All organisms respond to a sudden increase in temperature by the so-called heat shock response. This response results in the induction of a subset of genes, designated heat shock genes coding for heat shock proteins, which allow the cell to cope with the stress regimen. Research carried out during the last 10 years with eubacteria has revealed that the heat shock genes of a given species fall into different classes (regulons), where each class is regulated by a different transcriptional regulator, which could be an alternative sigma factor, a transcriptional activator, or a transcriptional repressor. All regulons of a single species constitute the heat shock stimulon. In Bacillus subtilis, more than 200 genes representing over 7% of the transcriptionally active genes are induced at least 3-fold in response to a heat shock. This response becomes apparent within the first minute after exposure to heat stress, is transient, and is coordinated by at least 5 transcriptional regulator proteins, including 2 repressors, an alternate sigma-factor, and a 2-component signal transduction system. A detailed analysis of the regulation of all known heat shock genes has shown that they belong to at least 6 regulons that together comprise the B subtilis heat shock stimulon. Potential thermosensors are discussed in this article.

The cochaperone GrpE functions as a nucleotide exchange factor to promote dissociation of adenosine 5′-diphosphate (ADP) from the nucleotide-binding cleft of DnaK. GrpE and the DnaJ cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of DnaK by regulating the nucleotide-bound state of DnaK. DnaJ stimulates nucleotide hydrolysis, and GrpE promotes the exchange of ADP for adenosine triphosphate (ATP) and also augments peptide release from the DnaK substrate-binding domain in an ATP-independent manner. The eukaryotic cytosol does not contain GrpE per se because GrpE-like function is provided by the BAG1 protein, which acts as a nucleotide exchange factor for cytosolic Hsp70s. GrpE, which plays a prominent role in mitochondria, chloroplasts, and bacterial cytoplasms, is a fascinating molecule with an unusual quaternary structure. The long α-helices of GrpE have been hypothesized to act as a thermosensor and to be involved in the decrease in GrpE-dependent nucleotide exchange that is observed in vitro at temperatures relevant to heat shock. This review describes the molecular biology of GrpE and focuses on the structural and kinetic aspects of nucleotide exchange, peptide release, and the thermosensor hypothesis.

BAG-1 (Bcl-2–associated athanogene) is a multifaceted protein implicated in the modulation of a large variety of cellular processes. Elucidating the molecular mechanisms that underlie the cellular functions of BAG-1 becomes an increasingly important task, particularly in light of the growing evidence connecting aberrant BAG-1 expression to certain human cancers. A common element of the remarkable functional diversity of BAG-1 appears to be the interaction with molecular chaperones of the Hsp70 family. In fact, BAG-1 functions as a nucleotide exchange factor of mammalian cytosolic Hsc70, thereby triggering substrate unloading from the chaperone. In addition, recent findings reveal an association of BAG-1 with the proteasome, which suggests a role in coordinating chaperone and degradation pathways.

We used sea urchin embryos as bioindicators to study the effects of exposure to sublethal cadmium concentrations on the expression of the metallothionein (MT) gene stress marker. For this purpose, the complete complementary deoxyribonucleic acid of the species Paracentrotus lividus (Pl) was cloned and sequenced. Northern blot analysis showed that basal levels of Pl-MT messenger ribonucleic acid, having an apparent size of 700 bases, are expressed in all developmental stages analyzed, from early cleavage to pluteus. However, when embryos were continuously cultured in sublethal CdCl₂ concentrations and harvested at cleavage, swimming blastula, late gastrula, and pluteus stages (6, 12, 24, and 48 hours after fertilization, respectively), a time- and dose-dependent increase in the transcription levels of the Pl-MT gene was observed. Interestingly, although microscopical inspection revealed the occurrence of abnormalities only after 24 hours of exposure to the pollutant, Northern blot and reverse transcriptase–polymerase chain reaction analyses revealed significant increases in Pl-MT expression levels already after 12 and 6 hours of exposure, respectively. Therefore, this study confirms the validity of MT as marker of exposure and provides evidence that Pl-MT and sea urchin embryos can be a potentially valuable and sensitive model for testing in very short periods of time seawaters heavily contaminated with cadmium.

