bluuebunny/crossref_metadata_2025_split huggingface dataset:
Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+
Authors: Meunier Frédéric A. , Mattei Cesar , Chameau Pascal , Lawrence Gary , Colasante Cesare , Kreger Arnold S. , Dolly J. Oliver , Molgó Jordi 
Published: April 2021

ABSTRACT
Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+influx through voltage-activated Ca2+channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.
Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model
Authors: Muraglia Anita , Cancedda Ranieri , Quarto Rodolfo 
Published: April 2021

ABSTRACT
Bone marrow stromal cells can give rise to several mesenchymal lineages. The existence of a common stem/progenitor cell, the mesenchymal stem cell, has been proposed, but which developmental stages follow this mesenchymal multipotent progenitor is not known. Based on experimental evidence, a model of mesenchymal stem cell differentiation has been proposed in which individual lineages branch directly from the same progenitor. We have verified this model by using clonal cultures of bone marrow derived stromal fibroblasts. We have analyzed the ability of 185 non-immortalized human bone marrow stromal cell clones to differentiate into the three main lineages: osteo-, chondro- and adipogenic. All clones but one differentiated into the osteogenic lineage. About one third of the clones differentiated into all three lineages analyzed. Most clones (60-80%) displayed an osteo-chondrogenic potential. We have never observed clones with a differentiation potential limited to the osteo-adipo- or to the chondro-adipogenic phenotype, nor pure chondrogenic and adipogenic clones. How long the differentiation potential of a number of clones was maintained was assessed throughout their life span. Clones progressively lost their adipogenic and chondrogenic differentiation potential at increasing cell doublings.
Our data suggest a possible model of predetermined bone marrow stromal cells differentiation where the tripotent cells can be considered as early mesenchymal progenitors that display a sequential loss of lineage potentials, generating osteochondrogenic progenitors which, in turn, give rise to osteogenic precursors.
Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments
Authors: Bursch Wilfried , Hochegger Karin , Török Ladislaus , Marian Brigitte , Ellinger Adolf , Hermann Rolf Schulte 
Published: April 2021

ABSTRACT
Programmed cell death comprises several subtypes, as revealed by electron microscopy. Apoptosis or type I programmed cell death is characterized by condensation of cytoplasm and preservation of organelles, essentially without autophagic degradation. Autophagic cell death or type II programmed cell death exhibits extensive autophagic degradation of Golgi apparatus, polyribosomes and endoplasmatic reticulum, which precedes nuclear destruction. In the present study, we analysed the fate of cytokeratin and F-actin during autophagic cell death in the human mammary carcinoma cell line MCF-7 because recent studies suggest that an intact cytoskeleton is necessary for autophagocytosis. Programmed cell death was induced by 10−6M tamoxifen. For quantitative light microscopic analysis, autophagic vacuoles were visualized by monodansyl cadaverin, which stains autophagic vacuoles as distinct dot-like structures. In control cultures, the number of monodansylcadaverin-positive cells did not exceed 2%. Tamoxifen induced a dramatic increase 2-4 days after treatment to a maximum of 60% monodansylcadaverin-positive cells between days 5 and 7. Cell death, as indicated by nuclear condensation, increased more gradually to about 18% of all cells on day 7. In cells with pyknotic nuclei cytokeratin appeared disassembled but retained its immunoreactivity; actin was still polymerized to filaments, as demonstrated by its reaction with phalloidin. Western blot analysis showed no significant cleavage of the monomeric cytokeratin fraction. For comparison, apoptotic or type I cell death was studied using the human colon cancer cell HT29/HI1 treated with the tyrosine kinase inhibitor tyrphostin A25 as a model. Cleavage of cytokeratin was already detectable in early morphological stages of apoptosis. F-actin was found to depolymerize; its globular form could be detected by antibodies; western blot analysis revealed no products of proteolytic cleavage. In conclusion, in our model of apoptosis, early stages are associated with depolymerization of actin and degradation of intermediate filaments. In contrast, during autophagic cell death intermediate and microfilaments are redistributed, but largely preserved, even beyond the stage of nuclear collapse. The present data support the concept that autophagic cell death is a separate entity of programmed cell death that is distinctly different from apoptosis.
Bidirectional transcytosis of IgG by the rat neonatal Fc receptor expressed in a rat kidney cell line: a system to study protein transport across epithelia
Authors: McCarthy Kevin M. , Yoong Yinlee , Simister Neil E. 
Published: April 2021

