bluuebunny/crossref_metadata_2025_split huggingface dataset:
Platelet activating factor receptor (PAF-R) is found in a large endosomal compartment in human umbilical vein endothelial cells
Authors: Ihida Kaori , Predescu Dan , Czekay Ralf-Peter , Palade George E. 
Published: April 2021

ABSTRACT
In previous studies, we have localized the platelet activating factor receptor (PAF-R) in situ on the surface of the endothelium in a number of microvascular beds without providing information on its intracellular location. In the present study, we used human umbilical vein cells (HUVECs) as a model to immunolocalize PAF-R by light and electron microscopic procedures. We raised two different polyclonal antibodies against synthetic peptides of the C- and N-terminal of PAF-R and used them for immunolocalization studies. By immunofluorescence, we found that the anti-C-terminal antibody (CPAF-R) stains an extensive intracellular tubular network. By electron microscopy, using a preembedding staining procedure, we detected PAF-R on the surface of the plasmalemma in a staining pattern similar to that described on microvascular endothelia in situ, but at a considerably lower density. Immunogold labeling of thin frozen sections revealed the presence of PAF-R on the plasmalemma, and especially in an extensive network of tubular-vesicular elements and vesicles associated with it. No detectable amounts of PAF-R were found in the endoplasmic reticulum (ER) or in Golgi cisternae. Double immunofluorescence labeling with antibodies for compartment marker proteins and PAF-R revealed that PAF-R localizes in an endosomal compartment. Confocal microscopy showed that PAF-R colocalizes in this compartment together with the transferrin receptor (Tf-R) and the thrombin receptor (TH-R), but it also showed that the colocalization was partial rather than complete. These findings suggest that the endosomal network is either discontinuous or, conversely, that the proteins in its membrane do not have a fully randomized distribution.
Human primary fibroblasts in vitro express a purinergic P2X7 receptor coupled to ion fluxes, microvesicle formation and IL-6 release
Authors: Solini Anna , Chiozzi Paola , Morelli Anna , Fellin Renato , Di Virgilio Francesco 
Published: April 2021

ABSTRACT
We have investigated reponses to extracellular ATP in human fibroblasts obtained by skin biopsies. Our data show that these cells express a P2X7 purinergic receptor, as judged by (1) RT-PCR with specific primers, (2) reactivity with a specific anti-P2X7 antiserum, (3) activation by the selective P2X agonist benzoylbenzoylATP and (4) stimulation of transmembrane ion fluxes. Stimulation with benzoylbenzoylATP, and to a lesser extent with ATP, also caused striking morphological changes and increased formation of cytoplasmic microvesicles. These changes were fully reversible upon nucleotide removal. Two known blockers of P2X receptors, oxidised ATP and pyridoxalphosphate-6-azophenyl-2´,4´disulfonic acid, inhibited the morphological changes fully and the ion fluxes partially. The residual rise in intracellular Ca2+ levels and membrane depolarization observed in the presence of the inhibitors were dependent upon activation of a P2Y-type receptor exhibiting a peculiar pharmacological profile, in that CTP was the preferred agonist. ATP stimulation triggered release of the pro-inflammatory cytokine IL-6 in fibroblasts pre-treated with PMA and bacterial endotoxin. These observations reveal a novel pathway for fibroblast activation and for their recruitment in the inflammatory response.
Nonmuscle tropomyosin-4 requires coexpression with other low molecular weight isoforms for binding to thin filaments in cardiomyocytes
Authors: Helfman David M. , Berthier Christine , Grossman Jill , Leu Martin , Ehler Elisabeth , Perriard Evelyne , Perriard Jean-Claude 
Published: April 2021

ABSTRACT
Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle α-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV-or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This result was not obtained when TM-4 was co-transfected with high molecular weight (HMW) TMs (TM-1, TM-2 or skeletal muscle α-TM). These data demonstrate that the ability of TM-4 to bind to actin filaments can be specifically influenced by its interaction with other LMW TM isoforms. In addition, cells that incorporated the muscle or nonmuscle GFP-TMs into their sarcomeres continued to beat and exhibited sarcomeric contraction. These studies provide the first in vivo demonstration of synergistic effects between TM isoforms for binding to actin filaments. These results have important implications in understanding actin filament dynamics in nonmuscle cell systems, especially during development and in transformed cells, where alterations in the ratio of different LMW isoforms might lead to changes in their interactions with actin filaments. Furthermore, these studies demonstrate that GFP-TM can be used to study thin-filament dynamics in muscle cells and actin filament dynamics in nonmuscle cells.
SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation
Authors: Duprez Estelle , Saurin Andrew J. , Desterro Joana M. , Lallemand-Breitenbach Valerie , Howe Kathy , Boddy Michael N. , Solomon Ellen , Thé Hugues de , Hay Ronald T. , Freemont Paul S. 
Published: April 2021

