bluuebunny/crossref_metadata_2025_split huggingface dataset:
4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death
Authors: Liu Wei , Kato Masashi , Akhand Anwaral A, , Hayakawa Akemi , Suzuki Haruhiko , Miyata Toshio , Kurokawa Kiyoshi , Hotta Yoshihiro , Ishikawa Naohisa , Nakashima Izumi 
Published: April 2021

ABSTRACT
4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 μM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.
Tyrosine 766 in the fibroblast growth factor receptor-1 is required for FGF-stimulation of phospholipase C, phospholipase D, phospholipase A2, phosphoinositide 3-kinase and cytoskeletal reorganisation in porcine aortic endothelial cells
Authors: Cross Michael J. , Hodgkin Matthew N. , Roberts Sally , Landgren Eva , Wakelam Michael J. O. , Claesson-Welsh Lena 
Published: April 2021

ABSTRACT
Fibroblast growth factor-mediated signalling was studied in porcine aortic endothelial cells expressing either wild-type fibroblast growth factor receptor-1 or a mutant receptor (Y766F) unable to bind phospholipase C-γ. Stimulation of cells expressing the wild-type receptor resulted in activation of phospholipases C, D and A2and increased phosphoinositide 3-kinase activity. Stimulation of the wild-type receptor also resulted in stress fibre formation and a cellular shape change. Cells expressing the Y766F mutant receptor failed to stimulate phospholipase C, D and A2as well as phosphoinositide 3-kinase. Furthermore, no stress fibre formation or shape change was observed. Both the wild-type and Y766F receptor mutant activated MAP kinase and elicited proliferative responses in the porcine aortic endothelial cells. Thus, fibroblast growth factor receptor-1 mediated activation of phospholipases C, D and A2and phosphoinositide 3-kinase was dependent on tyrosine 766. Furthermore, whilst tyrosine 766 was not required for a proliferative response, it was required for fibroblast growth factor receptor-1 mediated cytoskeletal reorganisation.
Roles of caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies
Authors: Wang Jin , Lenardo Michael J. 
Published: April 2021

ABSTRACT
Caspases are a group of cysteine proteases critical for apoptosis of eukaryotic cells. Deletion of genes that encode murine caspases suggests that caspases are involved not only in apoptosis but also in cytokine maturation and cell growth and differentiation. Among them, caspase-1 and caspase-11 are primarily involved in the processing of pro-inflammatory cytokines. Caspase-3 and caspase-9 are essential for apoptosis during brain development. Caspase-8 is required for the development of heart muscle, cell proliferation in the hematopoietic lineage and death-receptor-mediated apoptosis. These studies suggest that caspases function in cell signaling events including apoptosis, cell growth and differentiation.
Calcium regulation of microtubule sliding in reactivated sea urchin sperm flagella
Authors: Bannai Hiroko , Yoshimura Misako , Takahashi Keiichi , Shingyoji Chikako 
Published: April 2021

ABSTRACT
The changes in the bending pattern of flagella induced by an increased intracellular Ca2+ concentration are caused by changes in the pattern and velocity of microtubule sliding. However, the mechanism by which Ca2+ regulates microtubule sliding in flagella has been unclear. To elucidate it, we studied the effects of Ca2+ on microtubule sliding in reactivated sea urchin sperm flagella that were beating under imposed head vibration. We found that the maximum microtubule sliding velocity obtainable by imposed vibration, which was about 170-180 rad/second in the presence of 250 μM MgATP and &lt;10−9 M Ca2+, was decreased by 10−6-10−5 M Ca2+ by about 15-20%. Similar decrease of the sliding velocity was observed at 54 and 27 μM MgATP. The Ca2+-induced decrease of the sliding velocity was due mainly to a decrease in the reverse bend angle. When the plane of beat was artificially rotated by rotating the plane of vibration of the pipette that held the sperm head, the asymmetric bending pattern also rotated at 10−5 M Ca2+ as well as at &lt;10−9 M Ca2+. The rotation of the bending pattern was observed at MgATP higher than 54 μM (∼100 μM ATP). These results indicate that the Ca2+-induced decrease of the sliding velocity is mediated by a rotatable component or components (probably the central pair) at high MgATP, but is not due to specific dynein arms on particular doublets. We further investigated the effects of a mild trypsin treatment and of trifluoperazine on the Ca2+-induced decrease in sliding velocity. Axonemes treated for 3 minutes with a low concentration (0.1 μg/ml) of trypsin beat with a more symmetrical waveform than before the treatment. Also, their microtubule sliding velocity and reverse bend angle were not affected by high Ca2+ concentrations. Trifluoperazine (25-50 μM) had no effect on the decrease of the sliding velocity in beating flagella at 10−5 M Ca2+. However, the flagella that had been ‘quiescent’ at 10−4 M Ca2+ resumed asymmetrical beating following an application of 10-50 μM trifluoperazine. In such beating flagella, both the sliding velocity and the reverse bend angle were close to their respective values at 10−5 M Ca2+. Trypsin treatment induced a similar recovery of beating in quiescent flagella at 10−4 M Ca2+, albeit with a more symmetrical waveform. These results provide first evidence that, at least at ATP concentrations higher than ∼100 μM, 10−6-10−5 M Ca2+ decreases the maximum sliding velocity of microtubules in beating flagella through a trypsin-sensitive regulatory mechanism which possibly involves the central pair apparatus. They also suggest that calmodulin may be associated with the mechanism underlying flagellar quiescence induced by 10−4 M Ca2+.
Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by α3β1, α6β1 and α6β4 integrins
Authors: Kikkawa Yamato , Sanzen Noriko , Fujiwara Hironobu , Sonnenberg Arnoud , Sekiguchi Kiyotoshi 
Published: April 2021