Cytokines play a major role in regulating both humoral and cell-mediated immune responses. Recent advances in our understanding of cell-mediated immune responses have focused on the antigen presentation machinery and the proteins in the endoplasmic reticulum (ER). These proteins help the formation and stabilization of the major histocompatibility complex (MHC)–peptide interaction. A 96-kDa, ER-resident glycoprotein (gp96) is being evaluated as a therapeutic agent in cancer because of its ability to associate with a vast number of cellular peptides irrespective of size or sequence. Because the antigen presentation complex is assembled in the ER and a number of ER-resident proteins are modulated by cytokines, it is important to examine the regulation of gp96 in response to immune cytokines interferon γ (IFN-γ), and interleukin 2 (IL-2). Defects in signaling pathway in either of the cytokines can result in suboptimal immune response. We examined the effect of the cytokines IFN-γ and IL-2 on the induction of gp96 in different cancer cell lines and examined the induction of DNA-binding proteins that recognize gamma interferon–activating sequence (GAS), present in the promoter region of gp96. The induction of GAS binding protein correlated with the induction of STAT 1 protein, a transcriptional regulator and mediator of IFN-γ–mediated gene expression. The use of cytokines in inducing gp96 levels may have significance in maintaining high levels of gp96 for a sustained immune response.

Hsp27 is considered a potential marker for cell differentiation in diverse tissues. Several aspects linked to the differentiation process and to the transition from high to low metastatic potential were analyzed in melanoma cells transfected with Hsp27. E-cadherin plays a central role in cell differentiation, migration, and normal development. Loss of expression or function of E-cadherin has been documented in a variety of human malignancies. We observed by fluorescence-activated cell sorter (FACS) as well as immunofluorescence (IF) analysis a pronounced expression of E-cadherin in Hsp27-transfected A375 melanoma cells compared with control melanoma cells. The expression of the adhesion molecule MUC18/MCAM correlates directly with the metastatic potential of melanoma cells. In contrast to wild-type and neotransfected melanoma cells, in Hsp27-transfected cells the expression of MUC18/MCAM could not be detected by FACS and IF analysis. The plasminogen activator (PA) system plays a central role in mediating extracellular proteolysis and also in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. Hsp27 transfectants revealed elevated messenger ribonucleic acid expression of the urokinase-type PA (uPA) and its inhibitor, PA inhibitor type 1, which might indicate a neutralization effect of the proteolytic activity of uPA. Control cells failed to express both these molecules. The influence of Hsp27 expression on uPA activity and the involvement of E-cadherin could be demonstrated by use of anti–E-cadherin–blocking antibody. Our data provide evidence for an inhibitory-regulatory role of Hsp27 in tumor progression as found in our system.

Lobster claw muscle undergoes atrophy in correlation with increasing ecdysteroid (steroid molting hormone) titers during premolt. In vivo molecular chaperone (constitutive heat shock protein 70 [Hsc70], heat shock protein 70 [Hsp70], and Hsp90) and polyubiquitin messenger ribonucleic acid (mRNA) levels were examined in claw and abdominal muscles from individual premolt or intermolt lobsters. Polyubiquitin gene expression was assayed as a marker for muscle atrophy. Both Hsc70 and Hsp90 mRNA levels were significantly induced in premolt relative to intermolt lobster claw muscle, whereas Hsp70 mRNA levels were not. Hsp90 gene expression was significantly higher in premolt claw muscle when compared with abdominal muscle. Polyubiquitin mRNA levels were elevated in premolt when compared with intermolt claw muscle and significantly elevated relative to premolt abdominal muscle.