ABSTRACT
PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL). PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs), which are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function. In a yeast two-hybrid screen, we first identified a ubiquitin-like protein named PIC1 (now known as SUMO-1), which interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IκBα and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation. The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown. Here we show that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain. Using site-specific mutagenesis, we have identified the primary PML-SUMO-1 modification site as being part of the nuclear localisation signal (Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuclear localisation as only nuclear PML is modified. The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and we have identified several other nuclear proteins which could also be targets for SUMO-1. We show that SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins. We also find that the APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA. Our results provide significant insights into the role of SUMO-1 modification of PML in both normal cells and the APL disease state.
Meiotic activation of rat pachytene spermatocytes with okadaic acid: The behaviour of synaptonemal complex components SYN1/SCP1 and COR1/SCP3
Authors: Tarsounas Madalina , Pearlman Ronald E. , Moens Peter B. 
Published: April 2021

ABSTRACT
The phosphatase inhibitor okadaic acid accelerates meiotic events in rodent germ cells in culture. Isolated pachytene spermatocytes treated with okadaic acid proceed to a metaphase I arrest in a few hours as opposed to the similar process in vivo, which requires several days. Leptotene/zygotene spermatocytes cannot be activated in this way, suggesting that okadaic acid enables cells to bypass a sensor of the meiotic progression, which is pachytene specific. We monitored the chromosome behaviour accompanying the transition to metaphase I in rat spermatocytes with antibodies against COR1/SCP3, a component of the meiotic chromosome cores, and against the synaptic protein, SYN1/SCP1. Okadaic acid induced a rapid synaptonemal complex dissolution and bivalent separation, followed by chromosome condensation and chiasmata formation, similar to the succession of events in untreated cells. The similarity between meiosis I induced with okadaic acid and the meiosis I events in vivo extends to the dissolution of the nuclear membrane and the disappearance of the microtubule network at the onset of metaphase I. This cell culture system provides a model for the in vivo transition from pachytene to metaphase I and therefore can be used in the study of this transition at the molecular level. The effect of okadaic acid is most likely mediated by the activation of tyrosine kinases, as addition of genistein, a general tyrosine kinase inhibitor, completely abolishes the observed effect of okadaic acid on chromosome metabolism. The okadaic acid-induced progression to the metaphase I arrest is not affected by the inhibition of protein synthesis. However, pachytene spermatocytes incubated in the presence of protein synthesis inhibitors for 6 hours show loss of synapsis which is abnormal in that it is not accompanied by chiasmata formation. The two meiosis-specific proteins, SYN1/SCP1 and COR1/SCP3, are efficiently phosphorylated in vitro by extracts from isolated pachytene cells. Extracts from cells that have reached metaphase I upon okadaic acid treatment, with concomitant displacement of SYN1/SCP1 and COR1/SCP3 from their chromosomes, do not have this capability. These data support the hypothesis that phosphorylation of SYN1/SCP1 and COR1/SCP3 targets their removal from the chromosomes and that activity of the kinases involved correlates with the presence of these two proteins on the chromosomes.
Induction of vascular smooth muscle cell tenascin-C gene expression by denatured type I collagen is dependent upon a β3 integrin-mediated mitogen-activated protein kinase pathway and a 122-base pair promoter element
Authors: Jones Peter Lloyd , Jones Frederick S. , Zhou Bin , Rabinovitch Marlene 
Published: April 2021

