Regulation Of Heat Shock Proteins Research Articles

Nonstructural 5A protein of hepatitis C virus regulates heat shock protein 72 for its own propagation

We identified heat shock protein 72 (Hsp72) as a host factor that was differentially expressed in cells expressing nonstructural 5A (NS5A) protein. To investigate how NS5A modulates Hsp72 in hepatitis C virus (HCV) life cycle, we examined the role of Hsp72 in HCV replication and virus production. NS5A specifically interacted with Hsp72. Both Hsp72 and nuclear factor of activated T cells 5 (NFAT5) levels were increased in cells expressing NS5A protein. Treatments of N-acetylcysteine and glutathione markedly reduced protein levels of both NFAT5 and Hsp72. Knockdown of NFAT5 resulted in decrease in Hsp72 level in cells expressing NS5A. Importantly, silencing of Hsp72 expression resulted in decrease in both RNA replication and virus production in HCV-infected cells. These data indicate that NS5A modulates Hsp72 via NFAT5 and reactive oxygen species activation for HCV propagation.

Regulation of heat shock protein 27 phosphorylation during microcystin-LR-induced cytoskeletal reorganization in a human liver cell line

Acute exposure to microcystin-LR (MC-LR) can induce the reorganization or disruption of the cytoskeleton, but proteins or enzymes correlated with this stress response have not been fully identified. Here, we report alterations to HSP27 during MC-LR-induced cytoskeletal reorganization in the human liver cell line HL7702. The cells incubated with MC-LR exhibited the rearrangement of filamentous actins and microtubules. The activity of protein phosphatase 2A was greatly decreased by MC-LR exposure. Furthermore, MC-LR markedly increased the level of HSP27 phosphorylation with the enhanced distribution of phosphorylated HSP27 to the cytoskeleton. To further determine the regulation of MC-LR-induced HSP27 phosphorylation, the activation of the MAPK superfamily was assessed. The result showed phospho-activation of p38 MAPK, JNK and ERK1/2 by MC-LR. Increases in HSP27 phosphorylation were suppressed by pretreating cells with SB203580 or SP600125, which are inhibitors of p38 MAPK or JNK, respectively. These data suggest that phosphorylated HSP27 is involved in cytoskeletal reorganization and is regulated by MAPKs, possibly as a consequence of PP2A inhibition. Moreover, the regulation of HSP27 phosphorylation may be important in MC-LR-induced cytoskeleton reassembly, which may provide helpful insights into the mechanism of MC-LR toxicity.

Heat shock protein 90 inhibition in murine macrophages prevents chronic alcohol induced TNF‐α production

Activation of toll-like receptor 4 (TLR-4) signaling on Kupffer cells is an important mediator of alcoholic liver disease. Chronic alcohol induced oxidative stress regulates heat shock protein 70 and 90 via HSF-1 activation in the liver. While hsp70 inhibits TLR4 signaling, hsp90 chaperones TLR4/CD14 receptor complex and its down-stream signaling molecules to regulate inflammatory cytokine TNF-α production in macrophages. It was hypothesized that the inhibition of hsp90 would decrease the inflammatory cytokine TNF-α and likely alleviates alcoholic liver injury. To test this hypothesis, murine macrophages RAW 264.7 were exposed to chronic alcohol at various concentrations and subsequently treated with 17-DMAG in the presence or absence of lipopolysaccharide (LPS). At the end of the six hour exposure, samples were analyzed for TNF-α. Total RNA was extracted at an earlier time point and analyzed for hsp70, which increased in cells treated with hsp90 inhibitors. Flow cytometry data show no significant alterations in surface expression of CD14 and TLR4 after inhibition of hsp90 chronic alcohol exposure. The results suggest that chronic alcohol exposure in RAW264.7 macrophages decreases TNF-α by inhibiting hsp90. Thus, these studies reveal hsp90 as a likely target to modulate inflammatory responses and alleviate alcoholic liver injury. Supported by NIH 5T34GM008411 and NIAAA/NIH RO1AA017986

