Influence of cell background on pharmacological rescue of mutant CFTR

2010 ◽  
Vol 298 (4) ◽  
pp. C866-C874 ◽  
Author(s):  
Nicoletta Pedemonte ◽  
Valeria Tomati ◽  
Elvira Sondo ◽  
Luis J. V. Galietta
Keyword(s):  
Direct Action ◽  
A549 Cells ◽  
Extreme Case ◽  
Cell Types ◽  
Biochemical Assays ◽  
Cftr Protein ◽  
Rat Thyroid ◽  
Cell Expression ◽  
Different Levels

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs the maturation and gating of the CFTR protein. Such defects may be corrected in vitro by pharmacological modulators named as correctors and potentiators, respectively. We have evaluated a panel of correctors and potentiators derived from various sources to assess potency, efficacy, and mechanism of action. For this purpose, we have used functional and biochemical assays on two different cell expression systems, Fischer rat thyroid (FRT) and A549 cells. The order of potency and efficacy of potentiators was similar in the two cell types considered, with phenylglycine PG-01 and isoxazole UCCF-152 being the most potent and least potent, respectively. Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. In contrast, corrector effect was strongly affected by cell background, with the extreme case of many compounds working in one cell type only. Our findings are in favor of a direct action of potentiators on CFTR, possibly at a common binding site. In contrast, most correctors seem to work indirectly with various mechanisms of action. Combinations of correctors acting at different levels may lead to additive F508del-CFTR rescue.

Novel Disulfide-Containing Poly(β-amino ester)-Functionalised Magnetic Nanoparticles for Efficient Gene Delivery

2016 ◽  
Vol 69 (3) ◽  
pp. 349 ◽  
Author(s):  
Yucheng Liu ◽  
Shufeng Li ◽  
Liandong Feng ◽  
Hao Yu ◽  
Xiaoliang Qi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Magnetic Nanoparticles ◽  
Disulfide Bonds ◽  
Transfection Efficiency ◽  
A549 Cells ◽  
Weight Ratio ◽  
Cell Types ◽  
Amino Ester ◽  
Gene Complexes

Poly(β-amino ester)s (PBAEs) have been proved to effectively transfer DNA to various cell types. However, PBAEs with high molecular weights also show considerable toxicities, partly resulting from inadequate degradation of their polyester backbone. In this study, we created novel poly(β-amino ester)s (SF-1, 2, 3, and 4; notation SFs refers to all the four polymers) which were characterised by the cleavable disulfide bonds. Moreover, a new technique, termed magnetofection that uses magnetic nanoparticles to enhance gene expression, has recently been well developed. The negatively charged magnetic nanoparticles (MNPs) with good biocompatibility in vitro were prepared here to subsequently combine with SFs and DNA via electrostatic interaction, leading to the formation of the magnetic gene complexes MNP/SFs/DNA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and transfection experiments were performed in A549 cells to investigate all the resulting complexes. Studies indicated that the synthesised PBAEs exhibited good biodegradation and regulated release of DNA as a result of the reductive cleavage of the disulfide bonds, giving higher transfection efficiency along with much lower cytotoxicity compared with commercially available transfection agent polyethylenimine (Mw 25 kDa). Furthermore, when MNP was involved at a MNP/DNA weight ratio of 0.5, the magnetic gene complexes MNP/SFs/DNA showed enhanced levels of gene expression while maintaining low cytotoxicity.

scholarly journals Novel cost-efficient protein-membrane based system for cells co-cultivation and modeling the intercellular communication

2021 ◽  
Author(s):  
Artem Minin ◽  
Igor Blatov ◽  
Valeria Lebedeva ◽  
Maxim Tuchai ◽  
Varvara Pozdina ◽  
...  
Keyword(s):  
Growth Factors ◽  
A549 Cells ◽  
Cell Types ◽  
Homogeneous Population ◽  
Paracrine Effects ◽  
Expression Of Genes ◽  
Custom Made ◽  
Cost Efficient ◽  
Different Cell Types

In vitro systems serve as compact and manipulate models to investigate interactions between different cell types. A homogeneous population of cells predictably and uniformly responds to external factors. In a heterogeneous cell population, the effect of external growth factors is perceived in the context of intercellular interactions. Indirect cell co-cultivation allows one to observe the paracrine effects of cells and separately analyze cell populations. The article describes an application of custom-made cell co-cultivation systems based on protein membranes separated from the bottom of the vessel by the 3d printed holder or kept afloat by a magnetic field. Using the proposed co-cultivation system, we analyzed the interaction of A549 cells and fibroblasts, in the presence and absence of growth factors. During co-cultivation of cells, the expression of genes of the activation for epithelial and mesenchymal transitions decreases. The article proposes the application of a newly available system for the co-cultivation of different cell types.

scholarly journals Alkaline proteinase localization in myoblasts.

