Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ³², was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.     

Two small heat shock protein genes in Apis cerana cerana: characterization,regulation, and developmental expression

In the present study, we identified and characterized two small heat shock protein genes from Apis cerana cerana, named AccHsp24.2 and AccHsp23.0. An alignment analysis showed that AccHsp24.2 and AccHsp23.0 share high similarity with other members of the α-crystallin/sHSP family, all of which contain the conserved α-crystallin domain. The recombinant AccHsp24.2 and AccHsp23.0 proteins were shown to have molecular chaperone activity by the malate dehydrogenase thermal aggregation assay. Three heat shock elements were detected in the 5′-flanking region of AccHsp24.2 and eleven in AccHsp23.0, and two Drosophila Broad-Complex genes for ecdysone steroid response sites were found in each of the genes. The presence of these elements suggests that the expression of these genes might be regulated by heat shock and ecdysone, which was confirmed by quantitative RT-PCR (RT-qPCR). The results revealed that the expression of the two genes could be induced by cold shock (4 °C) and heat shock (37 °C and 43 °C) in an analogous manner, and AccHsp24.2 was more susceptible than AccHsp23.0. In addition, the expression of the two genes was induced by high concentrations of ecdysone in vitro and in vivo. The accumulation of AccHsp24.2 and AccHsp23.0 mRNA was also detected in different developmental stages and tissues. In spite of the differential expression at the same stage, these genes shared similar developmental patterns, suggesting that they are regulated by similar mechanisms.     

Spatial control of developmental regulatory genes inAspergillus nidulans

ThebrlA andabaA genes ofAspergillus nidulans regulate stages of conidiophore development and are themselves regulated during development.brlA⁻ mutants produce conidiophore stalks devoid of vesicles, sterigmata, and spores.abaA⁻ mutants produce most of the conidiophore structures but fail to form conidia. To assess the spatial expression of these two genes, we fused the 5′ flanking region ofbrlA orabaA to theEscherichia coli lacZ gene.A. nidulans transformants with a single copy of either fusion gene integrated at a defined heterologus locus (argB) expressedβ-galactosidase during conidiophore development, parallelingbrlA andabaA mRNA accumulation. Controls lacking the fusion genes produced little or noβ-galactosidase activity. A method forin situ detection ofβ-galactosidase was devised. Hyphae or conidiophores were permeabilized by treatment with chloroform vapors and stained with 5-bromo-4-chloroindolyl-β-d-galactoside.β-Galactosidase activity was detected in specific conidiophore cell types.brlA- andabaA-directedβ-galactosidase accumulated in vesicles, sterigmata, and immature conidia. This procedure should be applicable for determining cellular specificities of gene expression in fungi for which transformation systems exist.     

Heat shock-induced development of infection structures by bean rust uredospore germlings

Germlings ofUromyces appendiculatus induced by exposure to 28.5°C heat for 1.5 h developed infection structures at nearly the same rate as those induced thigmotropically. Typically, the microtubules began to rearrange within 10 minutes of the start of heat treatment; nuclear migration followed. Mitosis started after 36 minutes. Appressorium development was accompanied by the appearance of six heat shock proteins, but not by the thigmotropic-specific proteins always observed during contact-induced development.     

Extensin and Phenylalanine Ammonia-Lyase Gene Expression Altered in Potato Tubers in Response to Wounding, Hypoxia, and Erwinia carotovora Infection

Potato (Solanum tuberosum L.) tubers are susceptible to infection by Erwinia carotovora, causal agent of bacterial soft rot, when wounded and subjected to wet, hypoxic environments. The expression of two putative plant defense genes, extensin and phenylalanine ammonia-lyase (PAL), was examined by monitoring their respective mRNA levels and cell wall hydroxyproline levels in tuber tissues under various conditions leading to susceptibility or resistance and after inoculation with E. carotovora in order to assess the possible roles of these genes and their products in this plant-pathogen interaction. Extensin and PAL mRNA levels as well as cell wall hydroxyproline levels accumulated markedly in response to wounding and subsequent aerobic incubation. Extensin and PAL mRNA levels as well as cell wall hydroxyproline levels decreased in response to wounding and subsequent anaerobic incubation; these changes were correlated with high susceptibility of tuber tissue to E. carotovora infection. Inoculation of wound sites with E. carotovora caused some additional accumulation of the wound-regulated extensin and PAL mRNAs under certain aerobic conditions, but never under anaerobic conditions.     

Heat Shock Modulates Neutrophil Motility in Zebrafish

Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.     

