Heat shock proteins of Toxoplasma gondii

We have investigated heat shock protein (HSP) expression in mouse-virulent and -avirulent strains of Toxoplasma gondii by performing Western blot analysis using a monoclonal antibody against HSP65 of Mycobacterium bovis and a polyclonal antiserum against HSP70 of Plasmodium falciparum as primary antibodies. We initially observed that murine macrophages express HSP65 when infected with either virulent or avirulent strains, a result which contradicts previous reports. Differential HSP expression consistent which virulence was observed between strains, with high levels of a 70kDa HSP (HSP70) only detected in virulent strains in vivo. This protein was not observed in virulent strains in the immunocompromised mouse or in vitro, suggesting induction by immunological stress. This protein was only poorly expressed in avirulent strains. A 65kDa protein was observed in all strains in vivo and in vitro, suggesting a shared epitope with HSP70. These results are consistent with the hypothesis that the induced expression of HSP70 in virulent strains of T. gondii by immunological stresses may provide protection for these strains against cell damage associated with invasion of the host, allowing the virulent strains to persist as tachyzoites without the requirement for the encystation observed in avirulent strains.     

Modulation of macrophage heat shock proteins (HSPs) expression in response to intracellular infection by virulent and avirulent strains of Leishmania donovani

We have examined the differential expression of heat shock proteins of murine macrophage-like cell line J774 G8 following infection with Leishmania donovani. In response to infection with virulent promastigotes, the up-regulation of HSP70 and 90 and a selective down-regulation of HSP60 was observed using monoclonal antibodies specific for host HSPs. However, infection with avirulent strain failed to alter the expression of host HSPs. The maximum alterations in HSPs expression were noted at 18 h post infection, a time period which coincided with the transformation of parasite from promastigote to the amastigote form. Data indicates that host HSPs may play a role in parasite differentiation/survival during infection with L. donovani.     

Heterogeneous patterns of constitutive and heat shock induced expression of HLA-linked HSP70-1 and HSP70-2 heat shock genes in human melanoma cell lines

The heat shock response, which is characterized by the induction of heat shock proteins, is known to affect the ability of tumour cells to cope with potentially adverse conditions such as hypoxia, glucose starvation and cytotoxic immune reactions. To assess the heat shock response of melanoma cells, spontaneous and heat shock induced expression of heat shock proteins was analysed in a panel of 17 human melanoma cell lines. Constitutive expression of HSP27, HSP70, HSC70, HSP90alphabeta and GRP94 proteins was found in all the melanoma cell lines, and HSP70 and HSC70 were also induced by heat shock. The major heat inducible HLA-linked HSP70-1 and HSP70-2 genes were analysed at the mRNA level. Basal expression and inducibility varied between the different melanoma cell lines. In addition, in situ hybridization demonstrated heterogeneous expression of these genes among single cells of a given cell line. In general, each melanoma cell line appears to exhibit an individual type of HSP70 expression that might reflect selection during tumour progression and therapy.     

Pressure-induced expression of heat shock protein 70 mRNA in adult rat heart is coupled both to protein kinase A-dependent and protein kinase C-dependent systems

BACKGROUND: Production of heat shock protein 70 (HSP70) in the heart is induced by hemodynamic stress, but its intracellular signal transduction system has not been elucidated well. OBJECTIVE: To investigate the hypothesis that protein kinase A (PKA)-dependent and protein kinase C (PKC)dependent systems are involved in the pressure-induced expression of HSP70 mRNA in perfused adult rat heart METHODS: Isolated tetrodotoxin-arrested Sprague-Dawley rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. Aortic pressure in rats of the pressure-overloaded group was elevated from 60 to 120 mmHg for 2-120 min. cAMP contents and rates of synthesis of protein were measured by radioimmunoassay and the incorporation of [14C]-phenylalanine into total heart protein, respectively. Expression of HSP70 mRNA was determined by Northern blot analysis. RESULTS: Elevation of aortic pressure significantly increased cAMP content after 2 min of perfusion (by 41%), significantly increased rates of synthesis of protein during the second hour of perfusion (by 41%), and induced expression of HSP70 mRNA maximally after 60 min of perfusion (2.7-fold the control value). Exposure to glucagon, forskolin or 1 -methyl-3-isobutylxanthine mimicked increases in these parameters caused by elevation of aortic pressure. Administration of a selective PKA inhibitor, H-89, significantly prevented induction of increases in expression of HSP70 mRNA and rates of synthesis of protein by a high pressure overload and exposure to agents that increase cAMP content. Furthermore, administration of phorbol ester induced expression of HSP70 mRNA. Administration of a PKC inhibitor, calphostin C, significantly prevented induction of increases in expression of HSP70 mRNA by a pressure overload and by exposure to phorbol ester. CONCLUSIONS: These results suggest that the pressure-induced induction of production of HSP70 is regulated both by PKA-dependent and by PKC-dependent systems during periods of active synthesis of protein in adult rat heart.     

Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.     

