scholarly journals Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression.

1988 ◽  
Vol 106 (4) ◽  
pp. 1105-1116 ◽  
Author(s):  
L A Mizzen ◽  
W J Welch
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Stress Proteins ◽  
Stress Protein ◽  
Shock Treatment ◽  
Heat Shock Treatment ◽  
Normal Medium ◽  
Growth Environment ◽  
Mammalian Cell Lines ◽  
C 30

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.

Synthesis of stress protein 70 (Hsp70) in rainbow trout (Oncorhynchus mykiss) red blood cells

1997 ◽  
Vol 200 (3) ◽  
pp. 607-614 ◽  
Author(s):  
S Currie ◽  
B Tufts
Keyword(s):  
Protein Synthesis ◽  
Rainbow Trout ◽  
Heat Shock ◽  
Oncorhynchus Mykiss ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Stress Proteins ◽  
Stress Protein ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Hsp70 Synthesis

Unlike enucleated mammalian red blood cells (rbcs), the nucleated rbcs of lower vertebrates are capable of protein synthesis and may, therefore, serve as a valuable model to investigate the adaptive significance of stress protein synthesis in cells. This study examined the synthesis of stress protein 70 (Hsp70) in rbcs of the temperature-sensitive rainbow trout Oncorhynchus mykiss in response to heat shock and anoxia. Through western blot analysis, we have demonstrated that rainbow trout rbcs synthesize Hsp70 both constitutively and in response to an increase in temperature. Radioisotopic labelling experiments indicated that the temperature at which Hsp70 synthesis was induced in fish acclimated to 10 °C was between 20 and 25 °C. Actinomycin D blocked de novo Hsp70 synthesis, implying that synthesis of Hsp70 is regulated at the level of transcription in rainbow trout rbcs. Since trout rbcs rely heavily on aerobic metabolism, but may also experience very low oxygen levels within the circulation, we also examined the relative importance of (1) anoxia as a stimulus for Hsp70 synthesis and (2) oxygen as a requirement for protein synthesis under control and heat-shock conditions. We found that trout rbcs were capable of protein synthesis during 2 h of anoxia, but did not increase Hsp70 synthesis. Moreover, rbcs subjected to combined anoxia and heat shock exhibited increases in Hsp70 synthesis that were similar in magnitude to those in cells exposed to heat shock alone. The latter results suggest that rainbow trout rbcs are (1) able to synthesize non-stress proteins during anoxia, (2) capable of tolerating periods of reduced oxygen availability without increased synthesis of stress proteins and (3) able to maintain the integrity of their heat-shock response even during periods of anoxia.

scholarly journals c-myc and c-myb protein degradation: effect of metabolic inhibitors and heat shock.

1988 ◽  
Vol 8 (6) ◽  
pp. 2504-2512 ◽  
Author(s):  
B Lüscher ◽  
R N Eisenman
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Ornithine Decarboxylase ◽  
Half Life ◽  
Shock Treatment ◽  
Metabolic Energy ◽  
Metabolic Inhibitors ◽  
Heat Shock Treatment ◽  
Myb Protein

The proteins encoded by both viral and cellular forms of the c-myc oncogene have been previously demonstrated to have exceptionally short in vivo half-lives. In this paper we report a comparative study on the parameters affecting turnover of nuclear oncoproteins c-myc, c-myb, and the rapidly metabolized cytoplasmic enzyme ornithine decarboxylase. The degradation of all three proteins required metabolic energy, did not result in production of cleavage intermediates, and did not involve lysosomes or ubiquitin. A five- to eightfold increase in the half-life of c-myc proteins, and a twofold increase in the half-life of c-myb proteins was detected after heat-shock treatment at 46 degrees C. In contrast, heat shock had no effect on the turnover of ornithine decarboxylase. Heat shock also had the effect of increasing the rate of c-myc protein synthesis twofold, whereas c-myb protein synthesis was decreased nearly fourfold. The increased stability and synthesis of c-myc proteins led to an overall increase in the total level of c-myc proteins in response to heat-shock treatment. Furthermore, treatments which reduced c-myc and c-myb protein turnover, such as heat shock and exposure to inhibitors of metabolic energy production, resulted in reduced detergent solubility of both proteins. The recovery from heat shock, as measured by increased turnover and solubility, was energy dependent and considerably more rapid in thermotolerant cells.

scholarly journals Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes.

1988 ◽  
Vol 106 (4) ◽  
pp. 1117-1130 ◽  
Author(s):  
W J Welch ◽  
L A Mizzen
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Stress Proteins ◽  
Intracellular Distribution ◽  
Shock Treatment ◽  
Heat Shock Treatment ◽  
Ribonucleoprotein Complexes ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
In Cells

Here we further characterize a number of properties inherent to the thermotolerant cell. In the preceding paper, we showed that the acquisition of the thermotolerant state (by a prior induction of the heat-shock proteins) renders cells translationally tolerant to a subsequent severe heat-shock treatment and thereby results in faster kinetics of both the synthesis and subsequent repression of the stress proteins. Because of the apparent integral role of the 70-kD stress proteins in the acquisition of tolerance, we compared the intracellular distribution of these proteins in both tolerant and nontolerant cells before and after a severe 45 degrees C/30-min shock. In both HeLa and rat embryo fibroblasts, the synthesis and migration of the major stress-induced 72-kD protein into the nucleolus and its subsequent exit was markedly faster in the tolerant cells as compared with the nontolerant cells. Migration of preexisting 72-kD into the nucleolus was shown to be dependent upon heat-shock treatment and independent of active heat-shock protein synthesis. Using both microinjection and immunological techniques, we observed that the constitutive and abundant 73-kD stress protein similarly showed a redistribution from the cytoplasm and nucleus into the nucleolus as a function of heat-shock treatment. We show also that other lesions that occur in cells after heat shock can be prevented or at least minimized if the cells are first made tolerant. Specifically, the heat-induced collapse of the intermediate filament cytoskeleton did not occur in cells rendered thermotolerant. Similarly, the disruption of intranuclear staining patterns of the small nuclear ribonucleoprotein complexes after heat-shock treatment was less apparent in tolerant cells exposed to a subsequent heat-shock treatment.

