scholarly journals Synthesis of Heat Shock Proteins in Rat Brain Cortex after Transient Ischemia

1986 ◽  
Vol 6 (4) ◽  
pp. 505-510 ◽  
Author(s):  
Gerald A. Dienel ◽  
Marika Kiessling ◽  
Michael Jacewicz ◽  
William A. Pulsinelli
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Stress Proteins ◽  
Two Dimensional ◽  
Vessel Occlusion ◽  
Two Dimensional Gel Electrophoresis ◽  
Messenger Ribonucleic Acid ◽  
Rat Neocortex ◽  
Shock Proteins

Cell-free protein synthesis and two-dimensional gel autoradiography were used to characterize early postischemic protein synthesis in rat neocortex. Severe forebrain ischemia was induced for 30 min (four-vessel occlusion model) and followed by 3 h of recirculation. Polysomes were isolated from the cerebral cortex, translated in vitro in a reticulocyte system, and analyzed by two-dimensional gel electrophoresis. The translation products of postischemic polysomes included a major new protein family (70 kDa) with multiple isoelectric variants that was found to comigrate with the 68- to 70-kDa “heat shock” protein synthesized from polysomes of hyperthermic rats. Two other stress proteins (93 and 110 kDa) also appeared to be synthesized in increased amounts after ischemia. A complement of proteins that was indistinguishable from that of controls was also synthesized after ischemia, indicating that messenger ribonucleic acid coding for most brain proteins is preserved after ischemia and is bound to polysomes.

scholarly journals The intracellular Ca2+-pump inhibitors thapsigargin and cyclopiazonic acid induce stress proteins in mammalian chondrocytes

1994 ◽  
Vol 301 (2) ◽  
pp. 563-568 ◽  
Author(s):  
T C Cheng ◽  
H P Benton
Keyword(s):  
Protein Synthesis ◽  
Stress Response ◽  
Heat Shock ◽  
Stress Proteins ◽  
Articular Chondrocytes ◽  
Primary Cultures ◽  
Cyclopiazonic Acid ◽  
Two Dimensional Gel Electrophoresis ◽  
Intracellular Ca ◽  
Shock Proteins

Primary cultures of mammalian articular chondrocytes respond to treatment with the intracellular Ca(2+)-pump inhibitors thapsigargin (TG) and cyclopiazonic acid by specific changes in protein synthesis consistent with a stress response. Two-dimensional gel electrophoresis of newly synthesized proteins confirmed that the response was consistent with the induction of glucose-regulated proteins. The effects of low-dose TG (10 nM), measured by changes in [35S]methionine labelling of newly synthesized proteins, can first be observed by 10 h and are maximal by 24 h. The pattern of changes induced by TG is shared with cyclopiazonic acid, but effects of both perturbants differ significantly from changes induced by heat shock. Upon removal of TG, normal protein synthesis is restored by 48 h. Immunoblots showed increased concentrations of the stress proteins HSP90, HSP72/73 and HSP60 in chondrocytes treated with TG, but induction of newly synthesized heat-shock proteins by TG was not apparent on [35S]methionine-labelled gels. The alterations in protein synthesis induced by Ca(2+)-pump inhibitors were unaffected by BAPTA-AM loading, which clamped cytosolic Ca2+ at resting levels. We conclude that inhibition of intracellular Ca(2+)-pump activity can elicit a stress response, which has important implications for the interpretation of chronic use of Ca(2+)-pump inhibitors. In particular, the activation of the cellular shock response should be considered in interpreting the regulation of protein synthesis and cell survival by Ca(2+)-pump inhibitors such as TG.

scholarly journals Integrity of mitochondria in a mammalian cell mutant defective in mitochondrial protein synthesis.

1981 ◽  
Vol 90 (1) ◽  
pp. 108-115 ◽  
Author(s):  
K G Burnett ◽  
I E Scheffler
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Two Dimensional ◽  
Mitochondrial Protein Synthesis ◽  
Two Dimensional Gel Electrophoresis ◽  
Translational Machinery ◽  
Sedimentation Behavior ◽  
Rrna Species

A defect in mitochondrial protein synthesis has previously been identified in the respiration-deficient Chinese hamster lung fibroblast mutant V79-G7. The present work extends the characterization of this mutant. A more sensitive analysis has shown that mutant mitochondria synthesize all mitochondrially encoded peptides, but in significantly reduced amounts. This difference is also seen when isolated mitochondria are tested for in vitro protein synthesis. To distinguish between a defect in the translational machinery and a defect in the transcription of mitochondrial DNA, we investigated the synthesis of the 16S and 12S mitochondrial rRNA species and found them to be made in normal amounts in G7 mitochondria. These rRNA species appear to be assembled into subunits whose sedimentation behavior is virtually indistinguishable from that of the wild-type subunits. We also examined the consequences of the defect in mitochondrial protein synthesis on mutant cells and their mitochondria-utilizing techniques of electron microscopy, two-dimensional gel electrophoresis and immunochemical analysis. G7 mitochondria have a characteristic ultrastructure distinguished by predominantly tubular cristae, but the overall biochemical composition of mitochondrial membrane and matrix fractions appears essentially unaltered except for the absence of a few characteristic peptides. Specifically, we identify the absence of two mitochondrially encoded subunits of cytochrome c oxidase on two-dimensional gels and demonstrate a drastic reduction of both cytoplasmically and mitochondrially synthesized subunits of enzyme in immunoprecipitates of G7 mitochondria.

scholarly journals Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

1981 ◽  
Vol 91 (2) ◽  
pp. 352-360 ◽  
Author(s):  
TW McKeithan ◽  
JL Rosenbaum
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Cold Temperature ◽  
Two Dimensional ◽  
Multiple Forms ◽  
Cytoplasmic Fraction ◽  
Two Dimensional Gel Electrophoresis ◽  
Brain Tubulin ◽  
Β Tubulin

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.