ABSTRACT
The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across cellular barriers between mother and offspring. FcRn also protects circulating IgG from catabolism, probably during transport across the capillary endothelium. Only one cell culture model of transcytosis has been used extensively, the transport of IgA from the basolateral to the apical surface of Madin-Darby canine kidney cells by the polymeric immunoglobulin receptor (pIgR). We report that rat inner medullary collecting duct (IMCD) cells transfected with DNA encoding the α subunit of rat FcRn specifically and saturably transport Fc when grown as polarized monolayers. Using this system, we have found that transcytosis by FcRn, like transcytosis by the pIgR, depends upon an intact microtubule system. FcRn differs most strikingly from the pIgR in its ability to transport its ligand in both the apical to basolateral and basolateral to apical directions. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibited basolateral to apical transport by FcRn more than apical to basolateral transport, suggesting that there are differences in the mechanisms of transport in the two directions. Lastly, we found that transcytosis by FcRn depends upon vesicular acidification. We anticipate that the IMCD cell culture model will allow further elucidation of the mechanism of IgG transport by FcRn.
The p120 catenin family: Complex roles in adhesion, signaling and cancer
Authors: Anastasiadis Panos Z. , Reynolds Albert B. 
Published: April 2021

ABSTRACT
p120 catenin (p120) is the prototypic member of a growing subfamily of Armadillo-domain proteins found at cell- cell junctions and in nuclei. In contrast to the functions of the classical catenins (α-catenin, β-catenin, and γ- catenin/plakoglobin), which have been studied extensively, the first clues to p120’s biological function have only recently emerged, and its role remains controversial. Nonetheless, it is now clear that p120 affects cell-cell adhesion through its interaction with the highly conserved juxtamembrane domain of classical cadherins, and is likely to have additional roles in the nucleus. Here, we summarize the data on the potential involvement of p120 both in promotion of and in prevension of adhesion, and propose models that attempt to reconcile some of the disparities in the literature. We also discuss the structural relationships and functions of several known p120 family members, as well as the potential roles of p120 in signaling and cancer.
Phospholipase A2 and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells
Authors: Shirai Yasuhito , Kashiwagi Kaori , Sakai Norio , Saito Naoaki 
Published: April 2021

ABSTRACT
The signal transduction involved in the purinergic stimuli- induced activation of protein kinase C (PKC) in CHO-K1 cells was investigated. Purinergic stimuli such as adenosine triphosphate and uridine triphosphate induced a transient translocation of PKC ε, γ, and δ from the cytoplasm to the plasma membrane. These translocations were blocked by an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), but not by an inhibitor of phosphatidylcholine- specific PLC. A diacylglycerol (DAG) analogue also induced reversible translocations of PKC γ, ε, and δ from the cytoplasm to the plasma membrane, while the calcium ionophore A23187 caused a similar translocation of only the γ subtype. These results confirm that the hydrolysis of phosphatidylinositol-2-phosphate by PLC and the subsequent generation of DAG and increase in Ca2+ are involved in the purinergic stimuli-induced translocation of PKC. A DAG antagonist, 1-o-hexadecyl-2-o-acetyl-glycerol, blocked the DAG analogue-induced translocations of all PKC subtypes tested but failed to inhibit the purinergic stimuli-induced translocations of PKC ε and γ. The DAG antagonist could not block the ATP- and UTP-induced translocation of PKC ε even in the absence of extracellular Ca2+. Co-application of the DAG antagonist and a phospholipase A2 (PLA2) inhibitor such as aristolochic acid, arachidonyltrifluoromethyl ketone, or bromoenol lactone inhibited the purinergic receptor-mediated translocation of PKC ε although each PLA2 inhibitor alone did not block the translocation. In contrast to the ε subtype, ATP-induced translocation of PKC γ was observed in the presence of both the PLA2 inhibitor and the DAG antagonist. However, it is noteworthy that re-translocation of PKC γ was hastened by the PLA2 inhibitor. Furthermore products of PLA2, such as lysophospholipids and fatty acids, induced the translocation of PKC γ and ε in a dose dependent manner, but not δ.
These results indicate that, in addition to PLC and DAG, PLA2 and its products are involved in the purinergic stimuli-induced translocation of PKC ε and γ in CHO-K1 cells. Each subtype of PKC in CHO-K1 cell is individually activated in response to a purinergic stimulation.
Human tau filaments induce microtubule and synapse loss in an in vivo model of neurofibrillary degenerative disease
Authors: Hall Garth F. , Chu Boyang , Lee Gloria , Yao Jun 
Published: April 2021

