Temperature adaptation of house keeping and heat shock gene expression in Neurospora crassa.

Adaptation of house keeping and heat shock gene expression was determined in Neurospora crassa during continuous exposure to different temperatures. Steady-state values of total protein synthesis differed little after incubation for 24 h at temperatures between 15 and 42 degreesC. Adaptation kinetics at 42 degreesC showed an initial, transient inhibition of total protein synthesis. Similar kinetics were observed with actin synthesis and tubulin mRNA. A priming 1-h heat shock of 42 degreesC 2 h prior to a second continuous exposure to 42 degreesC abolished the inhibitory effect of the second treatment and resulted in "acquired translational tolerance." Steady-state values of HSP70 synthesis rates revealed increasing levels with increasing temperatures after incubation for 24 h at different temperatures. Adaptation kinetics of the synthesis rates of different HSPs in vivo revealed maximal rates after 2 h and then a decrease to the elevated steady-state levels. The total amount of the major constitutive and inducible HSP70 isoform as determined by Western blots reached a maximum 2 h after the beginning of 42 degreesC exposure and only a slight decrease (25%) of the maximal value after 24 h. The inducible isoform of HSP70, in contrast, reached a maximum after 4-8 h and then decreased strongly after 24 h. HSP mRNAs reached maximal amounts 45-60 min after the beginning of 42 degreesC exposure and then declined after 8 h as determined by in vitro translation. Northern blots revealed maximal mRNA amounts of the inducible HSP70 after 30 min and zero amounts after 4 h exposure to 42 degreesC. After a shift to 42 degreesC HSP70 isoforms were immediately translocated into the nucleus and reshuttled into the cytoplasm during the following 6 h. The nuclear content of HSP70 remained elevated during the adapted steady state at 24 h. It is concluded that the adapted state after 24 h is based on enhanced amounts of constitutive isoforms in the cytoplasm and in the nucleus, whereas the inducible isoforms of HSP70 show faster adaptation kinetics.

Nuclear translocation of constitutive heat shock protein 70 during S phase in synchronous macroplasmodia of Physarum polycephalum.

The level of constitutive heat shock protein 70 (HSC70) in Physarum polycephalum was analyzed by means of Western blots during the synchronous cell cycle of macroplasmodia. Total amounts as well as nuclear and cytoplasmic contents were determined separately and evaluated densitometrically. A drastic increase of nuclear HSC70 was observed 10-40 min after the initiation of S phase (600% of the M phase value) and thereafter a slow decline toward the next M phase. Total HSC levels showed a slight (30%) increase during S phase whereas cytoplasmic HSC70 was about 30% lower during S phase compared to mitosis.

Stress response of lysosomal cysteine proteinases in rat C6 glioma cells.

Acid proteinases of C6 rat glioma cells were analyzed by means of gelatine polyacrylamide electrophoresis with respect to their responses to stress (heat shock and butanol). Proteinase activities on gelatine gels were characterized by their molecular masses. pH-optima, isoelectric points and reactions to inhibitors. Four bands of 25, 35 and 65/85 kDa most probably represent active and proforms as well as precursor complexes of lysosomal cysteine proteinases with pH optima between 4.0 and 5.0. The 25-kDa band seems to contain cathepsin L and B, the 35-kDa band proforms of cathepsin L and B and the 65/85-kDa bands possibly precursor complexes of cathepsin L and B. After 30-min heat shocks of different temperatures (40-50 degrees C), the 35-kDa activity increased, whereas the 65/85-kDa activity decreased after exposure to 42 and 44 degrees C, which also caused a strong increase in the level of the inducible heat shock protein of 68 kDa (HSP 68). The alterations of the proteinase activities and the increases of the HSP 68 levels occur at heat shock treatments that cause cell death in about 25-40% of the population as determined by Trypan blue staining. HSP 68 induction and proteinase activity changes were also observed 12 hr after a 1-hr treatment with different butanol concentrations (0.14-0.16 M). Kinetics of the response to a 30-min heat shock (44 degrees C) revealed a maximal decrease of the 35-kDa and a maximal increase of the 65/85-kDa activities after 12 hr recovery. When cells were exposed to repeated heat shocks (44 degrees C) at 12-hr intervals, the HSP 68 level further increased, whereas the 35-kDa and 65/85-kDa proteinase activities did not change. This result indicates a role of HSP 68 (or other HSPs) in the processing or stability of the putative cathepsin precursors (65/85-kDa complexes).

