scholarly journals Inhibiting coronavirus replication in cultured cells by chemical ER stress

2020 ◽  
Author(s):  
Mohammed Samer Shaban ◽  
Christin Müller ◽  
Christin Mayr-Buro ◽  
Hendrik Weiser ◽  
Benadict Vincent Albert ◽  
...  
Keyword(s):  
Er Stress ◽  
Cultured Cells ◽  
Specific Treatment ◽  
Cell Types ◽  
Data Sets ◽  
Human Pathogens ◽  
Essential Factor ◽  
Er Quality Control ◽  
Er Chaperone ◽  
Different Cell Types

AbstractCoronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. We found that the ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types, (partially) restores the virus-induced translational shut-down, and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide data sets revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including HERPUD1, an essential factor of ER quality control, and ER-associated protein degradation complexes. The data show that thapsigargin hits a central mechanism required for CoV replication, suggesting that thapsigargin (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.One Sentence Summary / Running titleSuppression of coronavirus replication through thapsigargin-regulated ER stress, ERQC / ERAD and metabolic pathways

scholarly journals Multi-level inhibition of coronavirus replication by chemical ER stress

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mohammed Samer Shaban ◽  
Christin Müller ◽  
Christin Mayr-Buro ◽  
Hendrik Weiser ◽  
Johanna Meier-Soelch ◽  
...  
Keyword(s):  
Er Stress ◽  
Specific Treatment ◽  
Cell Types ◽  
Autophagic Flux ◽  
Human Pathogens ◽  
Er Quality Control ◽  
Bronchial Epithelial ◽  
Infected Cells ◽  
Multi Level ◽  
Different Cell Types

AbstractCoronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

Cultured epithelial cells derived from human foetal pancreas as a model for the study of cystic fibrosis: further analyses on the origins and nature of the cell types

1988 ◽  
Vol 90 (1) ◽  
pp. 73-77
Author(s):  
A. Harris ◽  
L. Coleman
Keyword(s):  
Cystic Fibrosis ◽  
Tissue Culture ◽  
Epithelial Cells ◽  
Culture System ◽  
Cultured Cells ◽  
Cell Types ◽  
Cultured Epithelial Cells ◽  
Different Cell Types ◽  
Foetal Pancreas

The establishment of a tissue-culture system for epithelial cells derived from human foetal pancreas has recently been reported. Further analyses have now been made on these cells in vitro, together with parallel investigation of the distribution of different cell types within the intact foetal pancreas. Results support the view that the cultured cells are ductal in origin and nature. Pancreatic epithelial cell cultures have also been established from foetuses with cystic fibrosis.

scholarly journals Continuous spectrofluorometric measurements of uptake by cultured cells of 12-(1-pyrene)-dodecanoic acid from its complex with albumin

1988 ◽  
Vol 253 (2) ◽  
pp. 377-380 ◽  
Author(s):  
S Gatt ◽  
N Nahas ◽  
E Fibach
Keyword(s):  
Cultured Cells ◽  
Fluorescence Emission ◽  
Cell Types ◽  
Dodecanoic Acid ◽  
Direct Measurements ◽  
Fluorescence Emission Intensity ◽  
Covalently Linked ◽  
High Fluorescence ◽  
Different Cell Types ◽  
Very High

Aqueous dispersions of 12-(1-pyrene)-dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene has been covalently linked, shows a considerable increase in fluorescence when the probe is introduced into a hydrophobic environment. This enables the uptake of P12 by liposomes and cells to be followed directly in a spectrofluorometer, without separating the cells from the P12-containing medium. In the present study, we show that complexing P12 to albumin produced a very high fluorescence emission intensity. This made direct measurements of the uptake by cells of albumin-bound P12 impossible. Such direct measurements could, however, be made using albumin which had been interacted with trinitrobenzenesulphonic acid (TNBS). The yellow trinitrophenyl (TNP) residues, which were thereby covalently linked to the albumin, quenched the fluorescence of pyrene in the TNP-albumin/P12 complex. Upon release of the P12 molecules from this complex and their subsequent uptake by cells, fluorescence increased. This technique was utilized for the continuous monitoring of the uptake of P12 by different cell types and cells at various stages of maturation.

scholarly journals The Microtubule Plus-End Proteins EB1 and Dynactin Have Differential Effects on Microtubule Polymerization

2003 ◽  
Vol 14 (4) ◽  
pp. 1405-1417 ◽  
Author(s):  
Lee A. Ligon ◽  
Spencer S. Shelly ◽  
Mariko Tokito ◽  
Erika L.F. Holzbaur
Keyword(s):  
Cultured Cells ◽  
Cell Types ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Nucleation Effect ◽  
Microtubule Polymerization ◽  
The Individual ◽  
Different Cell Types ◽  
Dynactin Complex

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150Glued subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150Glued in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150Glued on microtubule polymerization, and they show that p150Gluedhas a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.

scholarly journals Detachment of cells from culture substrate by soluble fibronectin peptides.