Evaluation of stress-inducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis

In this study we have evaluated stress-inducible hsp90 mRNA accumulation as a potential molecular biomarker in Xenopus laevis. In order to obtain a probe for Northern blot analysis we employed a PCR-based approach using degenerate primers for the amplification and cloning of an hsp90 gene sequence from Xenopus laevis. The deduced amino acid sequence is 102 amino acids in length and exhibited the highest degree of identity with zebrafish and human hsp90 β genes. Futhermore, the putative intron and exon boundaries of this fragment are the same as hsp90 β in chicken, mouse and human, indicating that the fragment represents a Xenopus hsp90 β-like gene. Northern blot analyses revealed that this gene was constitutively expressed in cultured A6 cells. While heat shock and sodium arsenite exposure resulted in the increased accumulation of hsp90 mRNA in A6 cells, treatment with cadmium chloride and zinc chloride did not. Also, exposure of A6 cells to concurrent heat shock and sodium arsenite produced a mild synergistic response with respects to hsp90 mRNA levels in contrast to hsp70 mRNA levels which displayed a strong synergistic effect. Finally, hsp90 mRNA was detected consitutively throughout early embrogenesis but was heat-inducible only in late blastula and later stages of development. Given the normal abundance and limited stress-induced accumulation of hsp90 mRNA, it may not have a great deal of potential as a molecular biomarker compared to hsp70 and hsp30 mRNA. However, it may be useful in conjunction with other stress protein mRNAs to establish a set of biomarker profiles to characterize the cellular response to a stressful or toxic agent.     

Withaferin A Induces Proteasome Inhibition,Endoplasmic Reticulum Stress,the Heat Shock Response and Acquisition of Thermotolerance

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.     

Structure and Light-Induced Expression of a Small Heat-Shock Protein Gene of Pharbitis nil

To isolate genes that are regulated by a photoperiod that promotes flowering in Pharbitis nil, a cDNA library representing mRNA of induced cotyledons was screened by differential hybridization. The DNA sequence of one cDNA clone isolated by this approach, clone 12L, showed homology to plant small heat-shock protein (hsp) genes. P. nil genomic clones hybridizing to clone 12L were isolated, and the DNA sequences of two P. nil small hsp (shsp) genes, shsp-1 and shsp-2, were determined. The derived amino acid sequences of shsp-1 and shsp-2 showed maximum homology to the 17.9-kD soybean hsp, a member of the class II cytoplasmic hsps found in plants. A study of the expression of shsp-1 and shsp-2 genes by RNase protection assay indicated that shsp-1 is induced by photoperiod, by light treatment of dark-grown P. nil seedlings, and by heat shock, and that shsp-2 is induced only by heat shock. Analysis of the sequences of the nontranscribed region indicates that both genes contain multiple heat-shock elements. The shsp-1 gene, in addition, contains sequences homologous to the GT-1-binding site, which may play a role in its light-regulated expression.     

Constitutive and stress‐inducible expression of the endoplasmic reticulum heat shock protein 70 gene family member,immunoglobulin‐binding protein (BiP), during Xenopus laevis early development

We have characterized the constitutive and stress‐inducible pattern of immunoglobulin‐binding protein (BiP) gene expression during Xenopus early development. Whole mount in situ hybridization analysis revealed that BiP mRNA was detected in unfertilized eggs, cleavage and blastula stage embryos. In gastrulae, BiP mRNA was present across the surface of the embryo, while in neurulae BiP mRNA was enriched in the neural plate, neural fold, and around the blastopore. In early and late tailbud embryos, BiP mRNA was found primarily in the dorsal region. Tunicamycin and A23187, the calcium ionophore, enhanced BiP mRNA accumulation first at the neurula stage, while heat shock induced BiP mRNA accumulation first at the gastrula stage. Compared to control, A23187‐ and heat shock‐treated neurulae displayed relatively high levels of BiP mRNA in selected tissues, including the neural plate, neural folds, around the blastopore, and ectoderm. At the early tailbud stage, A23187 and heat shock enhanced BiP mRNA accumulation primarily in the head, somites, tail, and along the spinal cord. A similar situation was found with A23187‐ and heat shock‐treated late tailbud embryos, except that heat‐shocked embryos also displayed enhanced BiP mRNA accumulation in the epidermis. These studies demonstrate a preferential accumulation of BiP mRNA in selected tissues during development and in response to stress. Dev. Genet. 25:31–39, 1999. © 1999 Wiley‐Liss, Inc.     

Transformation by T-antigen and other oncogenes delays Hsp25 accumulation in heat shocked NIH 3T3 fibroblasts

We have recently reported that transformation of murine NIH 3T3 cells by v-fos oncogene interfered with Hsp70 and Hsp25 accumulation after heat shock. Here, we have investigated the effect mediated by other oncogenes on the accumulation of these stress proteins. We report that T-antigen transformation of NIH 3T3 cells delayed and reduced the accumulation of Hsp25 after heat shock and decreased the heat-mediated phosphorylation of this protein. This decreased level of Hsp25 correlated with a reduced accumulation of the corresponding mRNA and was related to T-antigen level. In contrast, T-antigen had no effect on the expression of the major stress protein Hsp70 nor did it interfere with the level of Hsp90 or Hsp60. We report also that v-src or Ha-ras oncogenes delayed Hsp25 accumulation after heat shock but that only v-src reduced the heat-induced phosphorylation of this protein. v-src, but not Ha-ras, interfered with Hsp70 expression and none of these oncogenes had an effect on Hsp60 or Hsp90 levels. Taken together, these observations suggest that an altered accumulation of Hsp25 after heat shock is a common characteristic of NIH 3T3 fibroblasts transformed by different oncogenes.