. They are functionally indistinguishable from embryonic myoblasts. The myogenic process includes the fusion of myoblasts into multinucleated myotubes, the biosynthesis of proteins specific for skeletal muscle and proteins that regulates glucose metabolism, the glucose transporters. We find that three isoforms of glucose transporter are expressed during fœtal myoblast differentiation: GLUT1, GLUT3 and GLUT4; their relative expression being dependent upon the stage of differentiation of the cells. GLUT1 mRNA and protein were abundant only in myoblasts from 19-day-old rat fœtuses or from adult muscles. GLUT3 mRNA and protein, detectable in both cell types, increased markedly during cell fusion, but decreased in contracting myotubes. GLUT4 mRNA and protein were not expressed in myoblasts. They appeared only in spontaneously contracting myotubes cultured on an extracellular matrix. Insulin or IGF-I had no effect on the expression of the three glucose transporter isoforms, even in the absence of glucose. The rate of glucose transport, assessed using 2-[3H]deoxyglu-cose, was 2-fold higher in myotubes than in myoblasts. Glucose deprivation increased the basal rate of glucose transport by 2-fold in myoblasts, and 4-fold in myotubes. The cellular localization of the glucose transporters was directly examined by immunofluorescence staining. GLUT1 was located on the plasma membrane of myoblasts and myotubes. GLUT3 was located intracellularly in myoblasts and appeared also on the plasma membrane in myotubes. Insulin or IGF-I were unable to target GLUT3 to the plasma membrane. GLUT4, the insulin-regulatable glucose transporter isoform, appeared only in contracting myotubes in small intracellular vesicles. It was translocated to the plasma membrane after a short exposure to insulin, as it is in skeletal muscle in vivo. These results show that there is a switch in glucose transporter isoform expression during myogenic differentiation, dependent upon the energy required by the different stages of the process. GLUT3 seemed to play a role during cell fusion, and could be a marker for the muscle’s ability to regenerate.
Cell attachment to extracellular matrix substrates is inhibited upon downregulation of expression of calreticulin, an intracellular integrin α-subunit-binding protein
Authors: Leung-Hagesteijn Chung Yee , Milankov Kosta , Michalak Marek , Wilkins John , Dedhar Shoukat 
Published: April 2021

ABSTRACT
We have demonstrated recently that calreticulin, an intra-cellular calcium-binding protein, can interact with the α-subunits of integrin receptors via the highly conserved KXGFFKR amino acid sequence present in the cytoplasmic domains of all integrin α-subunits (Rojiani et al. (1991)Biochemistry 30, 9859-9866). Here we demonstrate that calreticulin can be co-localized by immunofluorescence as well as co-purified with integrins, that recombinant calreticulin can also interact with integrins, and that the interaction occurs predominantly via the N-domain of calreticulin, to a much lesser extent with the C-domain, but not at all with the proline-rich P-domain. To demonstrate a physiological role for the interaction of calreticulin with integrins, calreticulin expression was downregulated by treating cells with antisense oligonucleotides designed to inhibit the initiation of translation of calreticulin. Antisense oligonucleotides, but not sense or non-sense oligonucleotides, inhibited attachment and spreading of cells cultured in the presence of fetal bovine serum, and also of cells plated on individual extracellular matrix substrates in the absence of serum. The antisense oligonucleotide inhibited cell proliferation of anchorage-dependent cells slightly, but there was no effect on cell viability. The effect on cell attachment was similar to that achieved by treating cells with an antisense oligonucleotide designed to inhibit translation of the integrin α3 subunit, which resulted in the inhibition of cell attachment to α3β1-specific substrates. The effect of the antisense calreticulin oligonucleotide on cell attachment was demonstrated to be integrin-mediated since antisense calreticulin treatment of Jurkat cells abrogated the stimulation of collagen cell attachment achieved by attachment-stimulating signalling anti-α2 (JBS2) and anti-β1 (21C8) antibodies. The oligonucleotides did not affect the rate of cell proliferation of these cells. These results demonstrate a fundamental role of calreticulin in cell-extracellular matrix interactions.
Retinoic acid as modulator of UVB-induced melanocyte differentiation: Involvement of the melanogenic enzymes expression
Authors: Roméro Christine , Aberdam Edith , Larnier Catherine , Ortonne Jean-Paul 
Published: April 2021