ABSTRACT
Laminin-10/11, the laminin isoforms containing the α5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin α3β1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin β1 antibody inhibited the adhesion of all cell types tested, anti-α3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-α3 and anti-α6 antibodies, suggesting that both α3β1 and α6β1 integrins function as laminin-10/11 receptors in these cells. To explore this possibility, we examined the adhesion of K562 leukemic cells transfected with integrin α3 or α6 subunit to laminin-10/11 or other laminin isoforms. Laminin-10/11 were potent adhesive ligands for both the α3β1 and α6β1 transfectants, whereas laminin-5 was the preferred ligand for the α3β1 transfectants. Upon stimulation with the activating anti-integrin β1 antibody, both transfectants became more adherent to the substratum regardless of the type of laminins coated, although their preference for laminin isoforms remained unaltered. K562 cells transfected with α6 and β4 subunits were also capable of adhering to laminin-10/11, indicating that integrin α6β4 is another receptor for laminin-10/11. Even with lung carcinoma cells, the α6-containing integrins partly contributed to adhesion to laminin-10/11 at higher coating concentrations, although non-integrin receptor(s) might also be involved under such conditions. These results indicated that laminin-10/11 are potent and versatile adhesive ligands in basement membranes capable of binding to both α3β1 and α6β1 integrins with high avidity and also to α6β4 integrin.
ARP1 in Golgi organisation and attachment of manchette microtubules to the nucleus during mammalian spermatogenesis
Authors: Fouquet Jean-Pierre , Kann Marie-Louise , Souès Sylvie , Melki Ronald 
Published: April 2021

ABSTRACT
Actin related protein of vertebrate, Arp1, is a major component of the dynactin complex. To characterise and localise Arp1 during mammalian spermatogenesis, polyclonal antibodies were raised against a human recombinant Arp1. Anti-Arp1 antibodies were used for western-immunoblotting, indirect immunofluorescence and immunoelectron microscopy. In round spermatids, Arp1 was detected at the centrosome and at the Golgi apparatus. In elongated spermatids, Arp1 was predominantly found along microtubules of the manchette and at their site of attachment to the nuclear envelope. In maturing spermatids, Arp1 was still present in the pericentriolar material, but in testicular spermatozoa it was not detectable. These various localisations of Arp1 and their changes during spermatid differentiation suggest that the dynactin complex in association with dynein might contribute to several activities: the functional organisation of the centrosome and of the Golgi apparatus and the shaping of the nucleus by manchette microtubules.
Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei
Authors: Li Cong-Jun , Bogan Joseph A. , Natale Darren A. , DePamphilis Melvin L. 
Published: April 2021

ABSTRACT
As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G1-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-β and β′) that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-β/β′ appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G1-phase of the cell division cycle, and their appearance did not depend on the presence of Xenopus Orc proteins. Therefore, hamster pre-replication complexes appear to be assembled at specific chromosomal sites during G1-phase. Third, selective activation of ori-β in late G1-nuclei depended on the ratio of Xenopus egg extract to nuclei, revealing that epigenetic parameters such as the ratio of initiation factors to DNA substrate could determine the number of origins activated.
Lysosome-endosome fusion and lysosome biogenesis
Authors: Luzio J. Paul , Rous Brian A. , Bright Nicholas A. , Pryor Paul R. , Mullock Barbara M. , Piper Robert C. 
Published: April 2021

ABSTRACT
Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.
Interferon γ regulates accumulation of the proteasome activator PA28 and immunoproteasomes at nuclear PML bodies
Authors: Fabunmi Rosalind P. , Wigley W. Christian , Thomas Philip J. , DeMartino George N. 
Published: April 2021

ABSTRACT
PA28 is an interferon γ (IFNγ inducible proteasome activator required for presentation of certain major histocompatibility (MHC) class I antigens. Under basal conditions in HeLa and Hep2 cells, a portion of nuclear PA28 is concentrated at promyelocytic leukemia oncoprotein (PML)-containing bodies also commonly known as PODs or ND10. IFNγ treatment greatly increased the number and size of the PA28- and PML-containing bodies, and the effect was further enhanced in serum- deprived cells. PML bodies are disrupted in response to certain viral infections and in diseases such as acute promyelocytic leukemia (APL). Like PML, PA28 was delocalized from PML bodies by expression of the cytomegalovirus protein, IE1, and in NB4 cells, an APL model line. Moreover, retinoic acid treatment, which causes remission of APL in patients and reformation of PML- containing bodies in NB4 cells, relocalized PA28 to this site. In contrast, the proteasome, the functional target of PA28, was not detected at PML bodies under basal conditions in HeLa and Hep2 cells, but IFNγ promoted accumulation of ‘immunoproteasomes’ at this site. These results establish PA28 as a novel component of nuclear PML bodies, and suggest that PA28 may assemble or activate immunoproteasomes at this site as part of its role in proteasome-dependent MHC class I antigen presentation.
The melanocortin 1 receptor is the principal mediator of the effects of agouti signaling protein on mammalian melanocytes
Authors: Abdel-Malek Zalfa A. , Scott M. Cathy , Furumura Minao , Lamoreux M. Lynn , Ollmann Michael , Barsh Greg S. , Hearing Vincent J. 
Published: April 2021