1987 ◽  
Vol 35 (1) ◽  
pp. 83-86 ◽  
Author(s):  
W T Stauber ◽  
V K Fritz ◽  
B Dahlmann ◽  
J Kay ◽  
R Heath ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cysteine Proteinase ◽  
Cell Types ◽  
Biochemical Assays ◽  
Strong Localization ◽  
Catabolic Potential ◽  
Chromatin Material ◽  
Immunohistochemical Techniques

Recent interest in elucidating the role of non-lysosomal proteases in intracellular protein catabolism in muscle has led to various investigations with three alkaline proteases: a trypsin-like, a chymotrypsin-like, and a high molecular weight cysteine proteinase. Although in vitro biochemical assays have revealed the catabolic potential of at least two of these proteases, confirmation of their presence in muscle cells has been difficult. In this study immunohistochemical techniques were employed to localize each of these proteases in rat myoblasts. Antisera against the trypsin-like and chymotrypsin-like proteinase (both serine proteinases) showed strong localization in the cytoplasm immediately around the nucleus. Both also stained chromatin material in the nucleus of these cells. Fluorescent localization of the high molecular weight cysteine proteinase (Proteinase I) also appeared to be cell-associated in the myoblasts. The use of myoblasts in cell culture sections of whole muscle was advantageous, since localization of the proteases could be assessed in the absence of other cell types.

In vitro evaluation of cobalt oxide nanoparticle-induced toxicity

2017 ◽  
Vol 33 (8) ◽  
pp. 646-654 ◽  
Author(s):  
Mahmoud Abudayyak ◽  
Tuba Altincekic Gurkaynak ◽  
Gül Özhan
Keyword(s):  
Nervous System ◽  
Cobalt Oxide ◽  
Cell Lines ◽  
Neuroblastoma Cells ◽  
A549 Cells ◽  
Cell Types ◽  
Carcinoma Cells ◽  
High Exposure

Cobalt oxide (Co3O4) nanoparticles have applications in nanomedicine and nanotechnology; therefore, any possible adverse effects require thorough investigation. The present study investigated the effects of Co3O4 nanoparticles on four different cell lines: liver, HepG2 hepatocellular carcinoma cells; lung, A549 lung carcinoma cells; gastrointestinal, Caco-2 colorectal adenocarcinoma cells; and nervous system, SH-SY5Y neuroblastoma cells. A difference was observed in cell sensitivity toward Co3O4 nanoparticles. Co3O4 nanoparticles were taken up by all the cell types. However, no cell death was observed in HepG2, Caco-2, or SH-SY5Y cells; only A549 cells showed cytotoxicity at relatively high exposure concentrations. Co3O4 nanoparticles did not induce DNA damage or apoptosis in the cell lines tested except in A549. Interestingly, Co3O4 nanoparticles induced cellular oxidative damage in all cell types except Caco-2, resulting in increased malondialdehyde and 8-hydroxydeoxyguanosine levels and decreased glutathione levels. According to our results, it could be indicated that high concentrations of Co3O4 nanoparticles affected the pulmonary system but were unlikely to affect the liver, nervous system, or gastrointestinal system. Co3O4 nanoparticles might be safely used for industrial, commercial, and nanomedical applications if dose rates are adjusted depending on the route of exposure. However, further in vivo and in vitro studies are required to confirm the safety of Co3O4 nanoparticles.

scholarly journals Renal and Hematological Effects of CLCF-1, a B-Cell-Stimulating Cytokine of the IL-6 Family

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Virginia J. Savin ◽  
Mukut Sharma ◽  
Jianping Zhou ◽  
David Gennochi ◽  
Timothy Fields ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Types ◽  
Urine Albumin ◽  
Stat3 Phosphorylation ◽  
Hematological Effects ◽  
Multiple Cell ◽  
Target Molecules ◽  
Cell Expression