Modulation of heat-shock protein 70 (HSP70) gene expression by sodium butyrate in U-937 promonocytic cells: relationships with differentiation and apoptosis

The administration of sodium butyrate at 0.75 mM induced the functional differentiation of U-937 human promonocytic leukemia cells with negligible cell mortality. However, the drug rapidly caused cell death with characteristics of apoptosis when used at concentrations of 5 mM and above. In addition, butyrate stimulated the expression of the stress-responsive heat-shock protein 70 (HSP70) gene when applied at both differentiation-inducing and apoptosis-inducing concentrations. The induction of HSP70 by butyrate was inhibited by the simultaneous addition of cAMP-increasing agents (dibutyryl cAMP or the combination of forskolin plus theophylline). However, these agents did not prevent differentiation and only partially reduced apoptosis. Moreover, the DNA topoisomerase II inhibitor etoposide, which provoked U-937 cell differentiation and apoptosis with the same or greater efficiency than butyrate, failed to stimulate HSP70 expression. Finally, it was observed that cAMP-increasing agents also abrogated the induction of HSP70 and reduced the apoptosis caused by cadmium chloride, a typical inducer of the stress response. Taken together, these results indicate that HSP70 expression is not required for differentiation of promonocytic cells, as earlier proposed, and that butyrate probably triggers the stress response in these cells.     

Bronchopulmonary dysplasia and oxidative stress: are we closer to an understanding of the pathogenesis of BPD?

OBJECTIVES: To determine if induction of heat shock protein 70 (HSP 70), a stress protein that plays a cytoprotective role and inhibits cell death in response to various stimuli, will protect thymocytes and T-cell clones from radiation-induced apoptosis, and to define the mechanism of such protection. DESIGN: Thymocytes from BALB/c mice or T-lymphocyte clones were incubated at 43 degrees C for 1 hour to induce HSP 70, then irradiated. Control cells were irradiated but not heated. Fragmentation of DNA was quantitated, and p53, bax, and bcl-2 expression was analyzed at various times by the Western blot method. RESULTS: Only heated cells expressed HSP 70. The induction of HSP 70 increased basal apoptosis but significantly decreased radiation-induced apoptosis. Furthermore, introduction of an HSP 70 antisense oligomer prior to heating reversed the protective effect of HSP 70. Induction of HSP 70 in T-cell clones with sodium arsenite had a similar protective effect against radiation-induced apoptosis. Irradiation induced p53 and markedly up-regulated bax. The expression of p53 peaked at 4 hours and preceded maximal bax induction. Induction of HSP 70 prior to irradiation suppressed p53 and significantly decreased bax levels. Levels of bcl-2 were unaffected. CONCLUSIONS: Our data show that HSP 70 induction protects thymocytes from radiation-induced apoptosis by down-regulating p53 and bax expression. The induction of HSP 70 may represent a novel mechanism by which the immunosuppressive effects and the associated infectious complications of radiation therapy can be minimized.     

One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase

Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.     

Evidence that GABA transmission mediates context-specific extinction of learned fear

Recent evidence has suggested that molecular chaperones participate in the conformational change between the normal cellular prion protein (PrPC) and its scrapie isoform (PrPSc). To study a role of PrPC in the regulation of expression of heat shock proteins (HSPs), a group of molecular chaperones, heat-induced expression of major HSPs (HSP105, HSP90alpha, HSP72, HSC70, HSP60, and HSP25) was investigated in cultured skin fibroblasts isolated from the mice homogeneous for a disrupted PrP gene (PrP-/- mice) by Western blot analysis and immunocytochemistry. Two lines of fibroblasts were established and designated SFK derived from the PrP-/- mice and SFH derived from the PrP+/+ mice, respectively. In both SFK and SFH cells, HSP105, HSP72, and HSP25 were expressed at low levels under unstressed conditions but they were induced markedly following exposure to heat stress (43 degreesC/20 min) at 3-72 h postrecovery. In both cell types, HSC70 and HSP60 were expressed at high levels under unstressed conditions and their levels remained unchanged after heat shock treatment. HSP90alpha was undetectable in both cell types under any conditions examined. The pattern of expression, induction, and subcellular location of HSP105, HSP72, HSC70, HSP60, and HSP25 was not significantly different between SFK and SFH cells under unstressed and heat-stressed conditions. Furthermore, the levels of constitutive expression of HSP105, HSC70, HSP60, and HSP25 were similar between the brain tissues isolated from the PrP-/- and PrP+/+ mice. These results indicate that HSP induction is not affected by either the existence or the absence of PrPC in the cells.     

