Growth and Macromolecular Synthesis in the 3T6 Mouse Fibroblast

1970 ◽  
Vol 7 (3) ◽  
pp. 671-682
Author(s):  
C. I. LEVENE ◽  
C. J. BATES
Keyword(s):  
Cell Proliferation ◽  
Ascorbic Acid ◽  
Protein Synthesis ◽  
Growth Medium ◽  
Cell Layer ◽  
Mouse Fibroblast ◽  
Detectable Effect ◽  
Glycosaminoglycan Synthesis ◽  
General Protein Synthesis ◽  
General Protein

The growth of the 3T6 mouse fibroblast and synthesis of macromolecules has been studied over the last 6 days of the cultures' 14-day life span. The effect of ascorbic acid was also tested. During this period, the cells synthesized collagen, as judged by the appearance of non-dialysable hydroxyproline, which was identified by chemical assay and by radioactive incorporation studies. A high proportion of the collagen in the cell layer was insoluble in 0.1 N acetic acid. Of the hydroxyproline synthesized in the presence of ascorbic acid, about 75% was eventually liberated into the growth medium, and about 25-30% of the liberated material behaved as free hydroxyproline. In the absence of ascorbic acid, the cell layer hydroxyproline was reduced to one-third, but the growth medium hydroxyproline was unaffected. The cells also synthesized glycosaminoglycans, as judged by the appearance of cetyl pyridinium-precipitable uronic acid, and the incorporation of labelled glucosamine into macro-molecules. A large proportion of this material has the properties of hyaluronic acid. Ascorbic acid had no detectable effect on overall glycosaminoglycan synthesis, in contrast to healing tendonectomy wounds in guinea-pigs. Cell proliferation and general protein synthesis were virtually unaffected by ascorbic acid. Whereas general protein synthesis, like cell proliferation, declined in the ageing culture, glycosaminoglycan synthesis and collagen synthesis continued at a steady or increasing rate.

scholarly journals Induction of carnitine acetyltransferase by clofibrate in rat liver

1981 ◽  
Vol 194 (1) ◽  
pp. 249-255 ◽  
Author(s):  
B Mittal ◽  
C K R Kurup
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Time Lag ◽  
Mitochondrial Protein ◽  
Carnitine Acetyltransferase ◽  
Enzyme Protein ◽  
General Protein Synthesis ◽  
Liver And Kidney ◽  
General Protein

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine O-acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.

scholarly journals Bmp Induces Osteoblast Differentiation through both Smad4 and mTORC1 Signaling

2016 ◽  
Vol 37 (4) ◽  
Author(s):  
Courtney M. Karner ◽  
Seung-Yon Lee ◽  
Fanxin Long
Keyword(s):  
Protein Synthesis ◽  
Intracellular Signaling ◽  
Osteoblast Differentiation ◽  
De Novo ◽  
Bmp Signaling ◽  
Endoplasmic Reticulum Stress Response ◽  
Versus Protein ◽  
Mtorc1 Signaling ◽  
General Protein Synthesis ◽  
General Protein

ABSTRACT The bone morphogenetic protein (Bmp) family of secreted molecules has been extensively studied in the context of osteoblast differentiation. However, the intracellular signaling cascades that mediate the osteoblastogenic function of Bmp have not been fully elucidated. By profiling mRNA expression in the bone marrow mesenchymal progenitor cell line ST2, we discover that BMP2 induces not only genes commonly associated with ossification and mineralization but also genes important for general protein synthesis. We define the two groups of genes as mineralization related versus protein anabolism signatures of osteoblasts. Although it induces the expression of several Wnt genes, BMP2 activates the osteogenic program largely independently of de novo Wnt secretion. Remarkably, although Smad4 is necessary for the activation of the mineralization-related genes, it is dispensable for BMP2 to induce the protein anabolism signature, which instead critically depends on the transcription factor Atf4. Upstream of Atf4, BMP2 activates mTORC1 to stimulate protein synthesis, resulting in an endoplasmic reticulum stress response mediated by Perk. Thus, Bmp signaling induces osteoblast differentiation through both Smad4- and mTORC1-dependent mechanisms.

scholarly journals Proline responding1 Plays a Critical Role in Regulating General Protein Synthesis and the Cell Cycle in Maize

2014 ◽  
Vol 26 (6) ◽  
pp. 2582-2600 ◽  
Author(s):  
Gang Wang ◽  
Jushan Zhang ◽  
Guifeng Wang ◽  
Xiangyu Fan ◽  
Xin Sun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Critical Role ◽  
General Protein Synthesis ◽  
General Protein

scholarly journals Multiple components of eIF4F are required for protein synthesis-dependent hippocampal long-term potentiation

2013 ◽  
Vol 109 (1) ◽  
pp. 68-76 ◽  
Author(s):  
Charles A. Hoeffer ◽  
Emanuela Santini ◽  
Tao Ma ◽  
Elizabeth C. Arnold ◽  
Ashley M. Whelan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Synaptic Plasticity ◽  
Translation Initiation ◽  
Translational Control ◽  
Late Phase ◽  
Long Term Potentiation ◽  
Term Potentiation ◽  
General Protein Synthesis ◽  
General Protein