Analysis of the temperature-dependent temporal pattern of heat-shock-protein synthesis in fish cells

1983 ◽  
Vol 3 (7) ◽  
pp. 647-658 ◽  
Author(s):  
Lashitew Gedamu ◽  
Beverly Culham ◽  
John J. Heikkila
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Temporal Pattern ◽  
Stress Proteins ◽  
Incubation Temperature ◽  
Stress Protein ◽  
In Vitro Translation ◽  
Continuous Exposure ◽  
Temperature Dependent

Continuous exposure of Chinook salmon embryo cells to an elevated incubation temperature of 24°C induces the transient expression of a set of heat-shock or stress proteins whereas maintenance of the cells at a higher incubation temperature of 28°C produces a continuous synthesis of these stress proteins. In vitro translation studies suggest that the temperature-dependent temporal pattern of stress-protein synthesis is correlated with the levels of stress-protein mRNA. This was verified using a recombinant-DNA probe complementary to the 70K heat-shock-protein mRNA. A transient increase in the level of the fish heat-shock 70K mRNA was observed in RNA samples isolated from cells continuously exposed at 24°C However, a constant increase in the level of this specific mRNA was found in RNA preparations obtained from cells maintained at 28°C Therefore, the temperature-dependent pattern of fish heat-shockprotein synthesis appears to be directly related to the level of heat-shock-protein mRNA.

c-myc and c-myb protein degradation: effect of metabolic inhibitors and heat shock

1988 ◽  
Vol 8 (6) ◽  
pp. 2504-2512
Author(s):  
B Lüscher ◽  
R N Eisenman
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Ornithine Decarboxylase ◽  
Half Life ◽  
Shock Treatment ◽  
Metabolic Energy ◽  
Metabolic Inhibitors ◽  
Heat Shock Treatment ◽  
Myb Protein

The proteins encoded by both viral and cellular forms of the c-myc oncogene have been previously demonstrated to have exceptionally short in vivo half-lives. In this paper we report a comparative study on the parameters affecting turnover of nuclear oncoproteins c-myc, c-myb, and the rapidly metabolized cytoplasmic enzyme ornithine decarboxylase. The degradation of all three proteins required metabolic energy, did not result in production of cleavage intermediates, and did not involve lysosomes or ubiquitin. A five- to eightfold increase in the half-life of c-myc proteins, and a twofold increase in the half-life of c-myb proteins was detected after heat-shock treatment at 46 degrees C. In contrast, heat shock had no effect on the turnover of ornithine decarboxylase. Heat shock also had the effect of increasing the rate of c-myc protein synthesis twofold, whereas c-myb protein synthesis was decreased nearly fourfold. The increased stability and synthesis of c-myc proteins led to an overall increase in the total level of c-myc proteins in response to heat-shock treatment. Furthermore, treatments which reduced c-myc and c-myb protein turnover, such as heat shock and exposure to inhibitors of metabolic energy production, resulted in reduced detergent solubility of both proteins. The recovery from heat shock, as measured by increased turnover and solubility, was energy dependent and considerably more rapid in thermotolerant cells.

scholarly journals Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment.

1985 ◽  
Vol 101 (4) ◽  
pp. 1198-1211 ◽  
Author(s):  
W J Welch ◽  
J P Suhan
Keyword(s):  
Stress Response ◽  
Heat Shock ◽  
Actin Filaments ◽  
Stress Proteins ◽  
Morphological Changes ◽  
Shock Treatment ◽  
Immunological Methods ◽  
Heat Shock Treatment ◽  
Rat Fibroblasts ◽  
Cytoskeletal Elements

Using both electron microscopy and immunological methods, we have characterized a number of changes occurring in rat fibroblasts after heat-shock treatment. Incubation of the cells for 3 h at 42 degrees-43 degrees C resulted in a number of changes within the cytoplasm including: a disruption and fragmentation of the Golgi complex; a modest swelling of the mitochondria and subtle alterations in the packing of the cristae; and alterations in cytoskeletal elements, specifically a collapse and aggregation of the vimentin-containing intermediate filaments around the nucleus. A number of striking changes were also found within the nuclei of the heat-treated cells: (a) We observed the appearance of rod-shaped bodies consisting of densely packed filaments. Using biochemical and immunological methods, these nuclear inclusion bodies were shown to be comprised of actin filaments. (b) Considerable alterations in the integrity of the nucleoli were observed after the heat-shock treatment. Specifically, there appeared to be a general relaxation in the condensation state of the nucleoli, changes in both the number and size of the granular ribonucleoprotein components, and finally a reorganization of the nucleolar fibrillar reticulum. These morphological changes in the integrity of the nucleoli are of significant interest since previous work as well as studies presented here show that two of the mammalian stress proteins, the major stress-induced 72-kD protein and the 110-kD protein, localize within the nucleoli of the cells after heat-shock treatment. We discuss these morphological changes with regards to the known biological and biochemical events that occur in cells after induction of the stress response.

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