ABSTRACT
Tenascin-C is an extracellular matrix glycoprotein, the expression of which is upregulated in remodeling arteries. In previous studies we showed that the presence of tenascin-C alters vascular smooth muscle cell shape and amplifies their proliferative response by promoting growth factor receptor clustering and phosphorylation. Moreover, we demonstrated that denatured type I collagen induces smooth muscle cell tenascin-C protein production via β3 integrins. In the present study, we examine the pathway by which β3 integrins stimulate expression of tenascin-C, and define a promoter sequence that is critical for its induction. On native collagen, A10 smooth muscle cells adopt a stellate morphology and produce low levels of tenascin-C mRNA and protein, whereas on denatured collagen they spread extensively and produce high levels of tenascin-C mRNA and protein, which is incorporated into an elaborate extracellular matrix. Increased tenascin-C synthesis on denatured collagen is associated with elevated protein tyrosine phosphorylation, including activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). β3 integrin function-blocking antibodies attenuate ERK1/2 activation and tenascin-C protein synthesis. Consistent with these findings, treatment with the specific MEK inhibitor, PD 98059, results in suppression of tenascin-C protein synthesis. To investigate whether β3 integrin-dependent activation of ERK1/2 regulates the tenascin-C promoter, we transfected A10 cells with a full-length (approx. 4 kb) mouse tenascin-C gene promoter-chloramphenicol acetyltransferse reporter construct and showed that, relative to native collagen, its activity is increased on denatured collagen. Next, to identify regions of the promoter involved, we examined a series of tenascinC promoter constructs with 5′ deletions and showed that denatured collagen-dependent promoter activity was retained by a 122-base pair element, located −43 to −165 bp upstream of the RNA start site. Activation of this element was suppressed either by blocking β3 integrins, or by preventing ERK1/2 activation. These observations demonstrate that smooth muscle cell binding to β3 integrins activates the mitogen activated protein kinase pathway, which is required for the induction of tenascin-C gene expression via a potential extracellular matrix response element in the tenascin-C gene promoter. Our data suggest a mechanism by which remodeling of type I collagen modulates tenascin-C gene expression via a β3 integrin-mediated signaling pathway, and as such represents a paradigm for vascular development and disease whereby smooth muscle cells respond to perturbations in extracellular matrix composition by altering their phenotype and patterns of gene expression.
The Ku70 autoantigen interacts with p40phox in B lymphocytes
Authors: Grandvaux Nathalie , Grizot Sylvestre , Vignais Pierre V. , Dagher Marie-Claire 
Published: April 2021

ABSTRACT
Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.
Intracellular localization and membrane topology of 11-cis retinol dehydrogenase in the retinal pigment epithelium suggest a compartmentalized synthesis of 11-cis retinaldehyde
Authors: Simon András , Romert Anna , Gustafson Anne-Lee , McCaffery J. Michael , Eriksson Ulf 
Published: April 2021

ABSTRACT
11-cis retinol dehydrogenase (EC 1.1.1.105) catalyses the last step in the biosynthetic pathway generating 11-cis retinaldehyde, the common chromophore of all visual pigments in higher animals. The enzyme is abundantly expressed in retinal pigment epithelium of the eye and is a member of the short chain dehydrogenase/reductase superfamily. In this work we demonstrate that a majority of 11-cis retinol dehydrogenase is associated with the smooth ER in retinal pigment epithelial cells and that the enzyme is an integral membrane protein, anchored to membranes by two hydrophobic peptide segments. The catalytic domain of the enzyme is confined to a lumenal compartment and is not present on the cytosolic aspect of membranes. Thus, the subcellular localization and the membrane topology of 11-cis retinol dehydrogenase suggest that generation of 11-cis retinaldehyde is a compartmentalized process.
Endothelial cell integrin α5β1 expression is modulated by cytokines and during migration in vitro
Authors: Collo Ginetta , Pepper Michael S. 
Published: April 2021

ABSTRACT
Alterations in endothelial cell-extracellular matrix interactions are central to the process of angiogenesis. We have investigated the effect of wound-induced two-dimensional migration, basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF-β1) and leukemia inhibitory factor (LIF) on expression of the α5β1 integrin in endothelial cells. In multiple-wounded monolayers of bovine microvascular endothelial (BME) cells, an increase in mRNA and total protein for both α5 and β1 subunits was observed, and this could be correlated with a reduction in cell density but not proliferation, both of which are induced following wounding. Although as previously reported, the α5 subunit was increased when cells were exposed to TGF-β1 alone, co-addition of bFGF and TGF-β1 resulted in a striking synergistic induction of α5, with no significant changes in the expression of β1. In contrast, the α5 subunit was decreased by LIF in bovine aortic endothelial but not in BME cells. These findings suggest that quantitative alterations in α5 and β1 integrin subunit expression modulate the adhesive and migratory properties of endothelial cells during angiogenesis.
Phosphatidylinositol 3-kinase translocation to the nucleus is induced by interleukin 1 and prevented by mutation of interleukin 1 receptor in human osteosarcoma Saos-2 cells
Authors: Bavelloni Alberto , Santi Spartaco , Sirri Alessandra , Riccio Massimo , Faenza Irene , Zini Nicoletta , Cecchi Stefania , Ferri Alberto , Auron Philip , Maraldi Nadir M. , Marmiroli Sandra 
Published: April 2021

