scholarly journals Conserved function of the lysine-based KXD/E motif in Golgi retention for endomembrane proteins among different organisms

2015 ◽  
Vol 26 (23) ◽  
pp. 4280-4293 ◽  
Author(s):  
Cheuk Hang Woo ◽  
Caiji Gao ◽  
Ping Yu ◽  
Linna Tu ◽  
Zhaoyue Meng ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Transgenic Arabidopsis ◽  
Morphological Changes ◽  
Retention Mechanism ◽  
Loss Of Function ◽  
Expression Systems ◽  
Conserved Motif ◽  
Ultrastructural Studies ◽  
Golgi Retention

We recently identified a new COPI-interacting KXD/E motif in the C-terminal cytosolic tail (CT) of Arabidopsis endomembrane protein 12 (AtEMP12) as being a crucial Golgi retention mechanism for AtEMP12. This KXD/E motif is conserved in CTs of all EMPs found in plants, yeast, and humans and is also present in hundreds of other membrane proteins. Here, by cloning selective EMP isoforms from plants, yeast, and mammals, we study the localizations of EMPs in different expression systems, since there are contradictory reports on the localizations of EMPs. We show that the N-terminal and C-terminal GFP-tagged EMP fusions are localized to Golgi and post-Golgi compartments, respectively, in plant, yeast, and mammalian cells. In vitro pull-down assay further proves the interaction of the KXD/E motif with COPI coatomer in yeast. COPI loss of function in yeast and plants causes mislocalization of EMPs or KXD/E motif–containing proteins to vacuole. Ultrastructural studies further show that RNA interference (RNAi) knockdown of coatomer expression in transgenic Arabidopsis plants causes severe morphological changes in the Golgi. Taken together, our results demonstrate that N-terminal GFP fusions reflect the real localization of EMPs, and KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes.

scholarly journals Antileishmanial Activity of a Linalool-Rich Essential Oil from Croton cajucara

2003 ◽  
Vol 47 (6) ◽  
pp. 1895-1901 ◽  
Author(s):  
Maria do Socorro S. Rosa ◽  
Ricardo R. Mendonça-Filho ◽  
Humberto R. Bizzo ◽  
Igor de Almeida Rodrigues ◽  
Rosangela Maria A. Soares ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Essential Oil ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Peritoneal Macrophages ◽  
Transmission Electron ◽  
Low Concentrations ◽  
Mouse Peritoneal Macrophages ◽  
Croton Cajucara

ABSTRACT The in vitro leishmanicidal effects of a linalool-rich essential oil from the leaves of Croton cajucara against Leishmania amazonensis were investigated. Morphological changes in L. amazonensis promastigotes treated with 15 ng of essential oil per ml were observed by transmission electron microscopy; leishmanial nuclear and kinetoplast chromatin destruction, followed by cell lysis, was observed within 1 h. Pretreatment of mouse peritoneal macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these macrophages and L. amazonensis, with a concomitant increase by 220% in the level of nitric oxide production by the infected macrophages. Treatment of preinfected macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these cells and the parasites, which led to a 60% increase in the amount of nitric oxide produced by the preinfected macrophages. These results provide new perspectives on the development of drugs with activities against Leishmania, as linalool-rich essential oil is a strikingly potent leishmanicidal plant extract (50% lethal doses, 8.3 ng/ml for promastigotes and 8.7 ng/ml for amastigotes) which inhibited the growth of L. amazonensis promastigotes at very low concentrations (MIC, 85.0 pg/ml) and which presented no cytotoxic effects against mammalian cells.

scholarly journals The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia.

1996 ◽  
Vol 16 (9) ◽  
pp. 5069-5080 ◽  
Author(s):  
R Kozma ◽  
S Ahmed ◽  
A Best ◽  
L Lim
Keyword(s):  
Mammalian Cells ◽  
Phorbol Esters ◽  
Morphological Changes ◽  
Neuroblastoma Cells ◽  
Leading Edge ◽  
Dominant Negative ◽  
Gtpase Activating Protein ◽  
Simultaneous Formation ◽  
Gdp Dissociation Inhibitor

n-Chimaerin is a GTPase-activating protein (GAP) mainly for Rac1 and less so for Cdc42Hs in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1(T17N) and Cdc42Hs(T17N), respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding was ineffective in inducing morphological changes, while a mutant lacking GAP activity alone was effective. Microinjected n-chimaerin colocalized in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases.

