Measuring Protein Synthesis in Cultured Cells and Mouse Tissues Using the Non‐radioactive SUnSET Assay

2020 ◽  
Vol 133 (1) ◽  
Author(s):  
Venkatraman Ravi ◽  
Aditi Jain ◽  
Sneha Mishra ◽  
Nagalingam Ravi Sundaresan
Keyword(s):  
Protein Synthesis ◽  
Cultured Cells ◽  
Mouse Tissues

Characterization of a factor that stimulates hydrolysis of GTP bound to ras gene product p21 (GTPase-activating protein) and correlation of its activity to cell density

1988 ◽  
Vol 8 (10) ◽  
pp. 4169-4173
Author(s):  
M Hoshino ◽  
M Kawakita ◽  
S Hattori
Keyword(s):  
Gene Product ◽  
Cell Density ◽  
Cultured Cells ◽  
Specific Activity ◽  
Gtpase Activating Protein ◽  
Ras Gene ◽  
Cell Extracts ◽  
Mouse Tissues ◽  
Almost All ◽  
Hydrolysis Of

The postmicrosomal fraction of the extract from NIH 3T3 and BALB/c 3T3 cells stimulated the hydrolysis of GTP bound to H-ras gene product p21 by severalfold. The stimulation was observed with normal p21 but not with p21 with valine as the 12th residue. This specificity is similar to that of GTPase-activating protein (GAP) for N-ras p21 described by M. Trahey and F. McCormick (Science 238:542-545, 1987). Consistent with this specificity, analysis of p21-bound nucleotides in living cells revealed that almost all normal p21 bound GDP, whereas oncogenic mutant p21s bound both GTP and GDP. Similar activity was also found in various mouse tissues, with brain tissue showing the highest specific activity. When cell extracts were prepared from cultured cells, there was a linear relationship between GAP activity and cell density. These results suggest the factor is involved in the regulation of cell proliferation.

The Effect of Insulin on the Growth and Metabolism of the Human Diploid Cell, Wi-38

1970 ◽  
Vol 7 (2) ◽  
pp. 575-585
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Glucose Uptake ◽  
Rna Synthesis ◽  
Cultured Cells ◽  
Cell Sheet ◽  
High Energy ◽  
Contact Inhibition ◽  
Contributory Factor ◽  
Membrane Area

In a confluent culture of WI-38 cells the membrane area available for nutrient uptake is greatly reduced and the possibility exists that this reduction in uptake capacity of the cell is a contributory factor in contact inhibition. Insulin has been reported by many authors to facilitate glucose uptake and also to stimulate protein, DNA and RNA synthesis, glycolysis, pino-cytosis and growth in cultured cells. The effect of insulin on WI-38 cells was determined, therefore, to find out whether it enabled the cell to escape from contact inhibition of growth. The action of insulin was found to be dependent upon medium composition. Growth and protein synthesis were stimulated in Eagle's minimal essential medium, but not when this medium was supplemented with glucose and glutamine. Apparently insulin is only effective when high-energy compounds become limiting. Whilst insulin did not induce any post-confluent division, the protein content of cells was increased by 30%, and this was correlated with an increased rate of protein synthesis. Despite this increased activity in protein metabolism, the utilization of amino acids was less in the presence of insulin indicating that a control mechanism for more economical utilization of amino acids for protein synthesis was activated by insulin. Insulin had no effect on RNA synthesis, and only a slight inhibitory effect on DNA synthesis. Evidence was produced suggesting that insulin blocked cell division and encouraged differentiation. Glucose uptake and incorporation into the cell was stimulated by insulin, and this was especially noticeable after the cell sheet became confluent. The turnover of labelled glucose and derivatives was also enhanced by insulin and this was accompanied by a much higher rate of lactic acid production. It is concluded that insulin does not overcome contact inhibition and permit post-confluent division, but that it does enable the cell to take up and utilize nutrients more efficiently in confluent cultures with a resultant increase in metabolic activity and cell size.

CAN PROTEIN SYNTHESIS IN CULTURED CELLS BE REGULATED BY MEMBRANE-MEDIATED EVENTS?

1975 ◽  
pp. 345
Author(s):  
G. Koch ◽  
P. Bilello ◽  
R. Mittelstaedt ◽  
A. Hoffman ◽  
L. Fisher
Keyword(s):  
Protein Synthesis ◽  
Cultured Cells

scholarly journals Quantitative relationships betweenlacZmutant frequency and DNA adduct frequency in Muta™Mouse tissues and cultured cells exposed to 3-nitrobenzanthrone

Mutagenesis ◽  
2017 ◽  
pp. gew067 ◽  
Author(s):  
Paul A. White ◽  
George R. Douglas ◽  
David H. Phillips ◽  
Volker M. Arlt
Keyword(s):  
Cultured Cells ◽  
Dna Adduct ◽  
Mouse Tissues

scholarly journals High-resolution visualization and quantification of nucleic acid–based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS)

