Heat shock stress in Bacteroides fragilis

The response to heat shock was investigated in the obligate anaerobe Bacteroides fragilis. The cells responded quickly to stress and synthesised seven heat shock proteins immediately upon exposure to heat. The apparent molecular weights of the seven proteins differed from the apparent molecular weights of the proteins induced by UV irradiation, O₂ and H₂O₂. Heat shock did not induce phage reactivation whereas UV irradiation, O₂ and H₂O₂ did induce phage reactivation systems. Ethanol did not elicit the heat shock response. Two heat resistant B. fragilis mutants were isolated. Both mutants lost the ability to synthesise the same two heat shock proteins. It is concluded that the heat shock response and the responses to UV irradiation, O₂ and H₂O₂ represent two independent groups of stress responses in B. fragilis.     

Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72

Various stresses activate the c-Jun N-terminal kinase (JNK), which is involved in the regulation of many aspects of cellular physiology, including apoptosis. Here we demonstrate that in contrast to UV irradiation, heat shock causes little or no stimulation of the JNK-activating kinase SEK1, while knocking out the SEK1 gene completely blocks heat-induced JNK activation. Therefore, we tested whether heat shock activates JNK via inhibition of JNK dephosphorylation. The rate of JNK dephosphorylation in unstimulated cells was high, and exposure to UV irradiation, osmotic shock, interleukin-1, or anisomycin did not affect this process. Conversely, exposure of cells to heat shock and other protein-damaging conditions, including ethanol, arsenite, and oxidative stress, strongly reduced the rate of JNK dephosphorylation. Under these conditions, we did not observe any effects on dephosphorylation of the homologous p38 kinase, suggesting that suppression of dephosphorylation is specific to JNK. Together, these data indicate that activation of JNK by protein-damaging treatments is mediated primarily by inhibition of a JNK phosphatase(s). Elevation of cellular levels of the major heat shock protein Hsp72 inhibited a repression of JNK dephosphorylation by these stressful treatments, which explains recent reports of the suppression of JNK activation by Hsp72.     

Pairing of heterochromatin in response to cellular stress

We previously reported that exposure of human cells to DNA-damaging agents (X-rays and mitomycin C (MMC)) induces pairing of the homologous paracentromeric heterochromatin of chromosome 9 (9q12-13). Here, we show that UV irradiation and also heat shock treatment of human cells lead to similar effects. Since the various agents induce very different types and frequencies of damage to cellular constituents, the data suggest a general stress response as the underlying mechanism. Moreover, local UV irradiation experiments revealed that pairing of heterochromatin is an event that can be triggered without induction of DNA damage in the heterochromatic sequences. The repair deficient xeroderma pigmentosum cells (group F) previously shown to fail pairing after MMC displayed elevated pairing after heat shock treatment but not after UV exposure. Taken together, the present results indicate that pairing of heterochromatin following exposure to DNA-damaging agents is initiated by a general stress response and that the sensing of stress or the maintenance of the paired status of the heterochromatin might be dependent on DNA repair.     

Differential Responses to Environmental Conditions by Fungal Cells Sensitized by Heat Shock or UV Irradiation

Cells of the ascomycele Ophiostoma multianulatum were sensitized to the supra-optimal temperature of 30°C either by heat shock or by UV irradiation. At this incubation temperature the death rate of the heat-shocked cells was higher than that of the irradiated cells. This difference was increased if hydrolysed casein was added to the incubation medium. The heat-shocked cells were also killed faster at 30°C, if nitrogen instead of air was bubbled through the cell suspension. Heat shock, in contrast to UV irradiation, strongly increased the sensitivity to a high concentration of sodium chloride.     

Augmented expression of HSP72 protein in normal human fibroblasts irradiated with ultraviolet light.

Normal human fibroblasts synthesized heat shock protein (HSP) 72 constitutively and its expression was augmented 6 hours after UV irradiation. Maximum induction of HSP72 was obtained at 12 hours and HSP72 showed a punctuated distribution in nucleus. While unscheduled DNA synthesis was almost completed 12 hours after UV irradiation, the S phase fraction decreased immediately and recovered after 6 hours. Thus, HSP72 augmentation was occurred coincidentally with the recovery of S phase, and suggested that HSP72 had some function during the recovery of DNA replication inhibited after UV irradiation.     

Magnetic field exposure stimulates transposition through the induction of DnaK/J synthesis

Like some naturally occurring environmental stress factors such as heat shock and UV irradiation, magnetic field exposure is also stimulatory to transposition activity. This feature could be illustrated by a bacterial conjugation study using an Escherichia coli strain that carries the transposable element Tn5 as the donor. When the donor cultures were exposed to a low-frequency (50 Hz) magnetic field of 1.2 mT, Tn5 located on the bacterial chromosome was stimulated to transpose and settled on the extrachromosomal episome, and eventually transferred to the recipient cell through conjugation. Such transposition activity stimulation was mediated by the induced synthesis and accumulation of the heat shock proteins DnaK/J.     

Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block

We report here that importin alpha accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin alpha appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin alpha. In addition, nuclear retention and the importin beta/Ran-independent import of importin alpha also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin alpha from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin alpha is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin alpha can be involved in a common physiological response to various stress conditions.     

Functional HSF1 requires aromatic-participant interactions in protecting mouse embryonic fibroblasts against apoptosis via G2 cell cycle arrest

The present study highlighted the aromatic-participant interactions in in vivo trimerization of HSF1 and got an insight into the process of HSF1 protecting against apoptosis. In mouse embryonic fibroblasts (MEFs), mutations of mouse HSF1 (W37A, Y60A and F104A) resulted in a loss of trimerization activity, impaired binding of the heat shock element (HSE) and lack of heat shock protein 70 (HSP70) expression after a heat shock. Under UV irradiation, wild-type mouse HSF1 protected the MEFs from UV-induced apoptosis, but none of the mutants offered protection. We found that normal expression of HSF1 was essential to the cell arrest in G2 phase, assisting with the cell cycle checkpoint. The cells that lack normal HSF1 failed to arrest in the G2 phase, resulting in the process of cell apoptosis. We conclude that the treatment with UV or heat shock stresses appears to induce the approach of HSF1 monomers directly via aromatic-participant interactions, followed by the formation of a HSF1 trimer. HSF1 protects the MEFs from the stresses through the expression of HSPs and a G2 cell cycle arrest.     

Requirement of the Escherichia coli dnaK gene for thermotolerance and protection against H2O2

Thermotolerance in Escherichia coli is induced by exposing cells to a brief heat shock (42 degrees C for 15 min). This results in resistance to the lethal effect of exposure to a higher temperature (50 degrees C). Mutants defective in the recA, uvrA and xthA genes are more sensitive to heat than the wild-type. However, after development of thermotolerance these mutants are like the wild-type in their heat sensitivity. This suggests that thermotolerance is an inducible response capable of protecting cells from the lethal effects of heat, independently of recA, uvrA and xthA. Thermotolerance does not develop in a dnaK mutant. In addition, the dnaK mutant is sensitive to heat and H2O2, but is resistant to UV irradiation. This implies that the E. coli heat-shock response includes a mechanism that protects cells from heat and H2O2, but not from UV.     

Increase in UV Mutagenesis by Heat Stress on UV-Irradiated E. coli Cells

When leu- auxotrophs of Escherichia coli, after UV irradiation, were grown at temperatures between 30 and 47°C, the frequency of UV-induced mutation from leu- to leu+ revertant increased as the UV dose and the temperature increased. For cells exposed to a UV dose of 45 J/m2, the mutation frequency at 47°C was 1.9 times that at 30°C; for a dose of 90 J/m2, it was 3.25 times; and for 135 J/m2, it was 4.8 times. Similar enhancement of reversion frequency was observed when the irradiated cells were grown at 30°C in the presence of a heat shock inducer, ethanol (8% v/v). Heat shock-mediated enhancement of UV mutagenesis did not occur in an E. coli mutant sigma 32 (heat shock regulator protein), but sigma 32 overexpression in the mutant strain (transformed with a sigma 32-bearing plasmid) increased the UV-induced mutation frequency. These results suggest that heat stress alone has no mutagenic property, but when applied to UV-damaged cells, it enhances the UV-induced frequency of cell mutation.     

The extracellular protein of Luteococcus japonicus subsp casei reactivates cells inactivated by ultraviolet radiation or heating

The culture liquid of Luteococcus japonicus subsp. casei was found to be able to reactivate cells of this bacterium inactivated by UV irradiation or heat shock. The antistress activity of the culture liquid was due to the presence of an extracellular exometabolite of a protein nature with a molecular mass of more than 10 kDa. When the bacterium was grown in nutrient broth or glucose-containing mineral medium, the antistress protein was secreted by cells in the logarithmic growth phase. The reactivating effect of the antistress protein was inversely proportional to the survival rate of stressed cells.     

Induction of protein synthesis in response to ultraviolet light,nalidixic acid and heat shock in the cyanobacterium Phormidium laminosum

Synthesis of three polypeptides (43, 34 and 26 kDa) was enhanced or induced in the thermophilic cyanobacterium Phormidium laminosum following irradiation with UV light. Synthesis of several polypeptides was also enhanced/induced following treatment with nalidixic acid. Three had similar molecular masses to those induced by UV. One of these (43 kDa) was also apparently enhanced following prolonged heat shock, as were a number of other polypeptides with a wide range of molecular masses. The specific induction of the 34 and 26 kDa polypeptides following DNA damage and inhibition of replication suggests that they may be involved in DNA repair and, possibly, also in recombination.     

Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis

Exposure of mammalian cells to UV irradiation leads to activation of the c-Jun NH(2)-terminal protein kinase (JNK) pathway, which is associated with cell apoptosis. However, the molecular mechanism for JNK activation by UV exposure is not fully understood. We show here an essential role of a multisubstrate adapter, Gab1, in this signaling cascade. Gab1-deficient mouse fibroblast cells were defective in induction of JNK activity by UV exposure or heat shock, and this defect was rescued by reintroduction of Gab1 into Gab1(-/-) cells. Consistently, Gab1(-/-) cells displayed reduced caspase 3 induction and apoptotic cell death in response to UV irradiation. Gab1 was constitutively complexed with JNK and became tyrosine phosphorylated in UV-irradiated cells. Genetic and pharmaceutical analyses suggest the involvement of c-Met and the Src family tyrosine kinases in mediating UV-induced Gab1 phosphorylation as well as JNK activation. In aggregate, these observations identify a new function of Gab1 in the response of mammalian cells to UV light.     

Heat-shock-induced enhanced reactivation of UV-irradiated Herpesvirus

The objective of this study was to compare the ability of heat shock (HS) with that of another type of cellular stress, UV irradiation, to cause the induction of enhanced viral reactivation, a process that may represent an SOS-type repair process in mammalian cells. Studies performed to evaluate the effect of HS on growth of Vero cells revealed that HS at 45 degrees C for 45 min caused inhibition of cell growth similar to that caused by UV irradiation at 12 J/m2, but this inhibition was not observed at HS treatment for 5-15 min, or at a UV fluence of 2 J/m2. Enhanced reactivation of UV-irradiated Herpesvirus was observed in cells which had been pretreated by HS for greater than 30 min or UV at 12 J/m2. The synthesis of new proteins following HS for 15 and 45 min and UV at 12 J/m2 was examined by [35S]methionine-labeling experiments. The new synthesis of two HS proteins with molecular weights of 46 000 and 78 000 was induced by both levels of HS, but to a much greater extent at the high dose. These proteins were not detected in response to UV irradiation. These results indicate that, like UV irradiation, HS at levels inhibitory to cell growth induced enhanced viral reactivation in Vero cells. The results also suggest that at least two proteins in the HS protein family are not necessary for this response to occur.     

Formation and Stability of the Bacteriophage λ Replication Complexes in UV-Irradiated Escherichia coli

Bacteriophage λ replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter λ DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage λ, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the λ replication complex. We found that UV irradiation of the host cells prevented formation of the stable λ replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable λ replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of λ replication complexes is possible. Both stable and unstable λ replication complexes are functional because replication of λ DNA under conditions preventing formation of the stable complex proceeds efficiently. Received: 18 January 2000 / Accepted: 2 March 2000     

Heat-induced relocalization of protein kinase CK2. Implication of CK2 in the context of cellular stress

Among various other roles described so far, protein kinase CK2 has been involved in cell cycle, proliferation, and development. Here, we show that in response to specific stresses (heat shock or UV irradiation), a pool of the cellular CK2 content relocalizes in a particular nuclear fraction, increasing the activity of the kinase there. Electron microscopic analysis shows that upon heat shock, CK2alpha and CK2beta subunits are both detected in similar speckle structures occurring in the interchromatin space but are differentially targeted inside the nucleolus. This CK2 relocalization process takes place in a time- and dose-dependent manner and is reversible upon recovery at 37 degrees C. Altogether, this work suggests CK2 be involved in the response to physiological stress in higher eukaryotic cells.     

Cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways

Human immunodeficiency virus-1 (HIV-1) infection leads to changes in cellular gene expression, which in turn tend to modulate viral gene expression and replication. Cellular heat shock proteins (HSPs) are induced upon heat shock, UV irradiation and microbial or viral infections. We have reported earlier Nef-dependent induction of HSP40 leading to increased HIV-1 gene expression; however, the mechanism of induction remained to be elucidated. As expression of HSPs is regulated by heat shock factors (HSFs), we have now studied the role of HSF1 not only in Nef-dependent HSP40 induction but also in HIV-1 gene expression. Our results show that HSF1 is also induced during HIV-1 infection and it positively regulates HIV-1 gene expression by two distinct pathways. First, along with Nef it activates HSP40 promoter which in turn leads to increased HIV-1 gene expression. Second, HSF1 directly interacts with newly identified HSF1 binding sequence on HIV-1 LTR promoter and induces viral gene expression and replication. Thus, the present work not only identifies a molecular basis for HSF1-mediated enhancement of viral replication but also provides another example of how HIV-1 uses host cell machinery for its successful replication in the host.     

The Extracellular Protein of Luteococcus japonicus subsp. casei Reactivates Cells Inactivated by UV Irradiation or Heat Shock

The culture liquid of Luteococcus japonicus subsp. casei was found to be able to reactivate cells of this bacterium inactivated by UV irradiation or heat shock. The antistress activity of the culture liquid was due to the presence of an extracellular exometabolite of a protein nature with a molecular mass of more than 10 kDa. When the bacterium was grown in a nutrient broth or glucose-containing mineral medium, the antistress protein was secreted by cells in the logarithmic growth phase. The reactivating effect of the antistress protein was inversely proportional to the survival rate of stressed cells.