Extracellular vesicles of Trypanosoma cruzi tissue-culture cell-derived trypomastigotes: Induction of physiological changes in non-parasitized culture cells

The usual method of scraping or trypsinization to detach tissue culture cell sheets from their glass substrate for further pelletization and processing for electron microscopy introduces objectionable morphological alterations. It is also impossible under these conditions to study a particular area or individual cell which have been preselected by light microscopy in the living state.Several schemes which obviate centrifugation and allow the embedding of nondetached tissue culture cells have been proposed. However, they all preserve only a small part of the cell sheet and make use of inverted gelatin capsules which are in this case difficult to handle.We have evolved and used over a period of several years a technique which allows the embedding of a complete cell sheet growing at the inner surface of a tissue culture roller tube. Observation of the same cell by light microscopy in the living and embedded states followed by electron microscopy is performed conveniently.

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Facilitated nuclear transport of calmodulin in tissue culture cells.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1527 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1527-1536 ◽

Cited By ~ 48

Author(s):

M Pruschy ◽

Y Ju ◽

L Spitz ◽

E Carafoli ◽

D S Goldfarb

Keyword(s):

Tissue Culture ◽

Nuclear Transport ◽

Culture Cell ◽

Atp Depletion ◽

Tissue Culture Cell ◽

Permeabilized Cell ◽

Permeabilized Cells ◽

Culture Cells ◽

Cytosolic Factor ◽

Dependent Mechanism

Calmodulin (CaM) potentiates Ca(2+)-dependent signaling pathways in both the cytoplasm and nucleus. We have investigated the mechanism of CaM nuclear transport using tissue culture cell microinjection and a permeabilized cell import assay. The inhibition of CaM import by the translocation inhibitor wheat germ agglutinin (WGA) and by chilling, indicates that CaM import is facilitated, but because ATP depletion does not affect CaM import, the mechanism does not appear to be active. Chilling and WGA arrest persist in ATP-depleted cells, indicating that CaM is not retained in the cytoplasm by an ATP-dependent mechanism. In permeabilized cells, both Ca(2+)-CaM and Ca(2+)-free CaM are sensitive to extract-dependent WGA and chilling import inhibition. Titration experiments in microinjected and permeabilized cells indicate that a saturable cytosolic factor(s) mediates chilling and WGA arrest.

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Tissue-culture cell fractionation. Fractionation of membranes from tissue-culture cells homogenized by glycerol-induced lysis

Biochemical Journal ◽

10.1042/bj1820157 ◽

1979 ◽

Vol 182 (1) ◽

pp. 157-164 ◽

Cited By ~ 2

Author(s):

J M Graham ◽

J K Sandall

Keyword(s):

Tissue Culture ◽

Culture Cell ◽

Enzyme Analysis ◽

Tissue Culture Cell ◽

Cell Fractionation ◽

Culture Cells ◽

Tissue Culture Cells ◽

Mitochondrial Fractions ◽

Complete Cell ◽

Yield And Purity

1. The disruption of various types of tissue-culture cells by (a) incubation in solutions of 1.2 M-glycerol and (b) transfer of the glycerol-loaded cells to relatively hypo-osmotic solutions of 0.25 M-sucrose was studied. 2. Bivalent cations (2mM-Mg2+) were generally included to preserve the nuclei, but some cells (polyoma-virus-transformed baby-hamster kidney cells) failed to be disrupted adequately under these conditions. 3. Other cells (mouse-embryo fibroblasts) required additional gentle Dounce homogenization to effect complete cell breakage. 4. Purification of the whole homogenate was carried out by a combination of differential centrifugation and sedimentation or flotation through sucrose gradients. 5. Enzyme analysis showed that plasma-membrane, endoplasmic-reticulum and mitochondrial fractions were obtained in good yield and purity.

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Tolevamer, an Anionic Polymer, Neutralizes Toxins Produced by the BI/027 Strains of Clostridium difficile

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00041-08 ◽

2008 ◽

Vol 52 (6) ◽

pp. 2190-2195 ◽

Cited By ~ 27

Author(s):

Paul L. Hinkson ◽

Carol Dinardo ◽

Daniel DeCiero ◽

Jeffrey D. Klinger ◽

Robert H. Barker

Keyword(s):

Tissue Culture ◽

Clostridium Difficile ◽

Bacterial Flora ◽

Culture Cell ◽

Binary Toxin ◽

Tissue Culture Cell ◽

Normal Flora ◽

Anionic Polymer ◽

Culture Cells

ABSTRACTClostridium difficile-associated diarrhea (CDAD) is caused by the toxins the organism produces when it overgrows in the colon as a consequence of antibiotic depletion of normal flora. Conventional antibiotic treatment of CDAD increases the likelihood of recurrent disease by again suppressing normal bacterial flora. Tolevamer, a novel toxin-binding polymer, was developed to ameliorate the disease without adversely affecting normal flora. In the current study, tolevamer was tested for its ability to neutralize clostridial toxins produced by the epidemic BI/027 strains, thereby preventing toxin-mediated tissue culture cell rounding. The titers of toxin-containingC. difficileculture supernatants were determined using confluent cell monolayers, and then the supernatants were used in assays containing dilutions of tolevamer to determine the lowest concentration of tolevamer that prevented ≥90% cytotoxicity. Tolevamer neutralized toxins in the supernatants of allC. difficilestrains tested. Specific antibodies against the large clostridial toxins TcdA and TcdB also neutralized the cytopathic effect, suggesting that tolevamer is specifically neutralizing these toxins and that the binary toxin (whose genes are carried by the BI/027 strains) is not a significant source of cytopathology against tissue culture cells in vitro.

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Biophysical and Biochemical Comparison of Extracellular Vesicles Produced by Infective and Non-Infective Stages of Trypanosoma cruzi

International Journal of Molecular Sciences ◽

10.3390/ijms22105183 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5183

Author(s):

Lissette Retana Moreira ◽

Alexa Prescilla-Ledezma ◽

Alberto Cornet-Gomez ◽

Fátima Linares ◽

Ana Belén Jódar-Reyes ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Extracellular Vesicles ◽

Cell Communication ◽

Lipid Vesicles ◽

Eukaryotic Cell ◽

Culture Cell ◽

Etiologic Agent ◽

Tissue Culture Cell ◽

Physicochemical Composition ◽

Biochemical Comparison

Extracellular vesicles (EVs) are small lipid vesicles released by either any prokaryotic or eukaryotic cell, or both, with a biological role in cell-to-cell communication. In this work, we characterize the proteomes and nanomechanical properties of EVs released by tissue-culture cell-derived trypomastigotes (mammalian infective stage; (TCT)) and epimastigotes (insect stage; (E)) of Trypanosoma cruzi, the etiologic agent of Chagas disease. EVs of each stage were isolated by differential centrifugation and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy and atomic force microscopy (AFM). Measurements of zeta-potential were also included. Results show marked differences in the surface molecular cargos of EVs between both stages, with a noteworthy expansion of all groups of trans-sialidase proteins in trypomastigote’s EVs. In contrast, chromosomal locations of trans-sialidases of EVs of epimastigotes were dramatically reduced and restricted to subtelomeric regions, indicating a possible regulatable expression of these proteins between both stages of the parasite. Regarding mechanical properties, EVs of trypomastigotes showed higher adhesion compared to the EVs of epimastigotes. These findings demonstrate the remarkable surface remodeling throughout the life cycle of T. cruzi, which shapes the physicochemical composition of the extracellular vesicles and could have an impact in the ability of these vesicles to participate in cell communication in completely different niches of infection.

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