mammalian cell cultures
Recently Published Documents





I. Martínez‐Monge ◽  
C. Martínez ◽  
M. Decker ◽  
I. A. Udugama ◽  
I. Marín de Mas ◽  

10.1645/20-63 ◽  
2021 ◽  
Vol 107 (4) ◽  
David S. Lindsay ◽  
S. K. Verma ◽  
J. P. Dubey ◽  
David Scott ◽  
Alexa Rosypal von Dohlen

2021 ◽  
Sohei Yamada ◽  
Yasumasa Bessho ◽  
Yasuyuki Fijita ◽  
Yoichiroh Hosokawa ◽  
Takaaki Matsui

When oncogenic transformed or damaged cells appear within an epithelial sheet, they are apically extruded by surrounding cells. Recently, using cultured mammalian epithelial cells and zebrafish embryonic epithelial cells, we found that a calcium (Ca2+) wave propagates from RasV12-transformed cells and laser-irradiated damaged cells to surrounding cells and promotes apical extrusion by inducing polarized movements of the surrounding cells. In mammalian cell cultures, we reported that the inositol trisphosphate (IP3) receptor, gap junctions, and the mechanosensitive Ca2+ channel TRPC1 are involved in Ca2+ wave-mediated polarized movements. However, which molecules regulate Ca2+ wave-mediated polarized movements in zebrafish and whether the Ca2+ wave can generate a force remain unknown. In this study, we aimed to answer these questions. By performing pharmacological and gene knockout experiments, we showed that a Ca2+ wave induced by the IP3 receptor and trpc1 led to formation of cryptic-lamellipodia and polarized movements of surrounding cells toward extruding cells in zebrafish. By using an in vivo force measurement method, we found that the Ca2+ wave generated approximately 1 kPa of force toward extruding cells. Our results reveal a previously unidentified molecular mechanism underlying the Ca2+ wave in zebrafish and demonstrate that the Ca2+ wave generates a force during cell extrusion.

2021 ◽  
Taniya Bhardwaj ◽  
Kundlik Gadhave ◽  
Shivani Krishna Kapuganti ◽  
Prateek Kumar ◽  
Zacharias Faidon Brotzakis ◽  

The phenomenon of protein aggregation is widespread and associated with a wide range of human diseases. Our knowledge on the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated the distribution of aggregation-prone regions in the the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions in these proteomes, and revealed an enhanced aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone regions in the of SARS-CoV-2 proteome, including the signal sequence peptide and fusion peptide 1 of the spike protein, a peptide from the NSP6 protein (NSP6-p), the ORF10 protein, and the NSP11 protein. Our re-sults show that these peptides and proteins form aggregates via a nucleation-dependent mecha-nism. Moreover, we demonstrated that the aggregates of NSP11 are toxic to mammalian cell cultures. These findings provide evidence about the aggregation of proteins in the SARS-CoV-2 proteome.

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Michael J. Cohen ◽  
Brianne Philippe ◽  
Peter N. Lipke

ABSTRACT In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100 μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells. IMPORTANCE Eukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate nonconventional secretion more generally.

2020 ◽  
Vol 48 (22) ◽  
pp. 12804-12816
Amer Elias ◽  
Hala Kassis ◽  
Suha Abd Elkader ◽  
Natasha Gritsenko ◽  
Alessio Nahmad ◽  

Abstract HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active ‘attB’ sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native ‘attB’ sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native ‘attB’ sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.

MethodsX ◽  
2020 ◽  
pp. 101197
Bhavna Dixit ◽  
Shon Vanhoozer ◽  
Nana Abena Anti ◽  
Matthew S. O'Connor ◽  
Amutha Boominathan

Marzieh Marzbany ◽  
Mahsa Rasekhian ◽  

The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3′-untranslated region (3′UTR) from RNA viral genome to modulate mRNA stability. The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

Sign in / Sign up

Export Citation Format

Share Document