Persistent forms of synaptic plasticity are widely thought to require the synthesis of new proteins. This feature of long-lasting forms of plasticity largely has been demonstrated using inhibitors of general protein synthesis, such as either anisomycin or emetine. However, these drugs, which inhibit elongation, cannot address detailed questions about the regulation of translation initiation, where the majority of translational control occurs. Moreover, general protein synthesis inhibitors cannot distinguish between cap-dependent and cap-independent modes of translation initiation. In the present study, we took advantage of two novel compounds, 4EGI-1 and hippuristanol, each of which targets a different component of the eukaryotic initiation factor (eIF)4F initiation complex, and investigated their effects on long-term potentiation (LTP) at CA3-CA1 synapses in the hippocampus. We found that 4EGI-1 and hippuristanol both attenuated long-lasting late-phase LTP induced by two different stimulation paradigms. We also found that 4EGI-1 and hippuristanol each were capable of blocking the expression of newly synthesized proteins immediately after the induction of late-phase LTP. These new pharmacological tools allow for a more precise dissection of the role played by translational control pathways in synaptic plasticity and demonstrate the importance of multiple aspects of eIF4F in processes underlying hippocampal LTP, laying the foundation for future studies investigating the role of eIF4F in hippocampus-dependent memory processes.

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts

1986 ◽  
Vol 6 (4) ◽  
pp. 1088-1094
Author(s):  
R B Widelitz ◽  
B E Magun ◽  
E W Gerner
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mrna Levels ◽  
Hsp 70 ◽  
Mrna Accumulation ◽  
General Protein Synthesis ◽  
Rat Fibroblasts ◽  
General Protein ◽  
Rat Embryonic Fibroblasts

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.

The Turnover of Collagen in Fibroblast Cultures

1973 ◽  
Vol 12 (1) ◽  
pp. 217-234
Author(s):  
J. STEINBERG
Keyword(s):  
Stationary Phase ◽  
Growth Medium ◽  
Specific Activity ◽  
Cell Layer ◽  
Gel Filtration ◽  
Mouse Fibroblast ◽  
Growth Media ◽  
Precursor Pool ◽  
Fibroblast Cultures ◽  
Cell Layers

Collagen turnover was studied in mouse fibroblast cultures (3T6) by radioactive labelling and compartmental analyses. The incorporation of [14C]proline into protein during continuous labelling rapidly reached a maximum value which was directly proportional to the medium specific activity. Radioactivity appeared more slowly in hydroxyproline, and gradually accumulated as cultures became enriched in collagen and its breakdown products. In relation to total new protein synthesis, the proportional synthesis of collagen, as measured by the formation of [14C]hydroxyproline, was less in logarithmically growing than in stationary-phase cultures, and little was deposited in the cell layer. Newly synthesized hydroxyproline was consistently present in all growth media. In stationary-phase cultures, media contained as much as 60% of the total [14C]hydroxyproline in a form soluble in 0.5 M perchloric acid. Gel filtration chromatography confirmed that this was predominantly free hydroxyproline, only 30% appearing in small peptides whose degree of hydroxylation suggested their origin from larger collagen molecules. This acid-soluble compartment was taken as a convenient index of collagenolysis, which proved to be significant in both growth states, but was proportionately more important throughout logarithmic growth. Reincubation of prelabelled cultures in fresh medium containing an excess of non-radioactive proline (‘chase’ medium) was followed by the degradative loss of labelled cell layer protein. The released radioactivity could be quantitatively recovered in the growth medium for periods up to 6 days; the rate of its appearance was little influenced by the frequency of feeding. Despite extensive dilution of the proline precursor-pool specific activity, synthesis of [14C]hydroxyproline continued in all chase cultures. The increment appeared largely as collagen breakdown products in the growth medium, and probably arose from 2 principal sources: (1) recently deposited collagen, and (2) the hydroxylation of peptidyl-[14C]proline residues in protocollagen. The balance between these contributions seemed to be dependent upon the extent to which ‘ageing’ of the cell layer collagen had occurred prior to initiating the chase. Radioactive hydroxyproline was rapidly lost from briefly prelabelled cell layers, but was well retained in a macromolecular form when the initial labelling period was sufficiently prolonged. It is proposed that the endogenous collagen-degradative apparatus attacks both young collagen and its polypeptide precursor, but that as the lability of the former substrate rapidly declines, enzyme activity continues to operate on protocollagen to yield [14C]hydroxyproline-containing breakdown products which gradually diminish as the latter substrate pool is exhausted.

scholarly journals Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

1986 ◽  
Vol 6 (4) ◽  
pp. 1088-1094 ◽  
Author(s):  
R B Widelitz ◽  
B E Magun ◽  
E W Gerner
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mrna Levels ◽  
Hsp 70 ◽  
Mrna Accumulation ◽  
General Protein Synthesis ◽  
Rat Fibroblasts ◽  
General Protein ◽  
Rat Embryonic Fibroblasts

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.

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