CLCF-1 is a cytokine known for B-cell stimulation and for neurotrophic properties. We have identified CLCF-1 as a potential injurious factor in the human renal disease focal segmental glomerulosclerosis (FSGS). We investigated its effects on renal cells and renal function inin vitroandin vivostudies. Methods include measurement of the effect of CLCF-1 on phosphorylation of target molecules of the JAK/STAT pathway, on cytoskeleton and cell morphology in cultured podocytes, on albumin permeability of isolated rat glomeruli, and on tissue phosphorylation and urine albumin after acute or chronic CLCF-1 injection. In addition, cell sorting was performed to determine the presence of cells expressing CLCF-1 in spleen and bone marrow of normal mice and the effect of CLCF-1 infusion on splenic B-cell populations. CLCF-1 increased phosphorylation of STAT3 in multiple cell types, activated podocytes leading to formation of lamellipodia and decrease in basal stress fibers, increased glomerular albumin permeability, and increased STAT3 phosphorylation of peripheral blood cells and renal cortex. CLCF-1 increased urine albumin/creatinine ratio in mice and increased B-cell expression of IgG in mouse spleen. We conclude that CLCF-1 has potentially important systemic effects, alters podocyte function, and may contribute to renal dysfunction and albuminuria.

scholarly journals Effect of Endothelin-1 on the Excitability of Rat Cortical and Hippocampal Slices In Vitro

2012 ◽  
pp. 215-219 ◽  
Author(s):  
R. KONOPKOVÁ ◽  
I. VILAGI ◽  
S. BORBELY ◽  
H. KUBOVÁ ◽  
J. OTÁHAL
Keyword(s):  
Direct Action ◽  
Hippocampal Slices ◽  
Cell Types ◽  
Evoked Response ◽  
Output Curve ◽  
Vascular Effects ◽  
Endothelin 1 ◽  
Acute Seizures

Endothelin-1 (ET-1) is a neuroactive protein produced in most brain cell types and participates in regulation of cerebral blood flow and blood pressure. In addition to its vascular effects, ET-1 affects synaptic and nonsynaptic neuronal and glial functions. Direct application of ET-1 to the hippocampus of immature rats results in cerebral ischemia, acute seizures, and epileptogenesis. Here, we investigated whether ET-1 itself modifies the excitability of hippocampal and cortical circuitry and whether acute seizures observed in vivo are due to nonvascular actions of ET-1. We used acute hippocampal and cortical slices that were preincubated with ET-1 (20 µM) for electrophysiological recordings. None of the slices preincubated with ET-1 exhibited spontaneous epileptic activity. The slope of the stimulus intensity-evoked response (input-output) curve and shape of the evoked response did not differ between ET-1-pretreated and control groups, suggesting no changes in excitability after ET-1 treatment. The threshold for eliciting an evoked response was not significantly increased in either hippocampal or cortical regions when pretreated with ET-1. Our data suggest that acute seizures after intrahippocampal application of ET-1 in rats are likely caused by ischemia rather than by a direct action of ET-1 on brain tissue.

scholarly journals Puumala and Tula Virus Differ in Replication Kinetics and Innate Immune Stimulation in Human Endothelial Cells and Macrophages

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 855 ◽  
Author(s):  
Bourquain ◽  
Bodenstein ◽  
Schürer ◽  
Schaade
Keyword(s):  
Endothelial Cells ◽  
A549 Cells ◽  
Hemorrhagic Fever ◽  
Cell Types ◽  
Innate Immune ◽  
Tula Virus ◽  
Lung Epithelial ◽  
Culture Models ◽  
Different Cell Types

Old world hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) upon zoonotic transmission to humans. In Europe, the Puumala virus (PUUV) is the main causative agent of HFRS. Tula virus (TULV) is also widely distributed in Europe, but there is little knowledge about the pathogenicity of TULV for humans, as reported cases are rare. We studied the replication of TULV in different cell types in comparison to the pathogenic PUUV and analyzed differences in stimulation of innate immunity. While both viruses replicated to a similar extent in interferon (IFN)-deficient Vero E6 cells, TULV replication in human lung epithelial (A549) cells was slower and less efficient when compared to PUUV. In contrast to PUUV, no replication of TULV could be detected in human microvascular endothelial cells and in macrophages. While a strong innate immune response towards PUUV infection was evident at 48 h post infection, TULV infection triggered only a weak IFN response late after infection of A549 cells. Using appropriate in vitro cell culture models for the orthohantavirus infection, we could demonstrate major differences in host cell tropism, replication kinetics, and innate immune induction between pathogenic PUUV and the presumably non- or low-pathogenic TULV that are not observed in Vero E6 cells and may contribute to differences in virulence.

scholarly journals Essential role of caldesmon in the actin filament reorganization induced by glucocorticoids.