ABSTRACT
The intracellular accumulation of tau protein and its aggregation into filamentous deposits is the intracellular hallmark of neurofibrillary degenerative diseases such as Alzheimer’s Disease and familial tauopathies in which tau is now thought to play a critical pathogenic role. Until very recently, the lack of a cellular model in which human tau filaments can be experimentally generated has prevented direct investigation of the causes and consequences of tau filament formation in vivo. In this study, we show that human tau filaments formed in lamprey central neurons (ABCs) that chronically overexpress human tau resemble the ‘straight filaments’ seen in Alzheimer’s Disease and other neurofibrillary conditions, and are distinguishable from neurofilaments by their ultrastructure, distribution and intracellular behavior. We also show that tau filament formation in ABCs is associated with a distinctive pattern of dendritic degeneration that closely resembles the cytopathology of human neurofibrillary degenerative disease. This pattern includes localized cytoskeletal disruption and aggregation of membranous organelles, distal dendritic beading, and the progressive loss of dendritic microtubules and synapses. These results suggest that tau filament formation may be responsible for many key cytopathological features of neurofibrillary degeneration, possibly via the loss of microtubule based intracellular transport.
ARVCF localizes to the nucleus and adherens junction and is mutually exclusive with P120ctn in E-cadherin complexes
Authors: Mariner Deborah J. , Wang Jue , Reynolds Albert B. 
Published: April 2021

ABSTRACT
ARVCF is a novel Armadillo repeat domain protein that is closely related to the catenin p120ctn. Using new ARVCF monoclonal antibodies, we have found that ARVCF associates with E-cadherin and competes with p120 for interaction with the E-cadherin juxtamembrane domain. ARVCF also localized to the nucleus in some cell types, however, and was significantly more nucleophilic than p120. Surprisingly, despite apparently ubiquitous expression, ARVCF was at least tenfold less abundant than p120 in a wide variety of cell types, and was difficult to detect by immunofluorescence unless overexpressed. Consequently, it is not likely to be abundant enough in adult tissues to functionally compete with p120. ARVCF also completely lacked the ability to induce the cell-branching phenotype associated with overexpression of p120. Expression of ARVCF/p120 chimeras confirmed previous results indicating that the branching activity of p120 maps to its Armadillo repeat domain. Surprisingly, the preferential localization of ARVCF to the nucleus required sequences in the amino-terminal end of ARVCF, suggesting that the sequences directing nuclear translocation of ARVCF are distinct from the predicted bipartite nuclear localization signal located between repeats 6 and 7. The dual localization of ARVCF to junctions and to nuclei suggests activities in different cellular compartments, as is the case for several other Armadillo repeat proteins including β-catenin, p120 and the plakophilins.
The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of βI-spectrin
Authors: Flick Matthew J. , Konieczny Stephen F. 
Published: April 2021

ABSTRACT
Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural integrity of the contractile apparatus. The ability of MLP to regulate myogenesis is particularly interesting since it exhibits multiple subcellular localizations, being found in both nuclear and cytoplasmic compartments. Despite extensive biochemical analyses on MLP, the mechanism(s) by which it influences the myogenic program remains largely undefined. To further examine the role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners. From this screen, the cytoskeletal protein βI-spectrin was isolated. Protein interaction assays demonstrate that MLP and βI-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions. βI-spectrin binds specifically to MLP but not to the MLP related proteins CRP1 and CRP2 or to other LIM domain containing proteins. The MLP:β-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of β-spectrin. Confocal microscopy studies also reveal that MLP co-localizes with β-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue. Given that β-spectrin is a known costamere protein, we propose that sarcolemma-associated MLP also serves as a key costamere protein, stabilizing the association of the contractile apparatus with the sarcolemma by linking the β-spectrin network to the α-actinin crosslinked actin filaments of the myofibril.
TNF-α stimulates activation of pro-MMP2 in human skin through NF-κB mediated induction of MT1-MMP
Authors: Han Yuan-Ping , Tuan Tai-Lan , Wu Huayang , Hughes Michael , Garner Warren L. 
Published: April 2021