ABSTRACT
Retinoic acid (RA) is a hormone-like agent involved in the control of cell differentiation. The most characteristic feature of melanocyte differentiation, melanogenesis, is stimulated by UV radiations. Excessive chronic sun exposure results in irregular skin hypermelanosis that can be partially corrected by topical RA. The basic mechanisms underlying this effect of RA are unknown. To determine whether RA can directly modulate excessive melanin synthesis, we analyzed the in vitro effect of cis- and trans-RA on UVB-induced melanogenesis in S91 mouse melanoma cells and in normal human melanocytes (NHM). In both cells types, the two RA isoforms significantly decreased the UVB-stimulated melanogenesis in term of tyrosinase activity and melanin neosynthesis. To correlate changes in melanogenesis with the expression of melanogenic enzymes, we determined the neosynthesis rate of tyrosinase, tyrosinase-related protein-1 (TRP-1/gp 75) and tyrosinase-related protein-2 (TRP-2/DOPAchrome tautomerase). Here we show that UVB-induced melanogenesis in NHM is related to an increased synthesis of tyrosinase and TRP-1 and to a dramatic decrease of TRP-2 expression. RA inhibition of UVB-induced melanogenesis acts at the post-transcriptional level leading to a decreased tyrosinase and TRP-1 synthesis. We also show that in NHM, inhibition of TRP-2 following UVB-treatment is significantly reversed by RA. This demonstrates a negative correlation between melanogenesis and TRP-2 expression.
Co-localization of 1,4-dihydropyridine receptor α2/δ subunit and N-CAM during early myogenesis in vitro
Authors: Vandaele Sylvie F. , Rieger François 
Published: April 2021

ABSTRACT
The surface distribution of the subunit of the 1,4-dihydropyridine receptor and its topographical relationship with the neural cell adhesion molecule (N-CAM) were investigated during early myogenesis in vitro, by double immunocytochemical labeling with the monoclonal antibody 3007 and an anti-N-CAM polyclonal antiserum. The monoclonal antibody 3007 has been previously shown to immunoprecipitate dihydropyridine receptor from skeletal muscle T-tubules. In further immunoprecipitation experiments on such preparations and muscle cell cultures, it was demonstrated here that the monoclonal antibody 3007 exclusively recognizes the α2/δ subunit of the 1,4-dihydropyridine receptor. In rabbit muscle cell cultures, the labeling for both α2/δ and N-CAM was first detected on myoblasts, in the form of spots on the membrane and per-inuclear patches. Spots of various sizes organized in aggregates were then found on the membrane of myotubes. At fusion (T0), aggregates of N-CAM spots alone were found at the junction between fusing cells. At T6and later stages, all α2/δ aggregates present on myotubes co-localized with N-CAM, while less than 3% of N-CAM aggregates did not co-localize with α2/δ. A uniform N-CAM staining also made its appearance. At T12, when myotubes showed prominent contractility, α2/δ–N-CAM aggregates diminished in size. Dispersed α2/δ spots of a small regular size spread over the whole surface of the myotubes and alignments of these spots became visible. Corresponding N-CAM spots were now occasionally seen, and uniform N-CAM staining was prominent. These results show that α2/δ and N-CAM are co-localized and that their distributions undergo concomitant changes during early myogenesis until the T-tubule network starts to be organized. This suggest that these two proteins might jointly participate in morphogenetic events preceding the formation of T-tubules.
Immuno-EM localization of the β1integrin subunit in wet-cleaved fibronectin-adherent fibroblasts
Authors: Meijne Alexandra M. L. , Casey Diane M. , Feltkamp Constance A. , Roos Ed 
Published: April 2021

ABSTRACT
Using immuno-EM, we have studied the distribution of the β1integrin subunit in chicken embryo fibroblasts allowed to adhere and spread for 3 hours on a fibronectin-coated surface in serum-free medium. The cells were wet-cleaved, which removed most of the cell body, yielding ventral plasma membranes with little, and sometimes virtually no, associated cytoskeleton.
The β1integrin subunit was detected with antibodies against the cytoplasmic domain. In immune fluorescence, it colocalized with adhesion plaques, in a punctate staining pattern, and often seemed to be at the periphery of the plaque. By immuno-EM, β1was in fact found in discrete clusters, not throughout the plaque. In deep-cleaved cells from which virtually all cytoskeleton was removed, clusters could often be seen to be located on fibronectin fibrils. Furthermore, β1was present in clusters at the cell margins, and isolated or in small groups at the very edge of the cell.
When fibronectin synthesis, and consequently fibril formation, was inhibited by cycloheximide, large adhesion plaque-like structures were formed at the cell margin. This phenotype was reversed by addition of soluble fibronectin, which was incorporated into fibrils. As in normal plaques, talin and vinculin were present, the plasma membrane was very close (10-20 nm) to the substratum and the fibronectin layer underneath was removed. These plaques did contain β1integrins but they were not in clusters.
These observations indicate that the talin-vinculin network of an adhesion plaque is normally anchored to the substratum at discrete β1integrin clusters that may be located on fibronectin fibrils, and that elsewhere the plaque is not necessarily attached to the substratum by interaction of integrins with matrix proteins. In the absence of fibronectin fibrils, adhesion plaque-like structures can be formed, but these are aberrant in size, location and fine structure.