ABSTRACT
The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with α-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E+/E+, e/e, or Eso/Eso mice to α-MSH and/or ASP. E+/E+ melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and Eso/Eso is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to α-melanocyte stimulating hormone. Mouse E+/E+ melanocytes, but not e/e or Eso/Eso melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E+/E+ melanocytes does agouti signaling protein abrogate the stimulatory effects of α-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.
At the crossroads of cell biology and immunology: DRiPs and other sources of peptide ligands for MHC class I molecules
Authors: Yewdell Jonathan W. , Schubert Ulrich , Bennink Jack R. 
Published: April 2021

ABSTRACT
CD8+ T cells are a critical element of vertebrate immune responses to viruses and other intracellular parasites. They roam the body, monitoring cells for the presence of foreign peptides associated with MHC class I molecules of the major histocompatibility complex (MHC). Although it is clear that most of these peptides are generated through the action of proteasomes, the nature of the substrates degraded by proteasomes is an open question. Recent findings indicate that the major pool of substrates consists of a heterogeneous subset of proteins that are degraded within minutes of their synthesis. Evidence suggests that the fraction of newly synthesized proteins targeted for destruction is remarkably high – 30% or more, depending on cell type – possibly because they are defective in some way and cannot reach their intended conformation or location cellular in a time frame deemed appropriate by cells.
Regulation of the differentiation and behaviour of extra-embryonic endodermal cells by basement membranes
Authors: Murray Patricia , Edgar David 
Published: April 2021

ABSTRACT
Both the extracellular matrix and parathyroid hormone-related peptide (PTHrP) have been implicated in the differentiation and migration of extra-embryonic endodermal cells in the pre-implantation mammalian blastocyst. In order to define the individual roles and interactions between these factors in endodermal differentiation, we have used embryoid bodies derived from Lamc1−/− embryonic stem cells that lack basement membranes. The results show that in the absence of basement membranes, increased numbers of both visceral and parietal endodermal cells differentiate, but they fail to form organised epithelia. Furthermore, although parietal endodermal cells only migrate away from control embryoid bodies in the presence of PTHrP, they readily migrate from Lamc1−/− embryoid bodies in the absence of PTHrP, and this migration is unaffected by PTHrP. Thus, the basement membrane between epiblast and extra-embryonic endoderm is required for the proper organisation of visceral and parietal endodermal cells and also restricts their differentiation to maintain the population of primitive endodermal stem cells. Moreover, this basement membrane inhibits migration of parietal endodermal cells, the role of PTHrP being to stimulate delamination of parietal endodermal cells from the basement membrane rather than promoting migration per se.
Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase
Authors: Campbell Michael S. , Chan Gordon K. T. , Yen Tim J. 
Published: April 2021

ABSTRACT
Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells. HsMad1 is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find HsMad1 associated with HsMad2. Association with p55CDC, a protein previously shown to bind HsMad2, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins. The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.
Assembly into chromatin and subtype-specific transcriptional effects of exogenous linker histones directly introduced into a living Physarum cell
Authors: Thiriet Christophe , Hayes Jeffrey J. 
Published: April 2021

ABSTRACT
The apparent diversity of linker histone subtypes may be related to their specific roles in defining functional states of chromatin in vivo. We have developed a novel method to study constitutive peptides throughout the cell cycle and have demonstrated that an exogenous linker histone could be introduced into a living cell of the slime mold Physarum polycephalum. Here, we have used this method to assess the functional differences between three somatic linker histone subtypes in vivo, and to demonstrate the general applicability of this method. Exogenous linker histone proteins H1°, H5 and H1 were directly absorbed into living cell segments of the naturally synchronous Physarum macroplasmodia at precise cell cycle stages. Fluorescence microscopy, native nucleoprotein gels and immunoblotting of nuclei and chromatin with subtype-specific antibodies revealed that exogenous linker histones were efficiently transported into nuclei and were integrated into chromatin. The immunoreactivity of a preparation of anti- H1° antibodies that are blocked from binding to specific H1° epitopes in native chromatin indicates that the exogenous linker histones were similarly associated into Physarum chromatin. Interestingly, linker histones were found to be less stably associated with Physarum chromatin during S-phase than during G2-phase. Furthermore, we show that exogenous linker histones incorporated in early G2-phase inhibited transcription and that the level of inhibition correlates with the apparent role of the linker histone subtype in regulating transcription in cells where it normally occurs.