2020 ◽  
Author(s):  
Cuiwen He ◽  
Michael T Migawa ◽  
Kai Chen ◽  
Thomas A Weston ◽  
Michael Tanowitz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Nucleic Acid ◽  
Secondary Ion Mass Spectrometry ◽  
Cultured Cells ◽  
Significant Advance ◽  
Mouse Tissues ◽  
Ion Mass Spectrometry ◽  
The Impact ◽  
Secondary Ion ◽  
In Cells

Abstract Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial—both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2′-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

Specific effect of oestrone sulphate on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium

1989 ◽  
Vol 123 (2) ◽  
pp. 233-NP ◽  
Author(s):  
G. Chaminadas ◽  
M. Alkhalaf ◽  
J. P. Rémy-Martin ◽  
A. Y. Propper ◽  
G. L. Adessi
Keyword(s):  
Protein Synthesis ◽  
Epithelial Cells ◽  
Progesterone Receptor ◽  
Guinea Pig ◽  
Cultured Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Effect ◽  
Cellular Proteins ◽  
Oestrone Sulphate ◽  
In Cells

ABSTRACT Patterns of induced protein synthesis and secretion in guinea-pig endometrial epithelial cell cultures in response to oestrone sulphate alone and oestrone sulphate plus progesterone were investigated. Epithelial cells were cultured for 3 days in growth medium, then washed three times in a steroid-free medium. For each experiment, anticytokeratin immunostaining was used to discriminate the epithelial cells from the stromal cells. Only experiments in which the control dishes displayed more than 80% of anticytokeratinimmunostained cells were further processed. After this period oestradiol-17β (20 nmol/l; control), oestradiol-17β (20 nmol/l) plus progesterone (0·5 μmol/l), oestrone sulphate (1 μmol/l) or oestrone sulphate (1 μmol/l) plus progesterone (0·5 μmol/l) were added to the medium for 48 h. An immunocytochemical progesterone receptor assay showed that oestradiol-17β increased the progesterone receptor content of cells, and progesterone added to cultured cells in the presence of oestradiol-17β induced a significant increase in oestrogen sulphotransferase activity assessing the hormone responsiveness of the cultured cells. In these culture conditions and after 16 h of incubation, oestradiol-17β induced a 1·7-fold increase in [3H]thymidine incorporation into DNA, and [35S]methionine incorporation into cellular proteins was linearly increased up to 8 h. Biochemical changes induced by the different hormone treatments were studied by labelling the proteins with a 6-h pulse of [35S]methionine. The proteins present in the medium and in cells were analysed by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. Addition of oestrone sulphate alone or with progesterone produced a change in the patterns of cellular and secreted proteins compared with those in cells cultured with either oestradiol-17β or oestradiol-17β plus progesterone. Three cellular proteins (Mr < 14 000, isoelectric point (pI) 5·2 and 5·3; Mr 75 000, pI 4·9) and one secreted protein (Mr 155 000, pI 5·6–5·9) were specifically induced and could serve as markers of oestrone sulphate action. Journal of Endocrinology (1989) 123, 233–241

Reevaluation of amino acid stimulation of protein synthesis in murine- and human-derived skeletal muscle cells assessed by independent techniques

2005 ◽  
Vol 288 (5) ◽  
pp. E1028-E1037 ◽  
Author(s):  
Britt-Marie Iresjö ◽  
Elisabeth Svanberg ◽  
Kent Lundholm
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Cultured Cells ◽  
Incorporation Rate ◽  
Synthesis Methods ◽  
Distribution Analysis ◽  
Western Blots ◽  
Initiation Factors ◽  
Initiation Of Translation

Murine L6 and human rhabdomyosarcoma cells were cultured standardized in low (0.28 mM) and normal (9 mM) amino acid (AA) concentrations to reevaluate by independent methods to what extent AA activate initiation of protein synthesis. Methods used were incorporation of radioactive AA into proteins, distribution analysis of RNA in density gradient, and Western blots on initiation factors of translation of proteins in cultured cells as well as in vivo (gastrocnemius, C57Bl mice) during starvation/refeeding. Incorporation rate of AA gave incorrect results in a variety of conditions, where phenylalanine stimulated the incorporation rate of phenylalanine into proteins, but not of tyrosine, and tyrosine stimulated incorporation of tyrosine but not of phenylalanine. Similar problems were observed when [35S]methionine was used for labeling of fractionated cellular proteins. However, the methods entirely independent of labeled AA incorporation indicated that essential AA activate initiation of translation, whereas nonessential AA did not. Branched-chain AA and glutamine, in combination with some other AA, also stimulated initiation of translation. Starvation/refeeding in vitro agreed qualitatively with results in vivo evaluated by initiation factors. Insulin at physiological concentrations (100 μM/ml) did not stimulate global protein synthesis at low or normal AA concentrations but did so at supraphysiological levels (3 mU/ml), confirmed by independent methods. Our results reemphasize that labeled AA should be used with caution for quantification of protein synthesis, since the precursor pool(s) for protein synthesis is not in complete equilibrium with surrounding AA. “Flooding” tracee experiments did not overcome this problem.