Stiffness detection and reduction in discrete stochastic simulation of biochemical systems

Typical multiscale biochemical models contain fast-scale and slow-scale reactions, where "fast" reactions fire much more frequently than "slow" ones. This feature often causes stiffness in discrete stochastic simulation methods such as Gillespie's algorithm and the Tau-Leaping method leading to inefficient simulation. This paper proposes a new strategy to automatically detect stiffness and identify species that cause stiffness for the Tau-Leaping method, as well as two stiffness reduction methods. Numerical results on a stiff decaying dimerization model and a heat shock protein regulation model demonstrate the efficiency and accuracy of the proposed methods for multiscale biochemical systems.

Expression of heat shock protein70 in oral submucous fibrosis and oral squamous cell carcinoma: An immunohistochemical study

Heat shock proteins are a highly conserved group of protective cellular proteins whose synthesis is increased in response to a variety of environmental or pathophysiological stresses. Heat shock proteins are useful biomarkers for carcinogenesis in tissues and signal the degree of differentiation and the aggressiveness of cancers. Regulation of heat shock protein 70 (HSP70) expression in oral submucous fibrosis is not known much, and the aim of this study was to evaluate HSP70 expression in oral submucous fibrosis and oral squamous cell carcinoma by immunohistochemical method and to understand the role of HSP70 in tumorigenesis. Immunohistochemical method was used to detect HSP70 expression in normal oral mucosa, oral submucous fibrosis (n=30) and oral squamous cell carcinoma (n=20). HSP70 immunoreactivity was correlated with histological and clinicopathological features. A significant increase in expression of HSP70 was observed (P<0.000) as the tissue progressed from oral submucous fibrosis towards oral squamous cell carcinoma. HSP70 is synthesized upon stress situations arising in cells of all living organisms. Expression of HSP70 indicates that stress plays an important role as a predisposing factor in oral submucous fibrosis and its subsequent progression to oral squamous cell carcinoma.

O-Linked β-N-acetylglucosamine (O-GlcNAc) Regulates Stress-induced Heat Shock Protein Expression in a GSK-3β-dependent Manner

To investigate the mechanisms by which O-linked β-N-acetylglucosamine modification of nucleocytoplasmic proteins (O-GlcNAc) confers stress tolerance to multiple forms of cellular injury, we explored the role(s) of O-GlcNAc in the regulation of heat shock protein (HSP) expression. Using a cell line in which deletion of the O-GlcNAc transferase (OGT; the enzyme that adds O-GlcNAc) can be induced by 4-hydroxytamoxifen, we screened the expression of 84 HSPs using quantitative reverse transcriptase PCR. In OGT null cells the stress-induced expression of 18 molecular chaperones, including HSP72, were reduced. GSK-3β promotes apoptosis through numerous pathways, including phosphorylation of heat shock factor 1 (HSF1) at Ser(303) (Ser(P)(303) HSF1), which inactivates HSF1 and inhibits HSP expression. In OGT null cells we observed increased Ser(P)(303) HSF1; conversely, in cells in which O-GlcNAc levels had been elevated, reduced Ser(P)(303) HSF1 was detected. These data, combined with those showing that inhibition of GSK-3β in OGT null cells recovers HSP72 expression, suggests that O-GlcNAc regulates the activity of GSK-3β. In OGT null cells, stress-induced inactivation of GSK-3β by phosphorylation at Ser(9) was ablated providing a molecular basis for these findings. Together, these data suggest that stress-induced GlcNAcylation increases HSP expression through inhibition of GSK-3β.