ABSTRACT
Although interleukin 1 (IL-1) functions have been extensively characterized, the mechanisms by which IL-1 signals are transduced from the plasma membrane to the nucleus are less known. Recent evidence indicates that phosphatidylinositol 3-kinase (PI3-kinase) could be activated by a direct association with the activated IL-1 receptor. In this study we analyzed the effects of IL-1 on the intracellular distribution of PI3-kinase in wild-type Saos-2 human osteosarcoma cells, and in cell clones overexpressing type I IL-1 receptor (IL-1RI). PI3-kinase intracellular distribution displays two distinct patterns. In quiescent cells, PI3-kinase is distributed through the cytoplasm, although a portion is present in the nucleus; following stimulation with IL-1, PI3-kinase is redistributed, increasing in the nuclear compartment. Both immunoblotting and immunofluorescence data indicate that IL-1 causes a rapid and transient translocation of PI3-kinase from the cytoplasm to the nucleus. This phenomenon is prevented by PI3-kinase inhibitors, suggesting that the maintenance of PI3-kinase activity is essential for IL-1-induced translocation. Indeed, in cell clones stably transfected with Y479F receptor mutant, in which the binding of the enzyme to the activated receptor is blocked, IL-1-induced PI3-kinase translocation to the nucleus is completely prevented. These data suggest that PI3-kinase translocation to the nucleus upon IL-1R activation is an early event in IL-1 signaling mechanism, and may be involved in transcriptional activation.
Bouquet formation in budding yeast: Initiation of recombination is not required for meiotic telomere clustering
Authors: Trelles-Sticken Edgar , Loidl Josef , Scherthan Harry 
Published: April 2021

ABSTRACT
Fluorescence in situ hybridization in combination with synaptonemal complex and spindle pole body immunostaining to both spread and structurally preserved nuclei from time course experiments disclosed prominent telomere clustering during meiotic prophase of the yeast Saccharomyces cerevisiae. It was found that centromere clustering, which dominates vegetative nuclear structure, is rapidly lost after induction of meiosis. Telomeres tightly clustered during leptotene/zygotene-equivalent stages in the vicinity of the spindle pole body, giving rise to a classical chromosomal bouquet arrangement. This arrangement dissolved later during prophase. Painting of chromosomes XI revealed that initially compacted chromosome territories adopt an outstretched morphology in bouquet nuclei. This conformational state was associated with alignment and pairing. Chromosome condensation during pachytene rendered condensed and compact bivalents, and dispersed telomeres. Both the spo11 and rad50S recombination mutants formed bouquets, demonstrating that bouquet formation is recombination and synapsis independent.
Cytological characterisation of the mutant phenotypes produced during early embryogenesis by null and loss-of-function alleles of the γTub37C gene in Drosophila
Authors: Llamazares Salud , Tavosanis Gaia , Gonzalez Cayetano 
Published: April 2021

ABSTRACT
We have studied the mutant phenotypes brought about during early embryogenesis by mutation in the γTub37C gene, one of the two isoforms of γ-tubulin that have been identified in Drosophila. We have focused our attention on fs(2)TW11 and fs(2)TW1RU34, a null and a hypomorph allele of this gene, whose sequences we report in this work. We have found that the abnormal meiotic figures observed in mutant stage 14 oocytes are not observed in laid oocytes or fertilised embryos, suggesting that these abnormal meiotic figures are not terminally arrested. We have also concluded that both null and hypomorph alleles lead to a total arrest of nuclear proliferation during early embryogenesis. This is in contrast to their effect on female meiosis-I where hypomorph alleles display a much weaker phenotype. Finally, we have observed that null and hypomorph alleles lead to some distinct phenotypes. Unfertilised laid oocytes and fertilised embryos deficient for γTub37C do not contain polar bodies and have a few bipolar microtubule arrays. In contrast, oocytes and embryos from weaker alleles do not have these microtubule arrays, but do contain polar bodies, or polar-body-like structures. These results indicate that γTub37C is essential for nuclear proliferation in the early Drosophila embryo.