scholarly journals Targeting of SET/I2PP2A oncoprotein inhibits Gli1 transcription revealing a new modulator of Hedgehog signaling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Iliana Serifi ◽  
Simoni Besta ◽  
Zoe Karetsou ◽  
Panagiota Giardoglou ◽  
Dimitris Beis ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Hedgehog Signaling ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Patterning Defect ◽  
Hh Signaling ◽  
Expression In Mammalian Cells

AbstractThe Hedgehog (Hh)/Gli signaling pathway controls cell proliferation and differentiation, is critical for the development of nearly every tissue and organ in vertebrates and is also involved in tumorigenesis. In this study, we characterize the oncoprotein SET/I2PP2A as a novel regulator of Hh signaling. Our previous work has shown that the zebrafish homologs of SET are expressed during early development and localized in the ciliated organs. In the present work, we show that CRISPR/Cas9-mediated knockdown of setb gene in zebrafish embryos resulted in cyclopia, a characteristic patterning defect previously reported in Hh mutants. Consistent with these findings, targeting setb gene using CRISPR/Cas9 or a setb morpholino, reduced Gli1-dependent mCherry expression in the Hedgehog reporter zebrafish line Tg(12xGliBS:mCherry-NLS). Likewise, SET loss of function by means of pharmacological inhibition and gene knockdown prevented the increase of Gli1 expression in mammalian cells in vitro. Conversely, overexpression of SET resulted in an increase of the expression of a Gli-dependent luciferase reporter, an effect likely attributable to the relief of the Sufu-mediated inhibition of Gli1. Collectively, our data support the involvement of SET in Gli1-mediated transcription and suggest the oncoprotein SET/I2PP2A as a new modulator of Hedgehog signaling.

scholarly journals In vitro screening of 65 mycotoxins for insecticidal potential

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248772
Author(s):  
Mieczysława Irena Boguś ◽  
Anna Katarzyna Wrońska ◽  
Agata Kaczmarek ◽  
Martyna Boguś-Sobocińska
Keyword(s):  
Cell Proliferation ◽  
Secondary Metabolites ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Animal Health ◽  
Economic Losses ◽  
In Vitro Screening ◽  
Cytochalasin E ◽  
Hc Toxin

The economic losses and threats to human and animal health caused by insects and the pathogens transmitted by them require effective and environmentally-friendly methods of controlling them. One such group of natural biocontrol agents which may be used as biopesticides is that of the entomopathogenic fungi and their toxic secondary metabolites (mycotoxins). The present in vitro work examined the insecticidal potential of 65 commercially-available mycotoxins against the insect Sf-9 cell line. Mammalian Caco-2 and THP-1 cell lines served as reference controls to select insecticidal mycotoxins harmless to mammalian cells. All tested mycotoxins significantly reduced the in vitro proliferation of the Sf-9 cells and evoked morphological changes. Ten of the mycotoxins found to strongly inhibit Sf-9 proliferation also had moderate or no effect on Caco-2 cells. The THP-1 cells were highly resistant to the tested mycotoxins: doses 103 times higher were needed to affect viability and morphology (1 μg/ml for THP-1 versus 1 ng/ml for Sf-9 and Caco-2). Nine mycotoxins significantly decreased Sf-9 cell proliferation with minor effects on mammalian cells: cyclosporins B and D, cytochalasin E, gliotoxin, HC toxin, paxilline, penitrem A, stachybotrylactam and verruculogen. These may be good candidates for future biopesticide formulations.

scholarly journals A novel role for kynurenine 3-monooxygenase in mitochondrial dynamics

PLoS Genetics ◽  
2020 ◽  
Vol 16 (11) ◽  
pp. e1009129
Author(s):  
Daniel C. Maddison ◽  
Mónica Alfonso-Núñez ◽  
Aisha M. Swaih ◽  
Carlo Breda ◽  
Susanna Campesan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Translational Regulation ◽  
Metabolic Flux ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Kynurenine Pathway ◽  
Loss Of Function ◽  
Disease Associated Genes