Heat shock and stress response of Taenia solium and T. crassiceps (Cestoda)

Parasitology ◽  
2001 ◽  
Vol 122 (5) ◽  
pp. 583-588 ◽  
Author(s):  
L. VARGAS-PARADA ◽  
C. F. SOLÍS ◽  
J. P. LACLETTE
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Stress Responses ◽  
Stress Proteins ◽  
Taenia Solium ◽  
Western Blots ◽  
Prolonged Incubation ◽  
Larval Stages ◽  
Shock Proteins

Heat shock and stress responses are documented for the first time in larval stages of the cestodes Taenia solium and Taenia crassiceps. Radioactive metabolic labelling after in vitro incubation of cysts at 43 °C, revealed the induction of heat shock proteins. In T. crassiceps, the major heat shock proteins were 80, 70 and 60 kDa. After prolonged incubation, a set of low molecular weight heat shock proteins (27, 31, 33 and 38 kDa), were also induced. In vitro incubation of cysts at 4 °C, induced the synthesis of stress proteins ranging from 31 to 80 kDa, indicating the parasite is also able to respond to cold shock. T. solium cysts exposure to temperature stress also resulted in an increased synthesis of 2 major heat shock proteins of 80 and 70 kDa. Western blots using the excretory–secretory products of T. solium showed that 2 heat shock proteins were recognized by antibodies in the sera of cysticercotic patients: one of 66 kDa and another migrating close to the run front. The T. solium 66 kDa protein was also recognized by specific antibodies directed to a 60 kDa bacterial heat shock protein, suggesting that it belongs to this family of proteins.

Heat and nutritional shock-induced proteins of the fungus Achlya are different and under independent transcriptional control

1984 ◽  
Vol 62 (9) ◽  
pp. 837-846 ◽  
Author(s):  
Herb B. LéJohn ◽  
Cleantis E. Braithwaite
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Transcriptional Control ◽  
Stress Proteins ◽  
Reproductive Development ◽  
Nutritional Stress ◽  
In Vitro Translation ◽  
Stressed Cells ◽  
Shock Proteins

When the temperature of exponentially growing cells of the coenocytic fungus Achlya klebsiana strain 1969 was suddenly elevated from 24 to 37 °C (thermal stress), synthesis of at least 12 preexisting proteins (heat-shock proteins, HSPs) was vigorously induced while synthesis of most other cell proteins declined transiently. After 2–3 h of thermal stress, the cells recovered and resumed normal protein synthesis. If the cells were first starved of nutrients (nutritional stress) before the temperature was raised to 37 °C, the same 12 HSPs were induced, but synthesis of both heat-shock-inducible and nonheat-shock proteins declined to trace levels after 4 h of thermal stress. Molecular weights (MW) of the HSPs were approximately 96 000a, 96 000b, 85 000, 72 000, 70 000, 69 000a, 69 000b, 68 000, 60 000, 52 000, 26 000a, and 26 000b, and they had similar isoelectric points (5.8–6.2). Nutritionally stressed cells showed an induced synthesis of some 28 proteins (nutritional stress proteins, NSPs), when they were not heat shocked, and an induced synthesis of 20 NSPs when heat shocked. In the presence of glutamine, nutritionally stressed cells induced the synthesis of 15 NSPs when they were not heat shocked and 17 NSPs when they were heat shocked. The NSPs and HSPs were electrophoretically different proteins. Glutamine did not affect the induction pattern of the HSPs, but it arrested reproductive development of starving cells while altering the pattern of NSP synthesis. Since actinomycin D inhibited the induced synthesis of HSPs and some NSPs, they may be under transcriptional control. In vitro translation of poly(A)+ RNAs from heat-shocked cells showed that these cells were rich in HSP mRNAs and poor in NSP mRNAs. We speculate that NSPs, but not HSPs, may play a role in reproductive development and sporulation in this fungus.

Exercising mammals synthesize stress proteins

1990 ◽  
Vol 258 (4) ◽  
pp. C723-C729 ◽  
Author(s):  
M. Locke ◽  
E. G. Noble ◽  
B. G. Atkinson
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Sprague Dawley ◽  
Spleen Cells ◽  
Sprague Dawley Rats ◽  
Electrophoretic Separations ◽  
Shock Proteins ◽  
Sufficient Stimulus

Spleen cells, peripheral lymphocytes, and soleus muscles were removed from male Sprague-Dawley rats that had been run on a treadmill (24 m/min) for either 20, 40, or 60 min or to exhaustion (86 +/- 41 min) and were labeled in vitro with [35S]methionine at 37 degrees C. Similar tissues from nonrunning control rats were labeled in vitro at either 37 or 43 degrees C (heat shock). Fluorographic analyses of one- and two-dimensional polyacrylamide gel electrophoretic separations of the proteins from cells and tissues of exercised rats demonstrate the new or enhanced synthesis of proteins of approximately 65, 72, 90, and 100 kDa. Although synthesis of these proteins is low or not detectable in tissues from control rats labeled at 37 degrees C, they are prominent products of similar tissues labeled under heat-shock conditions (43 degrees C) and, in fact, correspond in Mr and pI with the so-called heat-shock proteins. These results suggest that exercise is a sufficient stimulus to induce or enhance the synthesis of heat shock and/or stress proteins in mammalian cells and tissues.

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