Effect of microwave irradiation on expression of heat shock proteins family in primary cultured rat hippocampal neurons

To investigate the effects of microwave irradiation on the expression and regulation of heat shock proteins (HSPs) in primary cultured rat hippocampal neurons. Neurons were exposed to 90 mW/cm(2) microwave irradiation for 10 minutes. Western blot was used to determine the expression of HSP27, HSP70, HSP90 and heat shock factor 1 (HSF1) at 0, 3, 6, 12 and 24 hour respectively. Real-time RT-PCR was used to determine the mRNA expression of HSF1. DNA-binding activity of HSF1 was measured by electrophoretic mobility shift assay (EMSA). The protein expression of HSP27 was significantly increased by 22%, 36%, 18% at 3, 6, 12 h, respectively (P < 0.05). The protein expression of HSP70 was significantly increased by 23%, 32%, 26% at 3, 6, 12 h, respectively (P < 0.05, P < 0.01). The protein expression of HSP90 was significantly increased by 27%, 33% at 6, 12 h, respectively (P < 0.05, P < 0.01). The DNA-binding activity of HSF1 was stimulated, however, no significant change of the expression of HSF1 was observed on both the mRNA and protein levels. The transcriptional activity of HSF1 is activated by microwave irradiation, which promotes the expression of HSPs. Heat shock response which contributes to establish a cytoprotective state is induced by microwave irradiation in primary cultured rat hippocampal neurons.

Role of 4‐hydroxynonenal in oxidative stress induced signaling

ObjectivesWe have shown that 4‐HNE induces signaling for apoptosis in cells via death receptor (Fas) mediated extrinsic and p53‐mediated intrinsic pathways. However, the nature of interactions of 4‐HNE with the components of these signaling pathways are not understood. During present studies, we have elucidated the pathways of 4‐HNE induced signaling for apoptosis in HepG2 cells.ResultsOur results showed that 4‐HNE promoted apoptosis in HepG2 cells through activation of p53, Bax, p21, JNK, and caspase3. Our results also showed that exposure of HepG2 cells to 4‐HNE leads to the activation of Fas, Daxx, ASK1, JNK, and caspase3 and these effects of 4‐HNE could be inhibited by the over expression of hGSTA4‐4. 4‐HNE also induced translocation of Daxx from nucleus to cytoplasm which caused inhibition of apoptosis and the activation and the translocation of HSF1 to nucleus resulting in the up‐regulation of HSP70. We also demonstrate that 4‐HNE does indeed bind to Fas, Daxx, and p53 to affect Fas and p53 mediated signaling.ConclusionThese findings suggest that 4‐HNE generated during oxidative stress promotes apoptosis through multiple pathways, but upon sustained oxidative stress conditions, 4‐HNE may provide protection to cells by up regulation of heat shock proteins and by Daxx mediated inhibition of apoptosis.( NIH grants ES 012171, EY04396 (YCA) and CA 077495 (SA))

Advances and perspectives on the regulation and expression of piscine heat shock proteins

The focus of this review is on the regulation and expression of heat shock proteins (HSPs), in fish. Within the past decade, molecular studies pertaining to the isolation and characterization of fish HSP genes have been rapidly expanding with many gene sequences from many fish species being reported. Most of the gene sequences presently available belong to the HSP70 family but genes encoding constitutive and inducible members of the HSP90 family have also been identified as well as genes encoding HSP60, HSP47 and small HSP families. Environmental stressors such as alterations in environmental salinity, disease and chemical exposure are known to alter HSP expression and the regulation of HSPs by hormones has received much attention recently. Heat shock proteins are known to play key roles during embryonic development and recent findings have defined their ontogenetic profiles following hatching. Finally, we are gradually starting to add to our understanding as to the transcriptional regulation of HSPs especially the role and importance of the heat shock factor (HSF).