The enzyme kynurenine 3-monooxygenase (KMO) operates at a critical branch-point in the kynurenine pathway (KP), the major route of tryptophan metabolism. As the KP has been implicated in the pathogenesis of several human diseases, KMO and other enzymes that control metabolic flux through the pathway are potential therapeutic targets for these disorders. While KMO is localized to the outer mitochondrial membrane in eukaryotic organisms, no mitochondrial role for KMO has been described. In this study, KMO deficient Drosophila melanogaster were investigated for mitochondrial phenotypes in vitro and in vivo. We find that a loss of function allele or RNAi knockdown of the Drosophila KMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably, cinnabar genetically interacts with the Parkinson’s disease associated genes Pink1 and parkin, as well as the mitochondrial fission gene Drp1, implicating KMO in mitochondrial dynamics and mitophagy, mechanisms which govern the maintenance of a healthy mitochondrial network. Overexpression of human KMO in mammalian cells finds that KMO plays a role in the post-translational regulation of DRP1. These findings reveal a novel mitochondrial role for KMO, independent from its enzymatic role in the kynurenine pathway.

scholarly journals The Pseudomonas aeruginosa ExoY phenotype of high-copy-number recombinants is not detectable in natural isolates

Open Biology ◽  
2018 ◽  
Vol 8 (1) ◽  
pp. 170250 ◽  
Author(s):  
Antje Munder ◽  
Justin Rothschuh ◽  
Bastian Schirmer ◽  
Jens Klockgether ◽  
Volkhard Kaever ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Copy Number ◽  
Mammalian Cells ◽  
Real Life ◽  
High Copy Number ◽  
Infection Model ◽  
Loss Of Function ◽  
Natural Isolates

The nucleotidyl cyclase ExoY is an effector protein of the type III secretion system of Pseudomonas aeruginosa . We compared the cyclic nucleotide production and lung disease phenotypes caused by the ExoY-overexpressing strain PA103Δ exoUexoT::Tc pUCP exoY , its vector control strain PA103Δ exoUexoT::Tc pUCP18, its loss-of-function control PA103Δ exoUexoT::Tc pUCP exoY K81M and natural ExoY-positive and ExoY-negative isolates in a murine acute airway infection model. Only the P. aeruginosa carrier of the exoY- plasmid produced high levels of cUMP and caused the most severe course of infection. The pathology ascribed to ExoY from studies using the high-copy-number plasmid on mammalian cells in vitro and in vivo was not observed with natural P. aeruginosa isolates. This indicates that the role of ExoY during infection with real-life P. aeruginosa still needs to be resolved.

scholarly journals Ultrastructural Analysis of Differentiation in Legionella pneumophila

2002 ◽  
Vol 184 (24) ◽  
pp. 7025-7041 ◽  
Author(s):  
Gary Faulkner ◽  
Rafael A. Garduño
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Freeze Fracture ◽  
Physiological Adaptation ◽  
Developmental Cycle ◽  
Cytoplasmic Inclusions ◽  
Conserved Sequence

ABSTRACT Legionella pneumophila is an adaptive pathogen that replicates in the intracellular environment of fundamentally divergent hosts (freshwater protozoa and mammalian cells) and is capable of surviving long periods of starvation in water when between hosts. Physiological adaptation to these quite diverse environments seems to be accompanied by morphological changes (Garduño et al., p. 82-85, in Marre et al., ed., Legionella, 2001) and conceivably involves developmental differentiation. In following the fine-structural pathway of L. pneumophila through both in vitro and in vivo growth cycles, we have now discovered that this bacterium displays an unprecedented number of morphological forms, as revealed in ultrathin sections and freeze-fracture replicas for transmission electron microscopy. Many of the forms were identified by the obvious ultrastructural properties of their cell envelope, which included changes in the relative opaqueness of membrane leaflets, vesiculation, and/or profuse invagination of the inner membrane. These changes were best documented with image analysis software to obtain intensity tracings of the envelope in cross sections. Also prominent were changes in the distribution of intramembranous particles (clearly revealed in replicas of freeze-fractured specimens) and the formation of cytoplasmic inclusions. Our results confirm that L. pneumophila is a highly pleomorphic bacterium and clarify some early observations suggesting sporogenic differentiation in L. pneumophila. Since morphological changes occurred in a conserved sequence within the growth cycle, our results also provide strong evidence for the existence of a developmental cycle in L. pneumophila that is likely accompanied by profound physiological alterations and stage-specific patterns of gene expression.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

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