scholarly journals Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells

1993 ◽  
Vol 289 (1) ◽  
pp. 71-79 ◽  
Author(s):  
W L Wong ◽  
M A Brostrom ◽  
G Kuznetsov ◽  
D Gmitter-Yellen ◽  
C O Brostrom
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Extracellular Fluid ◽  
Mrna Translation ◽  
Protein Processing ◽  
Pituitary Cells ◽  
Translational Initiation ◽  
Early Protein ◽  
Inhibition Of Protein Synthesis

Thapsigargin, a tumour-promoting sesquiterpene lactone, selectively inhibits the Ca(2+)-ATPase responsible for Ca2+ accumulation by the endoplasmic reticulum (ER). Mobilization of ER-sequestered Ca2+ to the cytosol and to the extracellular fluid subsequently ensues, with concomitant alteration of cellular functions. Thapsigargin was found to serve as a rapid, potent and efficacious inhibitor of amino acid incorporation in cultured mammalian cells. At concentrations mobilizing cell-associated Ca2+ to the extracellular fluid, thapsigargin provoked extensive inhibition of protein synthesis within 10 min. The inhibition in GH3 pituitary cells involved the synthesis of almost all polypeptides, was not associated with increased cytosolic free Ca2+ concentration ([Ca2+]i), and was not reversed at high extracellular Ca2+. The transient rise in [Ca2+]i triggered by ionomycin was diminished by thapsigargin. Polysomes failed to accumulate in the presence of the drug, indicative of impaired translational initiation. With longer (1-3 h) exposures to thapsigargin, recovery of translational activity was observed accompanied by increased synthesis of the ER protein glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein (‘GRP78/BiP’) and its mRNA. Such inductions were comparable with those observed previously with Ca2+ ionophores which mobilize the cation from all intracellular sequestered sites. Actin mRNA concentrations declined significantly during such treatments. In HepG2 cells processing and secretion of the glycoprotein alpha 1-antitrypsin were rapidly suppressed by thapsigargin. Ca2+ sequestered specifically by the ER is concluded to be essential for optimal protein synthesis and processing. These rapid effects of thapsigargin on mRNA translation, protein processing and gene expression should be considered when evaluating potential mechanisms by which this tumour promoter influences cellular events.

Alterations of protein turnover underlying disuse atrophy in human skeletal muscle

2009 ◽  
Vol 107 (3) ◽  
pp. 645-654 ◽  
Author(s):  
S. M. Phillips ◽  
E. I. Glover ◽  
M. J. Rennie
Keyword(s):  
Protein Synthesis ◽  
Muscle Mass ◽  
Protein Turnover ◽  
Cultured Cells ◽  
Muscle Protein ◽  
Human Muscle ◽  
Muscle Loss ◽  
Disuse Atrophy ◽  
In Vivo Measurements

Unloading-induced atrophy is a relatively uncomplicated form of muscle loss, dependent almost solely on the loss of mechanical input, whereas in disease states associated with inflammation (cancer cachexia, AIDS, burns, sepsis, and uremia), there is a procatabolic hormonal and cytokine environment. It is therefore predictable that muscle loss mainly due to disuse alone would be governed by mechanisms somewhat differently from those in inflammatory states. We suggest that in vivo measurements made in human subjects using arterial-venous balance, tracer dilution, and tracer incorporation are dynamic and thus robust by comparison with static measurements of mRNA abundance and protein expression and/or phosphorylation in human muscle. In addition, measurements made with cultured cells or in animal models, all of which have often been used to infer alterations of protein turnover, appear to be different from results obtained in immobilized human muscle in vivo. In vivo measurements of human muscle protein turnover in disuse show that the primary variable that changes facilitating the loss of muscle mass is protein synthesis, which is reduced in both the postabsorptive and postprandial states; muscle proteolysis itself appears not to be elevated. The depressed postprandial protein synthetic response (a phenomenon we term “anabolic resistance”) may even be accompanied by a diminished suppression of proteolysis. We therefore propose that most of the loss of muscle mass during disuse atrophy can be accounted for by a depression in the rate of protein synthesis. Thus the normal diurnal fasted-to-fed cycle of protein balance is disrupted and, by default, proteolysis becomes dominant but is not enhanced.

scholarly journals Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells

1980 ◽  
Vol 188 (3) ◽  
pp. 941-944 ◽  
Author(s):  
P N Dalrymple ◽  
L L Houston
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
Indirect Immunofluorescence ◽  
Cultured Cells ◽  
Sodium Dithionite ◽  
Eukaryotic Cells ◽  
Protein Synthesis Inhibition ◽  
Inhibiting Protein ◽  
A Chain

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.

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