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O Bensaude

Publications and source records attributed to O Bensaude.

At least 55 records · Page 3Linked to original sources
PubMed1991

Heat-shock-induced denaturation of proteins. Characterization of the insolubilization of the interferon-induced p68 kinase.

Heat-shock stress causes inactivation and aggregation of various cellular proteins which become further insoluble. Previous studies have shown that the interferon-induced p68 kinase activity was greatly reduced in extracts of heat-shocked HeLa cells, and that the loss of activity was due to a decreased solubility of the enzyme. Here we show that the p68 kinase which is normally evenly distributed in the cytoplasm, aggregates as a thick ring around the nucleus in heat-shocked cells. The 70-kDa constitutive heat-shock proteins are major insolubilized proteins during stress and we find them to colocalize with the p68 kinase after stress. Treatments of cells with drugs which disrupt the cytoskeleton, such as colcemid and cytochalasin E, do not hinder the enzyme insolubilization during heat-shock. On the contrary, heat-protectors such as glycerol and deuterium oxide (D2O) keep the p68 kinase under a soluble and active form during heat-shock stress. Similarly, an attenuation of the insolubilization of this enzyme is observed in cells rendered thermo-tolerant by a previous heat-shock, suggesting that heat-shock proteins may also contribute to the protection. During the recovery period at normal temperature after heat-shock, resolubilization occurs and most of the enzyme is again recovered under an active soluble form.

Blotting, Western↗
PubMed1991

Binding activity of glucocorticoid receptors after heat shock.

The response of glucocorticoid receptors (GR) to heat was measured by the change in ligand binding activity both in control cells and in cells made tolerant to heat by a prior mild heat exposure. The study was prompted by earlier data showing that one of the heat shock proteins (HSP90) is an essential component of the GR complex and that treatment of mammalian cells with hydrocortisone induces resistance to heat damage. The GR rapidly loses binding activity after commencement of heating. There is a 50% loss of activity after 4 min at 45 degrees C, 8 min at 44 degrees C, or 17 min at 43 degrees C. The reduction in binding is due mainly to a reduction in affinity of binding to the ligand. The ability to bind glucocorticoid recovers quickly after heat treatment. Activity returns to levels 60-80% of normal by 2 h after a heat treatment that initially reduces binding to less than 20% of normal. However, complete restoration of binding activity takes approximately 3 days. The recovery of binding activity does not require protein synthesis. Pretreatment of cells with hydrocortisone, using conditions that induce heat resistance, reduces the activity to 10-20% of control, but residual receptors display a heat sensitivity similar to that of control cells. There was evidence for a limited degree of protection of GR from heat damage in thermotolerant cells.

Animals↗
PubMed1991

[IIa/IIo conversion of RNA polymerase II during heat shock].

Heat-shock treatment of cells activates a protein kinase which phosphorylates a heptapeptide analogous to the repeated motif of the C-terminal domain (CTD) of the large subunit of RNA polymerase II from mammalian cells. This is corroborated with a modification of the large subunit of this enzyme during thermal stress in HeLa cells. We have observed a shift from the IIa form (unphosphorylated) to the IIo form (phosphorylated) with a higher apparent molecular weight, during a heat-shock at 44, 45 or 46 degrees C and by a chemical stress induced by sodium arsenite. RNA polymerase II hyperphosphorylation, together with the activation of the heat-shock transcription factor, might contribute to the onset of the preferential transcription of heat-shock genes.

Arsenates↗
PubMed1990

Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts.

Changes in protein kinase activities are thought to contribute to the alteration of gene expression after heat shock and related stresses. In an attempt to identify enzymes which might be involved in both chromatin structure modification and transcriptional switch in heat-shocked cells, we have studied protein kinase activities in heat-shocked cell lysates with two exogenous substrates: a tetramer of a heptapeptide (heptapeptide 4) corresponding to the RNA polymerase II C-terminal domain (CTD), and the histone H1. Heat-shock and arsenite stress were found to stimulate strongly CTD kinase activity. H1 kinase activity was also stimulated but more weakly. Stimulation of CTD and H1 kinases occurs mainly at the early phase of recovery and by a process which is independent of protein synthesis. The stress-induced H1 kinase is shown to contain a molecule related to the mitotic-promoting factor (MPF) Cdc2 component. On the other hand, though Cdc2-related protein has also been reported to be part of a CTD kinase complex, we show that the stress-induced CTD kinase activity corresponds to a distinct entity. It is proposed that stress activation of CTD kinase might be involved in changing the specificity of RNA polymerase II.

Amino Acid Sequence↗
PubMed1989

Protein denaturation during heat shock and related stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase inactivation in mouse cells.