ABSTRACT
Tumor necrosis factor-alpha (TNF-α) is an important mediator during the inflammatory phase of wound healing. Excessive amounts of pro-inflammatory cytokines such as TNF-α are associated with inflammatory diseases including chronic wounds. Matrix metalloproteinases (MMPs) are involved in matrix re-modeling during wound healing, angiogenesis and tumor metastasis. As with pro- inflammatory cytokines, high levels of MMPs have been found in inflammatory states such as chronic wounds. In this report we relate these two phenomena. TNF-α stimulates secretion of active MMP-2, a type IV collagenase, in organ-cultured full-thickness human skin. This suggests a mechanism whereby excess inflammation affects normal wound healing.
To investigate this observation at the cellular and molecular levels, we examined TNF-α mediated activation of pro-MMP-2, induction of MT1-MMP, and the intracellular signaling pathways that regulate the proteinase in isolated human dermal fibroblasts. We found that TNF-α substantially promoted activation of pro- MMP-2 in dermal fibroblasts embedded in type-I collagen. In marked contrast, collagen or TNF-α individually had little influence on the fibroblast-mediated pro-MMP-2 activation. One well-characterized mechanism for pro- MMP-2 activation is through a membrane type matrix metalloproteinase, such as MT1-MMP. We report that TNF-α significantly induced MT1-MMP at the mRNA and protein levels when the dermal fibroblasts were grown in collagen. Although the intracellular signaling pathway regulating mt1-mmp gene expression is still obscure, both TNF-α and collagen activate the NF-κB pathway. In this report we provide three sets of evidence to support a hypothesis that activation of NF-κB is essential to induce MT1-MMP expression in fibroblasts after TNF-α exposure. First, SN50, a peptide inhibitor for NF-κB nuclear translocation, simultaneously blocked the TNF-α and collagen mediated MT1-MMP induction and pro-MMP-2 activation. Secondly, TNF-α induced IκB to breakdown in fibroblasts within the collagen lattice, a critical step leading to NF-κB activation. Lastly, a consensus binding site for p65 NF-κB (TGGAGCTTCC) was found in the 5′-flanking region of human mt1-mmp gene.
Based on these results and previous reports, we propose a model to explain TNF-α activation of MMP-2 in human skin. Activation of NF-κB signaling in fibroblasts embedded in collagen induces mt1-mmp gene expression, which subsequently activates the pro-MMP-2. The findings provide a specific mechanism whereby TNF-α may affect matrix remodeling during wound healing and other physiological and pathological processes.
Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins
Authors: Sun Dongming , Leung Conrad L. , Liem Ronald K. H. 
Published: April 2021

ABSTRACT
MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C- terminal domain that includes two EF-hand calcium- binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3′ end of the MACF cDNA; hence, we refer to it as MACF2. The C-terminal domain of mouse MACF2 was cloned and characterized. The microtubule-binding properties of MACF2 C-terminal domain are similar to that of MACF. The GAR domain was originally found in Gas 2 protein and here we show that it can associate with MTs in transfected cells. Plectin and desmoplakin have GSR-containing domains at their C- termini and we further demonstrate that the GSR- containing domain of plectin, but not desmoplakin, can bind to MTs in vivo.
A novel nucleolar G-protein conserved in eukaryotes
Authors: Park Jeong-Hyun , Jensen Bryan C. , Kifer Charles T. , Parsons Marilyn 
Published: April 2021

ABSTRACT
We describe here a novel, evolutionarily conserved set of predicted G-proteins. The founding member of this family, TbNOG1, was identified in a two-hybrid screen as a protein that interacts with NOPP44/46, a nucleolar phosphoprotein of Trypanosoma brucei. The biological relevance of the interaction was verified by co-localization and co-immunoprecipitation. TbNOG1 localized to the trypanosome nucleolus and interacted with domains of NOPP44/46 that are found in several other nucleolar proteins. Genes encoding proteins highly related to TbNOG1 are present in yeast and metazoa, and related G domains are found in bacteria. We show that NOG1 proteins in humans and Saccharomyces cerevisae are also nucleolar. The S. cerevisae NOG1 gene is essential for cell viability, and mutations in the predicted G motifs abrogate function. Together these data suggest that NOG1 may play an important role in nucleolar functions. The GTP-binding region of TbNOG1 is similar to those of Obg and DRG proteins, which, together with NOG, form a newly recognized family of G-proteins, herein named ODN. The ODN family differs significantly from other G-protein families, and shows several diagnostic sequence characteristics. All organisms appear to possess an ODN gene, pointing to the biological significance of this family of G-proteins.