scholarly journals Evidence of heterogeneity of protein-turnover states in cultured cells

1980 ◽  
Vol 190 (3) ◽  
pp. 673-683 ◽  
Author(s):  
J S Amenta ◽  
S C Brocher
Keyword(s):  
Protein Synthesis ◽  
Growth Medium ◽  
Cultured Cells ◽  
Cell Protein ◽  
Protein Pool ◽  
Distinct Cell ◽  
First Order Transition ◽  
G0 Phase ◽  
Fresh Growth Medium ◽  
Slow Turnover

Previous studies on L-cell cultures [Amenta & Sargus (1979) Biochem. J. 182, 847—859] have suggested: (a) that degradation of slow-turnover proteins occurs in a distinct cell state (D-state); (b) that cells randomly enter the D-state with a first-order transition constant, rapidly degrade cell protein, and return to a quiescent G0-state. In the present study we have tested the hypothesis that the putative D-state exists as a substate within A-state (non-replicating) fibroblasts. Rat-embryo fibroblasts were prelabelled with [14C]leucine and [3H]thymidine, ‘chased’ for 24 h, and then placed in fresh growth medium containing either vinblastine (10 microM) or colchicine (25 microM) for three successive 24 h periods. Cells trapped in mitosis were separated from the residual non-replicating cells and rates of protein synthesis, degradation and net accumulation were measured in both populations. We observed that significant protein degradation occurred only in the non-replicating population, although both populations showed equally high rates of protein synthesis induced by fresh growth medium. These data support the hypothesis that degradation of slow-turnover protein is heterogeneous, occurring only in A-state cells. A model that proposes a separate D-state within G0-phase successfully accounts for these observations and previous reports on this cell line [Amenta, Sargus & Baccino (1978) J. Cell. Physiol. 97, 267—283] showing no differences in degradation of the slow-turnover protein pool in growth-stimulated and stationary-phase fibroblast cultures.

scholarly journals Synthesis and secretion of plasma proteins by embryonic chick hepatocytes: changing patterns during the first three days of culture.

1978 ◽  
Vol 147 (6) ◽  
pp. 1806-1823 ◽  
Author(s):  
G Grieninger ◽  
S Granick
Keyword(s):  
Protein Synthesis ◽  
Plasma Protein ◽  
Plasma Proteins ◽  
Secretory Pathway ◽  
Culture Medium ◽  
Cultured Cells ◽  
Cell Mass ◽  
Cell Protein ◽  
Protein Levels ◽  
Regulatory Processes

A simple model system is described for studying synthesis of plasma proteins. The system is based on chick embryo hepatocytes in primary monolayer culture which synthesize a broad spectrum of plasma proteins and secrete them into the culture medium. The secreted proteins are stable and consist almost exclusively of plasma proteins. The cultured cells are nonproliferating hepatic parenchymal cells whose cell mass remains constant in culture. By a modification of Laurell's rocket immunoelectrophoresis, the secreted plasma proteins can be detected in nanogram amounts in 3 microliter of unconcentrated culture medium. Kinetics of secretion are obtained by sequential assay of proteins accumulating in the medium. In this system it is demonstrated that: (a) intracellular plasma protein levels are equivalent to less than 5% of the daily secretion; (b) synthesis and secretion are continuous; and (c) the overall half-time for plasma protein movement along the secretory pathway is less than 10 min. From these results, it follows that the rate at which the plasma proteins are secreted gives a valid estimate of their rate of synthesis. This feature of the culture and the sensitivity of the assay allow routine measurements of plasma protein synthesis without disruption of the cells and without the use of radioisotopes. It is shown, furthermore, that the overall rate of plasma protein synthesis in cultured hepatocytes is constant over a 3-day period and is similar to that of the intact liver. 3,000,000 cells, containing 1 mg cell protein, synthesize 0.2 mg of plasma proteins daily, amounting to one-fifth of hepatocellular protein synthesis. Under the conditions used, albumin synthesis steadily decreases with culture time whereas the synthesis of many other plasma proteins increases. The observed phenotypic changes and reorganization of plasma protein synthesis illustrate how the system may be exploited for studying the regulatory processes governing plasma protein synthesis.

Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium

1991 ◽  
Vol 260 (2) ◽  
pp. F185-F191 ◽  
Author(s):  
S. H. Ayo ◽  
R. A. Radnik ◽  
W. F. Glass ◽  
J. A. Garoni ◽  
E. R. Rampt ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Protein Synthesis ◽  
High Glucose ◽  
Mesangial Cells ◽  
Matrix Proteins ◽  
Cell Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Glomerular Mesangial Cells ◽  
Glomerular Mesangium

Nodular expansion of glomerular mesangium with increased amounts of extracellular matrix (ECM) material is pathognomic of diabetic nephropathy. The precise mechanisms involved in this accumulation are unknown. Recently, we reported using a solid-phase enzyme-linked immunosorbent assay (ELISA) technique that glomerular mesangial cells, the principal cell type residing in glomerular mesangium, accumulate 50–60% more fibronectin (FN), laminin (LM), and type IV collagen (T-IV) when cultured in medium containing high glucose (30 mM) (S. H. Ayo, R. A. Rodnik, J. Garoni, W. F. Glass II, and J. I. Kreiberg. Am. J. Pathol. 136: 1339-1348, 1990). ECM assembly is controlled by its rate of synthesis and degradation, as well as its binding and rate of incorporation into the ECM. To elucidate the mechanisms involved, pulse-chase experiments were designed to estimate ECM protein synthesis from the incorporation of Trans-35S [( 35S]methionine, [35S]cysteine) into immunoprecipitated FN, LM, and T-IV. mRNA levels were examined, and degradation rates were estimated from the disappearance of radioactivity from matrix proteins in mesangial cells previously incubated with Trans-35S. One week of growth in 30 mM glucose resulted in approximately 40–50% increase in the synthesis of all three matrix proteins compared with 10 mM glucose-grown cells. This was accompanied by a significant increase in the transcripts for all three matrix proteins (approximately twofold). The specific activity of the radiolabel in trichloroacetic acid-precipitable cell protein showed no difference between cells grown in 10 or 30 mM glucose, indicating that total protein synthesis was unchanged. After 1 wk, the rate of FN, LM, and T-IV collagen degradation was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Inhibition of Host Protein Synthesis and Degradation of Cellular mRNAs During Infection by Influenza and Herpes Simplex Virus