The presence of the soluble intracellular heat shock protein gp96 (an endoplasmic reticulum resident protein) at the surface of certain cell types is an intriguing phenomenon whose physiological significance has been unclear. We have shown that the active surface expression of gp96 by some immune cells is found throughout the vertebrate phylum including the Agnatha, the only vertebrate taxon whose members (lamprey, hagfish) lack an adaptive immune system. To determine whether gp96 surface expression can be modulated by pathogens, we investigated the effects of in vitro stimulation by purified lipopolysaccharide (LPS) and the heat-killed gram-negative bacteria, Escherischia coli and Aeromonas hydrophilia. Purified Xenopus B cells are readily activated and markedly proliferate in vitro in response to the heat-killed bacteria but not to purified LPS. Furthermore, messenger ribonucleic acid, and intracellular and surface protein expressions of both gp96 and immunoglobulin were upregulated only after activation of B cells by heat-killed bacteria. These data are consistent with an ancestral immunological role of gp96 as an antigen-presenting or danger-signaling molecule, or both, interacting directly with antigen-presenting cells, T cells, or natural killer cells, (or all), to trigger or amplify immune responses.

Extracellular heat-shock proteins (eHsp) such as those belonging to the 70-kDa family of Hsp (eg, Hsp72) have been hypothesized to act as a “danger signal” to immune cells, promote immune responses, and improve host defense. The current study tested this hypothesis. Adult male F344 rats were exposed to an acute laboratory stressor (100, 5-second, 1.6-mA inescapable tail shocks) and challenged with Escherichia coli. The number of colony-forming units (CFU) of bacteria at the site of injection, the levels of eHsp72, the immune response to eHsp72 and E coli–derived lipopolysaccharide (LPS), and the amount of time required to recover from in vivo bacterial challenge were measured. CFUs were reduced 2, 4, and 6 hours after injection of E coli in rats exposed to stress. Rats exposed to stress had elevated eHsp72 that was elevated rapidly (25 minutes) and remained elevated in the circulation and at the inflammatory site (2 hours after stressor termination). Both stressor exposure and eHsp72 administration in the absence of stress resulted in a facilitated pattern of recovery after bacterial inflammation induced by subcutaneous E coli injection. Rats exposed to acute restraint (100 minutes) did not demonstrate elevated circulating eHsp72 or a facilitated pattern of recovery after bacterial challenge. In vitro stimulation of rat splenocytes and macrophages with eHsp72 elevated nitric oxide (NO), tumor necrosis factor alpha (TNF-α), interleukin (IL)-1beta, and IL-6, and this effect was specific to eHsp72 because it was not diminished by polymyxin B and was reduced by earlier heat-denature treatment. Stimulation of cells with eHsp72 combined with LPS resulted in a greater NO and cytokine response than that observed after stimulation with eHsp72 or LPS alone. In vivo, at the inflammatory site, the bacterial-induced NO response was potentiated by stress, and NO inhibition (L-NIO) reduced the stress-induced facilitation but had no effect on the control kinetics of bacterial inflammation recovery. Thus, these results lend support to the hypothesis that intense stressor exposure increases eHsp72, which acts as a danger signal to potentiate the NO response to bacterial challenge and facilitate recovery from bacterial inflammation.

In pancreatic acinar cells, chaperonin Cpn60 is present in all the cellular compartments involved in protein secretion as well as in mitochondria. To better understand the role Cpn60 plays in pancreatic secretion, we have evaluated its changes under experimental conditions known to alter pancreatic secretion. Quantitative protein A–gold immunocytochemistry was used to reveal Cpn60 in pancreatic acinar cells. Cpn60 immunolabelings in cellular compartments involved in secretion were found to decrease in acute pancreatitis as well as upon stimulation of secretion and in starvation conditions. A major increase in Cpn60 was recorded in diabetic condition. This was normalized by insulin treatment. Although in certain situations changes in secretory enzymes and in Cpn60 correlate well, in others, nonparallel secretion seemed to take place. In contrast, expression of mitochondrial Cpn60 in acinar cells appeared to remain stable in all conditions except starvation, where its levels decreased. Expression of Cpn60 in the secretory pathway and in mitochondria thus appears to behave differently, and Cpn60 in the secretory pathway must be important for quality control and integrity of secretion.