1985 ◽  
Vol 100 (6) ◽  
pp. 1948-1954 ◽  
Author(s):  
E G Hayman ◽  
M D Pierschbacher ◽  
E Ruoslahti
Keyword(s):  
Cultured Cells ◽  
Cell Attachment ◽  
Cell Types ◽  
Soluble Form ◽  
Detectable Effect ◽  
Type I ◽  
Recognition Mechanism ◽  
Synthetic Cell ◽  
Attachment Mechanism ◽  
Different Cell Types

The synthetic cell attachment-promoting peptides from fibronectin (Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.)., 309:30-33) were found to detach cultured cells from the substratum when added to the culture in a soluble form. Peptides ranging in length from tetrapeptide to heptapeptide and containing the active L-arginyl-glycyl-L-aspartic acid (Arg-Gly-Asp) sequence had the detaching activity, whereas a series of different peptides with chemically similar structures had no detectable effect on any of the test cells. The Arg-Gly-Asp-containing peptides caused detachment of various cell lines of different species and histogenetic origin. Studies with defined substrates showed that the active peptides could inhibit the attachment of cells to vitronectin in addition to fibronectin, indicating that vitronectin is recognized by cells through a similar mechanism as fibronectin. The peptides did not inhibit the attachment of cells to collagen. However, cells cultured on collagen-coated plastic for 24-36 h, as well as cells with demonstrable type I or type VI collagen in their matrix, were susceptible to the detaching effect of the peptides. These results indicate that the recognition mechanism(s) by which cells bind to fibronectinand vitronectin plays a major role in the substratum attachment of cells and that collagens may not be directly involved in cell-substratum adhesion. Since vitronectin is abundant in serum, it is probably an important component in mediating the attachment of cultured cells. The independence of the effects of the peptide on the presence of serum and the susceptibility of many different cell types to detachment by the peptide show that the peptides perturb an attachment mechanism that is intrinsic to the cells and fundamentally significant to their adhesion.

scholarly journals ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish

2013 ◽  
Vol 24 (9) ◽  
pp. 1387-1395 ◽  
Author(s):  
Tokiro Ishikawa ◽  
Tetsuya Okada ◽  
Tomoko Ishikawa-Fujiwara ◽  
Takeshi Todo ◽  
Yasuhiro Kamei ◽  
...  
Keyword(s):  
Er Stress ◽  
The Body ◽  
Embryonic Lethality ◽  
Otic Vesicle ◽  
Medaka Fish ◽  
Double Knockout ◽  
Er Quality Control ◽  
Er Chaperone ◽  
Transcriptional Induction ◽  
Single Knockout

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

Visualization of Stat3 and Stat5 transactivation activity with specific response element dependent reporter constructs integrated into lentiviral gene transfer vectors

2010 ◽  
Vol 1 (3) ◽  
Author(s):  
Katrin Gäbel ◽  
Nadja Lydia Bednorz ◽  
Petra Klemmt ◽  
Vida Vafaizadeh ◽  
Corina Borghouts ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cancer Cells ◽  
Cultured Cells ◽  
Late Pregnancy ◽  
Cellular Transformation ◽  
Cell Types ◽  
Specific Response ◽  
Transactivation Activity ◽  
Transfer Vectors ◽  
Different Cell Types

Abstract: Signal transducer and activator of transcription 3 and 5 (Stat3 and Stat5) play important roles in cell differentiation, proliferation, apoptosis and inflammation. They are transiently activated by ligand-receptor interactions in normal cells but are often found to be constitutively active in cancer cells. Analysis of their activation pattern is therefore important for the description of developmental processes and the understanding of cellular transformation.: To visualize Stat3 and Stat5 transactivation activity in different cell types, we designed novel reporter constructs. These constructs comprise Stat3 or Stat5 specific promoter elements and reporter genes encoding β-galactosidase or fluorescent proteins. These constructs were integrated into lentiviral gene transfer vectors facilitating efficient transduction of most cell types.: The lentiviral reporter constructs were used to infect different cell types and their inducibility by activated Stat3 or Stat5 was measured. The Stat3-mCherry reporter was active in transduced tumor cells, which exhibit high levels of phosphorylated Stat3 and it was inducible in HepG2 liver cells by interleukin-6 treatment. The Stat5-LacZ reporter was active in cultured cells upon hormone induction of Stat5 and in primary mammary epithelial cells transplanted into cleared fat pads of mice during late pregnancy.: These novel reporter constructs are valuable tools to investigate and to distinguish between Stat3 and Stat5 activity in primary cells and cancer cells. They will also be useful in the discovery of drugs targeting Stat3 or Stat5. They can also be employed to generate transgenic mice and track Stat activity during development.