[Stress proteins: their growing significance in medicine]. / Stressproteine: Ihre wachsende Bedeutung in der Medizin.

In response to a variety of "emergencies", cells produce several stress proteins (heat shock proteins, HSP) that provide them with higher resistance and are able to repair protein damage. Their expression is regulated by heat shock transcription factors. Stress proteins are arousing growing interest in medicine, as major antigens in some infections and certain autoimmune diseases and also because of their possible involvement in the development and therapy of cancer. Elevated levels of stress proteins protect tissues and organs against ischemic injury and reduce infarct size. Stress proteins may also serve as a marker in diagnostic studies and in toxicology.

Close Correlation between Heat Shock Response and Cytotoxicity in Neurospora crassa Treated with Aliphatic Alcohols and Phenols.

In Neurospora crassa the aliphatic alcohols methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, ethylene glycol, glycerol, and allyl alcohol and the phenolic compounds phenol, hydroquinone, resorcinol, pyrogallol, phloroglucinol, sodium salicylate, and acetylsalicylic acid were analyzed with respect to their capacities to induce heat shock proteins (HSP) and to inhibit protein synthesis. Both the alcohols and phenols showed the greatest levels of HSP induction at concentrations which inhibited the overall protein synthesis by about 50%. The abilities of the different alcohols to induce the heat shock response are proportional to their lipophilicities: the lipophilic alcohol isobutanol is maximally inductive at about 0.6 M, whereas the least lipophilic alcohol, methanol, causes maximal induction at 5.7 M. The phenols, in general, show a higher capability to induce the heat shock response. The concentrations for maximal induction range between 25 mM (sodium salicylate) and 100 mM (resorcinol). Glycerol (4.1 M) shifted the concentration necessary for maximal HSP induction by hydroquinone from 50 to 200 mM. The results reveal that the induction of HSP occurs under conditions which considerably constrain cell metabolism. The heat shock response, therefore, does not represent a sensitive marker for toxicity tests but provides a good estimate for the extent of cell damage.

Heat shock inhibits and activates different protein degradation pathways and proteinase activities in Neurospora crassa.

In Neurospora crassa, heat shock treatment inhibits proteolytic activity. ATP-independent proteinases were analysed after polyacrylamide gel electrophoresis using renaturing gelatine gels. Proteinases of 24, 29, and 130 kDa were shown to be inhibited by heat shock and were further characterized as to their properties. A major part of the heat shock-induced inhibition is probably due to suppression of de novo synthesis of proteinases as deduced from experiments with cycloheximide. During several hours of recovery from heat shock, the inhibition of overall protein degradation and ATP-independent proteinases is reversed. Azocasein assays as well as pulse-chase experiments further showed that ATP-dependent protein degradation is only slightly affected by heat shock. Two ATP-binding proteinases of about 60 and 160 kDa even show an increased activity after heat shock. The degradation rate of heat shock proteins is inhibited by heat shock treatment, indicating that they are degraded by ATP-independent proteinases. Western blot analysis of a approximately 40-kDa degradation product of HSP70 containing its amino terminal portion revealed a reduction in the amount of this peptide after heat shock.

Heat shock response and cytotoxicity in C6 rat glioma cells: structure-activity relationship of different alcohols.

In C6 rat glioma cells, the n-alcohols methanol, ethanol, propanol, and butanol and the aromatic alcohol phenol all induce heat shock proteins (HSPs) of high molecular mass (68, 70, 90, and 110 kDa) when applied for 1 hr. The lowest alcohol concentrations that induce HSP synthesis cause about 20% cell death, as determined by neutral red assay. HSP induction thus occurs at alcohol concentrations close to the highest tolerable dose. The cytotoxicity and the potential of alcohols to induce the synthesis of HSPs increase with chain length and are correlated with the lipophilicity of the alcohols. A clear structure-activity relationship is observed for both parameters. A calculation of the putative membrane concentrations of these alcohols reveals that cytotoxic effects (50% cell death) occur at nearly the same membrane concentration (approximately 0.2 M). This also holds true for the lowest HSP 68-inducing alcohol concentrations, but at a lower concentration (approximately 0.12 M). The activities of major proteinases are affected by both heat shock and alcohols. The effects of alcohols also depend on the lipophilicity of the alcohols. Effective concentrations again are close to the highest tolerable dose. The stress reactions measured in terms of significant changes in HSP synthesis and proteinase activity provide information about the mechanisms by which toxic agents act on the cell.