In an attempt to question the toxic effect of heat shock and related stress, we have studied the activity of reporter enzymes during stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase were synthesized in mouse and Drosophila cells after transfection of the corresponding genes. Both enzymes are rapidly inactivated during hyperthermia. The corresponding polypeptides are not degraded but become insoluble even in the presence of non-ionic detergents. The heat inactivation is more dramatic in vivo within the living cell than in vitro, in a detergent-free crude cell lysate. The extent of enzyme inactivation at a given temperature depends on the cell type in which the enzyme is expressed. Luciferase is inactivated at lower temperatures within Drosophila cells than within mouse cells, whereas beta-galactosidase is inactivated at higher temperatures in E. coli than in mouse cells. A "priming" heat shock confers a transient increased resistance (thermotolerance) of cells against a second "challenging" heat shock. Enzyme inactivation during heat shock or exposure of the cells to ethanol is attenuated in heat shock-primed cells. A comparable thermoprotection is raised by a priming heat shock for both luciferase activity and protein synthesis. Thus, the study of reporter enzyme inactivation is a promising tool for understanding the molecular basis of the toxicity of heat shock and related stress as well as the mechanisms leading to thermotolerance.

Animals↗
PubMed1989

A heat shock-like protein from the human malaria parasite Plasmodium falciparum induces autoantibodies.

The humoral immune response to a 72-kDa heat shock-like protein of Plasmodium falciparum has been analyzed using mouse monoclonal antibodies (mAb) and human immune sera. Three regions of the molecule containing B cell epitopes were identified by screening a sublibrary encoding the COOH-terminal half of the antigen with the mAb. One B cell epitope mapped to a region poorly conserved between the parasite 72-kDa polypeptide and mammalian heat-shock proteins (Hsp 70). Another mAb, G10C9, reacted with an amino acid region that has a high degree of homology with mouse (87.5%) and human (81.2%) Hsp 70. Both mouse and human cells were recognized by this mAb when analyzed by indirect immunofluorescence and by two-dimensional immunoblots. Sera from humans infected with malaria also recognized the human Hsp 70. Thus, our results indicate that autoantibodies directed against host Hsp 70 can be induced by the homologous parasite protein.

Amino Acid Sequence↗
PubMed1989

High constitutive transcription of HSP86 gene in murine embryonal carcinoma cells.

In order to investigate HSP86 heat-shock gene expression in embryonal carcinoma cell lines (EC), a partial mouse HSP86 cDNA clone was isolated and characterized. As observed for the corresponding protein, HSP86 RNA is shown to be constitutively more abundant in PCC4 and undifferentiated F9 EC cells than in fibroblasts, while its amount decreases upon F9 differentiation. Although mRNA stabilization is suggested to account in part of the high constitutive expression of the heat-shock-like protein HSC73 in F9 cells, HSP86 RNA appears as stable in fibroblasts as in F9 cells. Using run-on experiments we have established that high HSP86 expression in undifferentiated F9 cells in mainly due to enhanced transcription of the gene. Possible mechanisms responsible for this high level of transcription are discussed.

Animals↗
PubMed1989

Unusual levels of heat shock element-binding activity in embryonal carcinoma cells.

In contrast to differentiated somatic cells, mouse embryonal carcinoma (EC) cell lines spontaneously express high levels of major members of the heat shock protein (HSP) family. In addition, some EC cell lines (noninducible) are not able to induce HSP gene transcription and HSP synthesis after a stress. However, after in vitro differentiation, constitutive HSP expression decreases and the differentiated derivatives become able to induce HSP gene transcription after a stress. These cells were tested by gel shift assays for the presence of an activity able to bind the heat shock element (HSE) before and after a stress. Control fibroblasts grown at 37 degrees C did not contain significant levels of HSE-binding activity, but heat shock dramatically increased the level of HSE-binding activity. In contrast to control fibroblasts, all EC cells contained significant levels of HSE-binding activity at 37 degrees C. In the inducible EC cell line F9, as in fibroblasts, heat shock strongly increased the level of HSE-binding activity. In the noninducible EC cells, however, HSE-binding activity markedly decreased upon heat shock. During in vitro differentiation of the noninducible cell line PCC7-S-1009, the constitutive HSE-binding activity found at 37 degrees C disappeared and heat induction of the HSE-binding activity appeared. Therefore, a good correlation exists between the high spontaneous expression of some members of the HSP family and the constitutive level of HSE-binding activity in EC cells at 37 degrees C. Heat induction of HSP gene transcription correlates with a strong increase in HSE-binding activity, whereas a deficiency in heat induction of HSP gene transcription is associated with a loss of HSE-binding activity upon heat shock.

Animals↗
PubMed1988

Phosphorylation of the 90 kDa heat shock protein in heat shocked HeLa cell lysates.

The 90 kDa heat shock protein (hsp 90) is a major phosphorylated protein under normal growth conditions. However, it does not incorporate detectable levels of phosphate by incubation of control HeLa cell lysates with [gamma-32P]ATP in vitro. In this paper we show that strong phosphorylation of hsp 90 occurs in lysates prepared from heat shocked HeLa cells. Possible involvement of the eukaryotic initiation factor 2 kinase of the heme-controlled repressor of translation is discussed.

HeLa Cells↗
PubMed1988

Regulation of the heat-shock response by interferon in mouse L cells.

