scholarly journals The Compound U18666A Inhibits the Intoxication of Cells by Clostridioides difficile Toxins TcdA and TcdB

2021 ◽  
Vol 12 ◽  
Author(s):  
Panagiotis Papatheodorou ◽  
Selina Kindig ◽  
Adriana Badilla-Lobo ◽  
Stephan Fischer ◽  
Ebru Durgun ◽  
...  
Keyword(s):  
Mode Of Action ◽  
Intracellular Transport ◽  
Cultured Cells ◽  
Clinical Symptoms ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Cholesterol Content ◽  
Host Cells ◽  
Membrane Cholesterol ◽  
Clostridioides Difficile

The intestinal pathogen Clostridioides (C.) difficile is a major cause of diarrhea both in hospitals and outpatient in industrialized countries. This bacterium produces two large exotoxins, toxin A (TcdA) and toxin B (TcdB), which are directly responsible for the onset of clinical symptoms of C. difficile-associated diseases (CDADs), such as antibiotics-associated diarrhea and the severe, life-threatening pseudomembranous colitis. Both toxins are multidomain proteins and taken up into host eukaryotic cells via receptor-mediated endocytosis. Within the cell, TcdA and TcdB inactivate Rho and/or Ras protein family members by glucosylation, which eventually results in cell death. The cytotoxic mode of action of the toxins is the main reason for the disease. Thus, compounds capable of inhibiting the cellular uptake and/or mode-of-action of both toxins are of high therapeutic interest. Recently, we found that the sterol regulatory element-binding protein 2 (SREBP-2) pathway, which regulates cholesterol content in membranes, is crucial for the intoxication of cells by TcdA and TcdB. Furthermore, it has been shown that membrane cholesterol is required for TcdA- as well as TcdB-mediated pore formation in endosomal membranes, which is a key step during the translocation of the glucosyltransferase domain of both toxins from endocytic vesicles into the cytosol of host cells. In the current study, we demonstrate that intoxication by TcdA and TcdB is diminished in cultured cells preincubated with the compound U18666A, an established inhibitor of cholesterol biosynthesis and/or intracellular transport. U18666A-pretreated cells were also less sensitive against TcdA and TcdB variants from the epidemic NAP1/027 C. difficile strain. Our study corroborates the crucial role of membrane cholesterol for cell entry of TcdA and TcdB, thus providing a valuable basis for the development of novel antitoxin strategies in the context of CDADs.

The Effect of Oxidized Cholesterol on the Aorta of Rabbits- Scanning Electron Microscopic Observations

1982 ◽  
Vol 40 ◽  
pp. 340-341
Author(s):  
S. K. Pena ◽  
C. B. Taylor ◽  
J. Hill ◽  
J. Safarik
Keyword(s):  
Cholesterol Biosynthesis ◽  
Cholesterol Content ◽  
Arterial Injury ◽  
Electron Microscopic ◽  
Aortic Smooth Muscle ◽  
Oxidized Cholesterol ◽  
Initial Event ◽  
Membrane Structure And Function ◽  
Scanning Electron Microscopic ◽  
And Function

Introduction: Oxidized cholesterol derivatives have been demonstrated in various cell cultures to be very potent inhibitors of 3-hvdroxy-3- methylglutaryl Coenzyme A reductase which is a principle regulator of cholesterol biosynthesis in the cell. The cholesterol content in the cells exposed to oxidized cholesterol was found to be markedly decreased. In aortic smooth muscle cells, the potency of this effect was closely related to the cytotoxicity of each derivative. Furthermore, due to the similarity of their molecular structure to that of cholesterol, these oxidized cholesterol derivatives might insert themselves into the cell membrane, alter membrane structure and function and eventually cause cell death. Arterial injury has been shown to be the initial event of atherosclerosis.

scholarly journals The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

2021 ◽  
Vol 22 (6) ◽  
pp. 2926
Author(s):  
Dinendra L. Abeyawardhane ◽  
Raquel Godoy-Ruiz ◽  
Kaylin A. Adipietro ◽  
Kristen M. Varney ◽  
Richard R. Rustandi ◽  
...  
Keyword(s):  
Host Cell ◽  
The Elderly ◽  
Cell Receptor ◽  
Host Cells ◽  
Binary Toxin ◽  
Clostridioides Difficile ◽  
Host Cell Receptor ◽  
Oligomeric Assembly ◽  
Nosocomial Disease ◽  
Binary Toxins

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

scholarly journals Nucleosome Positioning on Episomal Human Papillomavirus DNA in Cultured Cells

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 772
Author(s):  
Isao Murakami ◽  
Takashi Iwata ◽  
Tohru Morisada ◽  
Kyoko Tanaka ◽  
Daisuke Aoki
Keyword(s):  
Life Cycle ◽  
Cultured Cells ◽  
Basal Layer ◽  
Nucleosome Positioning ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Host Cells ◽  
Micrococcal Nuclease ◽  
Hpv Dna ◽  
Dna Replication And Repair

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that changes in nucleosome positions on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

scholarly journals Regulation of cholesterol biosynthesis in cultured cells by probable natural precursor sterols.

