Some effects of growth conditions on steady state and heat shock induced htpG gene expression in continuous cultures of Escherichia coli

Most of the data concerning heat shock gene expression reported in the literature are derived from batch culture experiments under substrate and nutrient sufficient conditions. Here, the effects of dilution rate and medium composition on the steady state and heat shock induced htpG gene expression have been investigated in continuous cultures of Escherichia coli, using a chromosomal htpG-lacZ gene fusion. During steady state growth temperature dependent patterns of the relative htpG expression were found to be largely similar, irrespective of the growth condition. However, nitrogen-limited growth resulted in a markedly reduced specific steady state htpG expression as compared to growth under carbon limitation or in complex medium, correlating qualitatively with the total cellular protein content. During heat shock, tight temperature controlled expression was evident. While the relative heat shock induced expression was largely identical at various dilution rates in a given growth medium, significantly different response patterns were observed in the three growth media at any give dilution rate. From these results a clearly temperature regulated htpG expression during both, steady and transient state growth in continuous culture is evident, which is further significantly affected by the growth condition used.     

Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70-kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several-fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70-related cDNA probes. Transient ischemia resulted in 5- to 10-fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the heat-shock response displayed by two Chaetomium species originating from different thermal environments.

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.     

Discordant expression of heat shock protein mRNAs in tissues of heat-stressed rats.

Although the induction of heat shock proteins (HSP) has been studied extensively in cultured cells, comparatively few studies have examined their expression in vivo. In this report, mRNA expression of two HSP families, HSP70 and HSP27, was investigated in brain, liver, lung, and skin of rats exposed to elevated ambient temperatures. The time course and relative magnitude of the heat-induced expression for these two HSP differed between tissues of the same animal. Even within the same tissue, HSP70 and HSP27 displayed differential kinetics of induction. In brain, lung, and skin, induction of HSP70 was dependent on the duration and temperature of the heat stress. This induction was transient with maximal HSP70 expression occurring at 1 h and returning to baseline 3 h after removal of the animals from heat stress. In liver, HSP70 expression did not show a direct relationship with temperature conditions and maximal induction did not occur until 6 h after heat stress. Heat-induced HSP27 expression was dependent on time and temperature of exposure in lung and skin but not in brain and liver. These findings demonstrate that the heat shock response in vivo lacks much of the coordinate control of expression characteristic of cultured cell populations and suggest that mechanisms controlling this cellular stress response are influenced by physiologic factors that cannot be studied in vitro.     

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.     

Characterization of cold-induced heat shock protein expression in neonatal rat cardiomyocytes

Cardiac surgery is usually performed under conditions of cardioplegicischemic arrest. To protect the heart during the ischemic period, themyocardium is exposed to varying degrees of hypothermia. Althoughhyperthermia is known to induce the heat shock response, the moleculareffects of hypothermia on the myocardium have not been investigated. We havestudied the effect of hypothermia on the induction of heat shock proteins inprimary cultures of neonatal cardiomyocytes. Cold stress in cardiomyocytesinduced a 6 fold increase in the heat shock protein HSP70 as compared tocontrol. Increased HSP70 protein levels correlated with induction of HSP70mRNAs. Maximal levels of HSP70 protein appeared 4-6 h following recoveryfrom cold shock, indicating the transient nature of the response. Inductionof HSP25 mRNA was also observed in cold-shocked cardiomyocytes, even thoughincreased HSP25 protein levels were not detected. Our results indicate thathypothermia is capable of inducing the heat shock response in neonatalcardiomyocytes.     

The effect of heat shock on amino acid transport and cell volume in 3T3 cells

Summary. In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response. Received June 30, 1999 Accepted July 27, 2000     

Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27

The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration). esophagus; esophagitis; gastroesophageal reflux disease; microvasculature; phosphatidylinositol 3-kinase/Akt; VCAM-1     

Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42°C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.     

大肠杆菌热激反应研究及其在重组蛋白表达中的应用

当大肠杆菌所处的环境温度忽然升高时, 细胞体内会激发热激反应, 体内会迅速合成多种热激蛋白, 由热激转录因子调控的热激蛋白主要包括一些分子伴侣、蛋白降解酶、折叠辅助蛋白等。热激蛋白可以促进蛋白正确折叠, 降解错误折叠的蛋白。主要介绍大肠杆菌热激蛋白的表达调控及其功能, 利用热激转录因子发展的新型温控分泌表达系统及其在蛋白可溶性表达中的应用, 以及热激分子伴侣与重组蛋白共表达的研究进展。     

Heat shock protects cultured neurons from glutamate toxicity.

Expression of heat shock proteins (HSPs) occurs in brain after ischemia and status epilepticus. We report that induction of the heat shock response in cortical cultures protects neurons from glutamate-induced excitotoxicity. Cultures heated to 42.2 degrees C for 20 min showed an overall decrease in protein synthesis but an increase in the synthesis of approximately 72 and approximately 85 kd proteins and in the levels of HSP70 mRNA. Heat shock inhibited excitotoxicity in cells exposed to glutamate at 3 or 24 hr following heat exposure, but not when the interval between heat and glutamate exposure was shortened to 15 min or lengthened to 48 hr. Protection due to heat shock required new protein synthesis, since it did not occur when protein or RNA synthesis inhibitors were added. By ameliorating excitotoxic processes, HSPs may attenuate brain injury in certain pathologic conditions.     

70-Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo

Induction of the 70-kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress-inducible species of hsp70-related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.     

Cold shock response in Escherichia coli

Sensing a sudden change of the growth temperature, all living organisms produce heat shock proteins or cold shock proteins to adapt to a given temperature. In a heat shock response, the heat shock sigma factor plays a major role in the induction of heat shock proteins including molecular chaperones and proteases, which are well-conserved from bacteria to human. In contrast, no such a sigma factor has been identified for the cold shock response. Instead, RNAs and RNA-binding proteins play a major role in cold shock response. This review describes what happens in the cell upon cold shock, how E. coli responds to cold shock, how the expression of cold shock proteins is regulated, and what their functions are.