Interferon (IFN) is not able to induce heat-shock protein (HSP) synthesis. However IFN pretreatment of mouse L cells has been shown to enhance the decrease of overall protein synthesis which follows a heat shock, and to stimulate the accumulation of HSPs. We show here that the synthesis of a protein (the hepatitis B virus surface antigen) under the control of a Drosophila HSP 70 promoter is also stimulated in IFN-pretreated cells. The regulation by IFN takes place at two levels: first, the rate of HSP gene transcription is increased in nuclei isolated from IFN-treated cells; second, the synthesis of HSPs is prolonged after pretreatment with IFN. Experiments performed in the presence of actinomycin D show that this effect is due to a stabilization by IFN of mRNAs coding for HSPs.

Animals↗
PubMed1988

Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells.

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.

Animals↗
PubMed1987

Deficient activation of heat shock gene transcription in embryonal carcinoma cells.

Heat shock protein (HSP) synthesis cannot be induced by stress in the cleavage stage embryos of many different species. For instance, no HSP synthesis can be induced in the mouse embryo before the formation of the blastocyst. Similarly, HSP synthesis is not stress inducible in some embryonal carcinoma (EC) cell lines such as PCC4 and PCC7 S-1009 (1009). We show that RNAs coding for the major stress inducible murine heat shock protein, HSP68, do not accumulate in PCC4 or 1009 EC cells in response to a stress. Using an in vitro nuclear transcription assay, we demonstrate that the transcription of the corresponding genes is not activated after a stress. A specific gene switch-off due to DNA methylation or chromatin conformation is unlikely to account for this result. Indeed, stress does not promote the activation of the heterologous Drosophila HSP70 heat shock promoter in transfection assays of these cells. In contrast, the same promoter, like endogenous HSP synthesis, becomes stress inducible in 1009 cells after in vitro differentiation. This suggests that, in contrast to differentiated cells, these EC cells, and maybe the very early mouse embryonic cells, could lack a transacting activating transcription factor or contain a repressor.

Actins↗
PubMed1987

Mouse 89 kD heat shock protein. Two polypeptides with distinct developmental regulation.

Unstressed early mouse embryos have been previously shown [1] to synthesize at very high rates 70 and 89 kD proteins belonging to the heat shock protein (HSP) family. But it was not clear whether expression of heat shock-inducible or non-inducible (cognate) genes accounted for this spontaneous synthesis. In this report we show that the 89 kD mouse HSP can be separated into two proteins by high resolution PAGE. These two components show distinct but related peptide pattern after limited proteolysis. They are synthesized from distinct mRNAs. One of these proteins--HSP89f--is synthesized at a high rate by unstressed cells and its synthesis is rather insensitive to stress, whereas synthesis of the other protein--HSP89s--is strongly stimulated by heat shock in fibroblasts. Both HSP89f and HSP89s are major proteins synthesized in unstressed mouse preimplantation embryos and embryonal carcinoma (EC) cells. After in vitro differentiation of the EC cells the spontaneous synthesis of HSP89s decreases. Thus spontaneous expression of a mammalian inducible HSP is developmentally regulated.

Animals↗
PubMed1986

Interferon pretreatment lowers the threshold for maximal heat-shock response in mouse cells.

Interferons (IFNs) are proteins which have antiviral and antiproliferative properties and are known to affect various immunological processes. Some of these activities have been shown to be potentiated by increased temperatures. When cells are subjected to a rise in temperature, the synthesis of the heat-shock proteins (HSPs) is 'switched on.' In this report we demonstrate a synergistic effect of IFN and stress (arsenite treatment or elevated temperature) on the heat-shock response. On the one hand, IFN pretreatment enhances the accumulation of HSP mRNAs and the corresponding protein synthesis after a mild stress and, on the other hand, it amplifies the decrease of the total protein synthesis after a severe stress. Thus in IFN pretreated cells the range of temperatures leading to the heat-shock response is shifted towards common physiological values.

Animals↗
PubMed1984

Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.

Animals↗
PubMed1983

Spontaneous high expression of heat-shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo.

When submitted to a heat-shock, mouse embryonal carcinoma (EC) and fibroblast cells show very different behavior. All the EC cells so far analyzed express very high levels of several heat-shock proteins (HSP) in the absence of stress and independent of their origin and culture conditions. In such cells, the 89-kd, 70-kd and 59-kd HSP are the most prominent proteins after actin. In addition, the 89-kd and 59-kd HSP are not stimulated by an arsenite shock in contrast to what is observed with fibroblasts or cells of the parietal yolk sac type. Arsenite induces the synthesis of a 105-kd polypeptide in fibroblasts but not in EC cells. In vitro differentiation of F9 cells induced by retinoic acid and dibutyryl cAMP is accompanied by a decrease in the spontaneous relative abundance of HSP and restores the arsenite-induced synthesis of the 105-kd polypeptide. EC cells are usually believed to be similar to inner cell mass cells of mouse blastocyst. Furthermore, data in the literature together with our own results suggest that the same three HSP are also spontaneously expressed in high amounts in the early mouse embryo.

Animals↗