Focal hyperexpression of hemeoxygenase-1 protein and messenger RNA in rat brain caused by cellular stress following subarachnoid injections of lysed blood

Induction of the hemeoxygenase-1 (ho-1) stress gene is of importance for rapid heme metabolism and protection against oxidative injury in vitro and in vivo. Although ho-1 expression is observed in glia following exposure to whole blood and oxyhemoglobin, expression is mild, and other stress genes are not induced simultaneously in this setting. Hemeoxygenase-1 can be induced by several other physiological stresses in addition to heme. In the brain, ho-1 induction has been observed in the penumbra following focal cerebral ischemia. Because lysed blood is a spasmogen, the authors studied focal hyperexpression of the ho-1 gene after injection of lysed blood, whole blood, or saline into the cisterna magna of adult rats. Immunocytochemical analysis of HO-1 was performed at 1, 2, 3, and 4 days after the injections. Because the 70-kD inducible heat shock protein (HSP70) is induced by cellular stress, alternate sections were immunostained for HSP70 to assess whether focal hyperexpression was a stress phenomenon. An oligonucleotide probe was also used for in situ hybridization to demonstrate that ho-1 messenger (m)RNA was present. Focal HO-1 immunostained areas were observed after lysed blood injection only and were located mainly in the basal cortex and cerebellar hemisphere, although focal hyperexpression was also found in many other regions. The intensity of staining and the number of regions were maximum at 1 day. Double-labeled immunofluorescence revealed that many HO-1-immunoreactive cells were microglia. The HSP70 immunostaining of adjacent sections from the same animals demonstrated focal regions of immunoreactivity whose topography corresponded exactly with the topography of the HO-1-immunostained areas. Conventional histology in regions of HO-1 hyperexpression was often normal. In situ hybridization using the same oligonucleotide demonstrated that ho-1 mRNA was induced in focal areas of forebrain and in large regions of cerebellum within 6 hours of injection. These results demonstrate that focal hyperexpression of the ho-1 stress gene occurs after lysed blood injection and appears to be an indicator of cellular stress and injury in regions in which infarction does not occur. These results also suggest that cellular injury that occurs after injection of lysed blood may go undetected using conventional histology. Although direct heme metabolism was not investigated, our results indicate that rapid metabolism of heme, both intracellular and extracellular, may prove to be beneficial after subarachnoid hemorrhage.     

Differential expression of Mr 70,000 heat shock protein in normal, premalignant, and malignant human uterine cervix

Transformed cells differ from normal cells in their pattern of heat shock protein expression. Here, we report differential expression of a Mr 70,000 heat shock protein (HSP70) in squamous cell carcinomas of the uterine cervix compared to normal or premalignant uterine cervix. Expression of HSP70 was measured using a mouse mAb against HSP70 by an ELISA. A significant increase in the level of expression of HSP70 was observed in fresh tumor cells as compared to normal or dysplastic ones (P < 0.001). The induction of HSP70 in tumor cells subjected to hyperthermia was less than that in normal or premalignant cells. Strong cytoplasmic and nuclear immunostaining was observed in cancerous tissues using anti-HSP70 mAb, biotinylated rabbit antimouse immunoglobulins, avidin combined in vitro with biotinylated horseradish peroxidase, and diaminobenzidine tetrachloride. HSP70 immunostaining was not detected in normal or dysplastic tissue sections. The results were confirmed by immunoprecipitation using anti-HSP70 mAb. These results are the first demonstration of overexpression of HSP70 in squamous cell carcinomas of the uterine cervix as compared to that in dysplastic or normal uterine cervix cells. We conclude that tumor cells differ from normal cells in their pattern of HSP70 expression, and increased levels of HSP70 correlate with an increase in tumor size.     

Elevated ovarian follicular apoptosis and heat shock protein-70 expression in white sucker exposed to bleached kraft pulp mill effluent

Exposure of feral fish populations to bleached kraft pulp mill effluent (BKME) results in a variety of negative impacts on reproductive fitness including reduced ovarian development, reduced egg size, decreased fecundity with age, delayed sexual maturation, and alterations in reproductive endocrine homeostasis at multiple sites along the pituitary-gonadal axis. The present study provides evidence of elevated apoptotic DNA fragmentation and increased expression of the 70-kDa heat shock protein (HSP70) in ovarian follicular cells from prespawning white sucker (Catostomus commersoni) exposed to BKME. Apoptosis is the molecular mechanism responsible for ovarian follicular atresia which is involved in various stages of vertebrate ovarian development such as follicular recruitment, growth, differentiation, and regression. In mammals, induction of HSP70 is associated with inhibition of hormone-sensitive steroidogenesis and mediation of luteal regression. The 3'-end labeling of isolated ovarian follicular cell DNA revealed a 10-fold increase in the extent of apoptosis in BKME-exposed white sucker in comparison to follicles collected from a nearby reference site. Western blotting for ovarian follicular HSP70 levels showed increased expression of this protein in fish exposed to BKME. The elevated ovarian cell apoptosis and increased HSP70 expression in BKME-exposed fish were associated with reduced ovary size, decreased plasma testosterone, and increased plasma 17 beta-estradiol concentrations, but not induction of hepatic ethoxyresorufin O-deethylase activity. It is not known whether increased ovarian HSP70 expression in BKME-exposed fish is related to elevated apoptosis or represents a general response to environmental stress. Since apoptosis is regulated by several hormonal factors and conserved gene products, these data suggest that certain components of BKME increase ovarian cell apoptosis in fish via stimulation of cell death signaling. However, it is unclear whether BKME stimulates ovarian cell apoptosis directly or if this response occurs secondarily as a result of altered reproductive endocrine homeostasis.