1982 ◽  
Vol 2 (12) ◽  
pp. 1644-1648 ◽  
Author(s):  
S. C. Inglis
Keyword(s):  
Protein Synthesis ◽  
Blot Hybridization ◽  
Host Protein ◽  
Cell Protein ◽  
Cellular Genes ◽  
Cellular Mrnas ◽  
The Stability ◽  
Simplex Virus ◽  
Host Protein Synthesis ◽  
Synthesis And Degradation

Cloned DNA copies of two cellular genes were used to monitor, by blot hybridization, the stability of particular cell mRNAs after infection by influenza virus and herpesvirus. The results indicated that the inhibition of host cell protein synthesis that accompanied infection by each virus could be explained by a reduction in the amounts of cellular mRNAs in the cytoplasm, and they suggested that this decrease was due to virus-mediated mRNA degradation.

scholarly journals Protein Kinase R Is Responsible for the Phosphorylation of eIF2α in Rotavirus Infection

2010 ◽  
Vol 84 (20) ◽  
pp. 10457-10466 ◽  
Author(s):  
Margarito Rojas ◽  
Carlos F. Arias ◽  
Susana López
Keyword(s):  
Protein Synthesis ◽  
Kinase Domain ◽  
Cell Protein ◽  
Protein Kinase R ◽  
Viral Protein Synthesis ◽  
Α Subunit ◽  
Rotavirus Infection ◽  
Eif2α Phosphorylation ◽  
Infected Cells ◽  
Interferon System

ABSTRACT The eukaryotic initiation translation factor 2 (eIF2) represents a key point in the regulation of protein synthesis. This factor delivers the initiator Met-tRNA to the ribosome, a process that is conserved in all eukaryotic cells. Many types of stress reduce global translation by triggering the phosphorylation of the α subunit of eIF2, which reduces the formation of the preinitiation translation complexes. Early during rotavirus infection, eIF2α becomes phosphorylated, and even under these conditions viral protein synthesis is not affected, while most of the cell protein synthesis is blocked. Here, we found that the kinase responsible for the phosphorylation of eIF2α in rotavirus-infected cells is PKR, since in mouse embryonic fibroblasts deficient in the kinase domain of PKR, or in MA104 cells where the expression of PKR was knocked down by RNA interference, eIF2α was not phosphorylated upon rotavirus infection. The viral component responsible for the activation of PKR seems to be viral double-stranded RNA, which is found in the cytoplasm of infected cells, outside viroplasms. Taken together, these results suggest that rotaviruses induce the PKR branch of the interferon system and have evolved a mechanism to translate its proteins, surpassing the block imposed by eIF2α phosphorylation.

Follicle cell protein synthesis in moth oöcytes

1971 ◽  
Vol 17 (2) ◽  
pp. 217-232 ◽  
Author(s):  
William J. Cruickshank
Keyword(s):  
Protein Synthesis ◽  
Follicle Cell ◽  
Cell Protein

Determination of protein replacement rates by deuterated water: validation of underlying assumptions

2007 ◽  
Vol 292 (5) ◽  
pp. E1340-E1347 ◽  
Author(s):  
Emmanuelle Belloto ◽  
Frédérique Diraison ◽  
Alexandra Basset ◽  
Gwenola Allain ◽  
Pauline Abdallah ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Peripheral Circulation ◽  
Deuterated Water ◽  
Deuterium Labeling ◽  
Kinetics Of ◽  
Glycolytic Rate ◽  
Slow Turnover ◽  
Correct Estimate

H2O administration has recently been proposed as a simple and convenient method to measure protein synthesis rates. 2H2O administration results in deuterium labeling of free amino acids such as alanine, and incorporation into proteins of labeled alanine can then be used to measure protein synthesis rates. We examined first whether during 2H2O administration plasma free alanine enrichment is a correct estimate of the enrichment in the tissue amino acid pools used for protein synthesis. We found that, after 2H2O administration, deuterium labeling in plasma free alanine equilibrated rapidly with body water, and stable enrichment values were obtained within 20 min. Importantly, oral administration of 2H2O induced no difference of labeling between portal and peripheral circulation except for the initial 10 min after a loading dose. The kinetics of free alanine labeling were comparable in various tissues (liver, skeletal muscle, heart) and in plasma with identical plateau values. We show next that increased glycolytic rate or absorption of unlabeled amino acids from ingested meals do not modify alanine labeling. Calculated synthesis rates of mixed proteins were much higher (20- to 70-fold) in plasma and liver than in muscle and heart. Last, comparable replacement rates of apoB100-VLDL were obtained in humans by using the kinetics of incorporation into apoB100 of infused labeled leucine or of alanine labeled by 2H2O administration. All of these results support 2H2O as a safe, reliable, useful, and convenient tracer for studies of protein synthesis, including proteins with slow turnover rate.

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