scholarly journals A modular microfluidic bioreactor to investigate plant cell–cell interactions

PROTOPLASMA ◽  
2021 ◽  
Author(s):  
T. Finkbeiner ◽  
C. Manz ◽  
M. L. Raorane ◽  
C. Metzger ◽  
L. Schmidt-Speicher ◽  
...  
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Cultured Cells ◽  
Abiotic Factors ◽  
Welding Process ◽  
Cell Types ◽  
Cell Interactions ◽  
Chemical Signalling ◽  
Different Cell Types ◽  
Cell Cell

AbstractPlants produce a wide variety of secondary metabolites, which often are of interest to pharmaceutical and nutraceutical industry. Plant-cell cultures allow producing these metabolites in a standardised manner, independently from various biotic and abiotic factors difficult to control during conventional cultivation. However, plant-cell fermentation proves to be very difficult, since these chemically complex compounds often result from the interaction of different biosynthetic pathways operating in different cell types. To simulate such interactions in cultured cells is a challenge. Here, we present a microfluidic bioreactor for plant-cell cultivation to mimic the cell–cell interactions occurring in real plant tissues. In a modular set-up of several microfluidic bioreactors, different cell types can connect through a flow that transports signals or metabolites from module to module. The fabrication of the chip includes hot embossing of a polycarbonate housing and subsequent integration of a porous membrane and in-plane tube fittings in a two-step ultrasonic welding process. The resulting microfluidic chip is biocompatible and transparent. Simulation of mass transfer for the nutrient sucrose predicts a sufficient nutrient supply through the membrane. We demonstrate the potential of this chip for plant cell biology in three proof-of-concept applications. First, we use the chip to show that tobacco BY-2 cells in suspension divide depending on a “quorum-sensing factor” secreted by proliferating cells. Second, we show that a combination of two Catharanthus roseus cell strains with complementary metabolic potency allows obtaining vindoline, a precursor of the anti-tumour compound vincristine. Third, we extend the approach to operationalise secretion of phytotoxins by the fungus Neofusicoccum parvum as a step towards systems to screen for interorganismal chemical signalling.

Transcriptional regulation of human genes for steroidogenic enzymes

1993 ◽  
Vol 39 (2) ◽  
pp. 333-340 ◽  
Author(s):  
D W Hum ◽  
W L Miller
Keyword(s):  
Cultured Cells ◽  
Cell Types ◽  
Steroidogenic Enzymes ◽  
Basal Expression ◽  
Chain Cleavage ◽  
Human Genes ◽  
Genes Encoding ◽  
Cleavage Enzyme ◽  
Adrenodoxin Reductase ◽  
Different Cell Types

Abstract Steroid hormones are synthesized from cholesterol in the adrenals, gonads, and placenta by a complex series of reactions. The human genes encoding each of these biosynthetic enzymes have been cloned, permitting study of their regulation. Tropic hormones, such as corticotropin and the gonadotropins, exert their chronic effects on steroidogenesis by increasing the amounts of steroidogenic enzymes; this in turn occurs primarily through increased gene transcription. Our studies have emphasized the cholesterol side-chain cleavage enzyme P450scc, which catalyzes the first and rate-limiting step in steroidogenesis, and P450c17, which determines what class of steroids is synthesized. By fusing the promoters of the genes for these enzymes to readily assayed reporter genes and transiently transfecting cultured cells with these constructions, we have identified the regions of each promoter that confer basal expression, induction by cAMP, and repression by activators of protein kinase C. Different segments of the P450scc promoter are used for each of these purposes in different cell types, indicating that the regulation of this gene is very complex. Transcription is not the only level at which steroidogenesis is regulated. The abundance of mRNA for adrenodoxin reductase, a flavoprotein needed for P450scc activity, is post-transcriptionally regulated by cAMP.

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