1980 ◽  
Vol 255 (2) ◽  
pp. 395-400 ◽  
Author(s):  
G.F. Gibbons ◽  
C.R. Pullinger ◽  
H.W. Chen ◽  
W.K. Cavenee ◽  
A.A. Kandutsch
Keyword(s):  
Cultured Cells ◽  
Cholesterol Biosynthesis ◽  
Natural Precursor

Sterol and lipid trafficking in mammalian cells

2006 ◽  
Vol 34 (3) ◽  
pp. 335-339 ◽  
Author(s):  
F.R. Maxfield ◽  
M. Mondal
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Vesicular Transport ◽  
Cholesterol Content ◽  
Cellular Functions ◽  
Lipid Trafficking ◽  
Sterol Transport ◽  
A Cell ◽  
Asymmetrically Distributed

The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.

The adrenal paraneurone: tubulin organization

1984 ◽  
Vol 62 (5) ◽  
pp. 502-511 ◽  
Author(s):  
M. F. Bader ◽  
F. Bernier-Valentin ◽  
B. Rousset ◽  
D. Aunis
Keyword(s):  
Cell Body ◽  
Intracellular Transport ◽  
Cultured Cells ◽  
Specific Binding ◽  
Secretory Granules ◽  
Immunofluorescent Staining ◽  
Chromaffin Granules ◽  
Two Dimensional Gel Electrophoresis ◽  
Granule Membrane

When chromaffin cells from the bovine adrenal medulla are maintained in culture, they develop neuritelike processes which end with growth-cone-like structures. Chromaffin granules were found to migrate from the cell body to the neurite endings. Thus, the intracellular transport of secretory granules, existing in vivo, seems to occur in an exaggerated way in the cultured cells. These cells offer an excellent model for studying the mechanism of transport, particularly the role of microtubules. By immunofluorescent staining, we observed that tubulin antibodies decorate a complex network visible along the neurites. Colchicine treatment induced the disappearance of this network followed by a return of granules in the cell body and a retraction of neurites. To test the presence of tubulin in the chromaffin granule membrane, we used two-dimensional gel electrophoresis and a radioimmunoassay. Our results indicate that tubulin is not a significant component of chromaffin granules. However, binding experiments show that granule membranes are able to bind tubulin through high affinity binding sites. These results show that microtubules appear involved in neurite formation and probably in granule transport. Tubulin is not an integral constituent of the granule membrane, but is present as a result of a reversible specific binding. This insertion of tubulin into the membrane might represent a step in the association between microtubules and secretory granules.

Inhibition of retroviral replication by anti-sense RNA

1986 ◽  
Vol 6 (12) ◽  
pp. 4758-4762
Author(s):  
R Y To ◽  
S C Booth ◽  
P E Neiman
Keyword(s):  
Steady State ◽  
Cultured Cells ◽  
Host Cells ◽  
Rna Molecules ◽  
Sense Orientation ◽  
Steady State Levels ◽  
Rna Inhibition ◽  
Retroviral Replication ◽  
Retroviral Infections ◽  
Neomycin Resistance

We tested the effect of anti-sense RNA on the replication of avian retroviruses in cultured cells. The replication of a recombinant retrovirus carrying a neomycin resistance gene (neor) in the anti-sense orientation was blocked when the cells expressed high steady-state levels of RNA molecules with neor in sequence in the sense was blocked when the cells expressed high steady-state levels of RNA molecules with neor sequences in the sense orientation, i.e., complementary to the viral sequence. Viral DNA bearing neor sequences was not detected specifically in host cells where this anti-sense RNA inhibition of viral replication occurred. These observations suggest that anti-sense RNA inhibition may be a useful strategy for the inhibition of retroviral infections.

TAMI-62. METHIONINE METABOLISM CLOSELY RELATED WITH SELF-RENEW, PLURIPOTENCY AND CELL DEATH IN GICS THROUGH MODIFICATION OF CHOLESTEROL BIOSYNTHESIS, RIBOSOMAL RNA AND AUTOPHAGY

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi211-vi211
Author(s):  
Kiyotaka Yokogami ◽  
Hideo Takeshima
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Ribosomal Rna ◽  
Cell Activation ◽  
Cultured Cells ◽  
Cholesterol Metabolism ◽  
Cholesterol Biosynthesis ◽  
Dna Demethylation ◽  
Methionine Metabolism ◽  
Starting Point

Abstract Glioma initiating cells (GICs) are the source of glioma cells that have the ability to self-renew and pluripotency, which are treatment-resistant, starting point for relapse and eventual death despite multimodality therapy. Since high accumulation is observed in 11cMet-PET at the time of recurrence, it is important to understand the mechanism of tumor cell activation caused by the reorganization of methionine metabolism. We cultured cells in methionine-deprived culture medium and performed a comprehensive analysis, and found that methionine depletion markedly decreased proliferation and increasing cell death of GICs. Decreased SAM, which is synthesized intracellularly catalyzed by methionine adenosyltransferase (MAT) using methionine, triggered the following: (i) global DNA demethylation, (ii) hyper-methylation of signaling pathways regulating pluripotentcy of stem cells, (iii) decreased expression of the core-genes and pluripotent marker of stem cells, (iv) decreased cholesterol synthesis and increased excretion mainly through decreased SREBF2 and FOXM1, (v) down-regulation of the large subunit of ribosomal protein configured 28S and ACA43, snoRNA guiding the pseudouridylation of 28S ribosomal RNA, which has crucial role for translation and (vi) possible connection between methionine metabolism and pluripotency, protein synthesis through cholesterol metabolism: SREBF2-FOXM1 and ACA43 axis, respectively. (vii) Disruption of autophagy by insufficient formation of macroautophagosomes. In conclusion, methionine metabolism closely related with self-renew, pluripotency and cell death in GICs through modification of cholesterol biosynthesis, ribosomal RNA and autophagy.

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