1995 ◽  
Vol 131 (5) ◽  
pp. 1223-1230 ◽  
Author(s):  
F Castellino ◽  
S Ono ◽  
F Matsumura ◽  
A Luini
Keyword(s):  
Actin Cytoskeleton ◽  
A549 Cells ◽  
Cell Types ◽  
Antisense Oligodeoxynucleotide ◽  
Similar Time ◽  
Calmodulin Binding ◽  
Time Courses ◽  
Induced Changes

Glucocorticoids induce the remodeling of the actin cytoskeleton and the formation of numerous stress fibers in a protein synthesis-dependent fashion in a variety of cell types (Castellino, F., J. Heuser, S. Marchetti, B. Bruno, and A. Luini. 1992. Proc. Natl. Acad. Sci. USA. 89:3775-3779). These cells can thus be used as models to investigate the mechanisms controlling the organization of actin filaments. Caldesmon is an almost ubiquitous actin- and calmodulin-binding protein that synergizes with tropomyosin to stabilize microfilaments in vitro (Matsumura, F., and Yamashiro, S. 1993. Current Opin. Cell Biol. 5:70-76). We now report that glucocorticoids (but not other steroids) enhanced the levels of caldesmon (both protein and mRNA) and induced the reorganization of microfilaments with similar time courses and potencies in A549 cells. A caldesmon antisense oligodeoxynucleotide targeted to the most abundant caldesmon isoform in A549 cells dramatically inhibited glucocorticoid-induced caldesmon synthesis and actin reorganization with similar potencies. Several control oligonucleotides were inactive. These results demonstrate that caldesmon has a crucial role in vivo in the organization of the actin cytoskeleton and suggest that hormone-induced changes in caldesmon levels mediate microfilament remodeling.

scholarly journals Development and Automation of a 384-Well Cell Fusion Assay to Identify Inhibitors of CCR5/CD4-Mediated HIV Virus Entry

2004 ◽  
Vol 9 (6) ◽  
pp. 516-524 ◽  
Author(s):  
Joe Bradley ◽  
Jasween Gill ◽  
Francois Bertelli ◽  
Sara Letafat ◽  
Romu Corbau ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Cell Lines ◽  
Cell Types ◽  
Hiv Virus ◽  
Biochemical Assays ◽  
Reagent Cost ◽  
Automated Screening ◽  
Hiv 1 ◽  
Fusion Efficiency

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/β-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of β-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturisation to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen ~650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z′ consistently above 0.6, which compares favourably with less complex biochemical assays.

Immunoenzymatic demonstration of the simultaneous presence of transferrin, hemopexin and albumin in a same hepatocyte and their ultrastructural localization

1978 ◽  
Vol 36 (2) ◽  
pp. 88-89
Author(s):  
M. Kraemer ◽  
J. Foucrier ◽  
J. Vassy ◽  
M.T. Chalumeau
Keyword(s):  
Plasma Proteins ◽  
Rat Hepatocytes ◽  
Ultrastructural Localization ◽  
Carrier Proteins ◽  
Normal Cells ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Monospecific Antisera ◽  
Different Levels

Some authors using immunofluorescent techniques had already suggested that some hepatocytes are able to synthetize several plasma proteins. In vitro studies on normal cells or on cells issued of murine hepatomas raise the same conclusion. These works could be indications of an hepatocyte functionnal non-specialization, meanwhile the authors never give direct topographic proofs suitable with this hypothesis.The use of immunoenzymatic techniques after obtention of monospecific antisera had seemed to us useful to bring forward a better knowledge of this problem. We have studied three carrier proteins (transferrin = Tf, hemopexin = Hx, albumin = Alb) operating at different levels in iron metabolism by demonstrating and localizing the adult rat hepatocytes involved in their synthesis.Immunological, histological and ultrastructural methods have been described in a previous work.

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