129 Down regulation of Heat Shock Protein 70 (HSP70) predicts responsiveness to neoadjuvant aromatase inhibitors in post-menopausal hormonal receptors expressing breast cancer

Cytotoxic chemotherapy remains the systemic therapy of choice for triple-negative breast cancer (TNBC), a poor-prognosis subtype of breast cancer. Growing data focusing on TNBC provides an opportunity to assess if we can tailor adjuvant chemotherapy based on patient and tumor characteristics. The standard of care for moderate-to-high risk TNBC remains a sequential anthracycline-taxane combination, with the potential for shorter and less toxic regimens in stage I disease. Platinums are promising in the neoadjuvant and metastatic settings but we await long-term outcome data before incorporation into standard regimens in the adjuvant setting. Specific subgroups within TNBC, such as BRCA mutation carriers, require special attention, and the role of platinums in these patients warrants further consideration. There is hope that in the future, further subdividing TNBC by gene expression profile, mutation, immune infiltrate, and others will reveal novel susceptibilities.

Regulation of heat shock protein 70-1 expression by androgen receptor and its signaling in human prostate cancer cells

Heat shock protein (hsp) 70-1 (hsp70-1) is overexpressed in human prostate cancer cells and may play important roles in prostate cancer resistance to conventional therapies. The purpose of this study was to investigate whether androgen receptor (AR) and its signaling regulate hsp70-1 expression. Several lines of AR-positive (LNCaP, LAPC-4, and 22Rv1) and -negative (PC-3, DU145, WPE1-NB14 and WPE1-NB-26) human prostatic cells were used in the study. Dihydrotestosterone (DHT) enhanced hsp70-1 expression in LNCaP cells. Expression of hsp70-1 in LNCaP cells was downregulated by the anti-androgens bicalutamide (Bic), and flutamide (Flut), and a newly identified AR signaling antagonist DL3. The downregulation of hsp70-1 by DL3 was also observed in LAPC-4 and 22Rv1 cells, but not in the four lines of AR-negative cells examined. Expression of hsp70-1 was also reduced by DL3 in PC-3 cells engineered with AR. On the other hand, knocking down AR in LNCaP cells by siRNA moderately reduced hsp70-1 level and abolished effects of DL3 on hsp70-1 expression. DL3 reduced hsp70-1 mRNA synthesis in cells and its in vitro gene transcription but did not significantly alter the stabilities of hsp70-1 mRNA and protein. Chromatin-immunoprecipitation (ChIP) assay showed that AR bound to the promoter region of HSPA1B gene, which was reduced in cells treated with DL3 or Bic. These data suggest that AR and its signaling regulate hsp70-1 expression in prostate cancer cells and that HSPA1B could be an AR target gene.

Regulation of heat shock protein 60 and 72 expression in the failing heart

Heart failure, a progressive, fatal disease of the heart muscle, is a state of chronic inflammation and injury. Heat shock protein (HSP) 72, a ubiquitous protective protein that is well-established as cardioprotective, is not increased in heart failure. In contrast, HSP60 levels are doubled in the failing heart. We hypothesized that HSF-1 is not activated in heart failure and that the increased expression of HSP60 was driven by NFκB activation. To test this hypothesis, we measured levels of heat shock factor (HSF) -1 and -2, the transcription factors controlling HSP expression, which were increased in heart failure. There was no increased phosphorylation of serine 230 or serine 303/307 in HSF-1, which are thought to regulate its activity; EMSA showed no increase in HSF binding activity with heart failure. Nonetheless, mRNA was increased for HSP60, but not HSP72. In contrast to HSF, NFκB activity was increased in heart failure. HSP60, but not HSP72, contained NFκB binding elements. ChIP assay demonstrated increased binding of NFκB to both of the NFκB binding elements in the heart failure HSP60 gene. TNFα treatment was used to test the role of NFκB activation in HSP60 expression in a cardiac cell line. TNFα increased HSP60 expression, and this could be prevented by pretreatment with siRNA inhibiting p65 expression. In conclusion, HSP72 is not increased in heart failure because HSF activity is not changed; increased expression of HSP60 may be driven by NFκB activation.

Regulation of the heat shock response under anoxia in the turtle, Trachemys scripta elegans

The effects of 20 h of anoxic submergence in cold water and 5 h of aerobic recovery on the heat shock response were analyzed in four organs of the anoxia-tolerant turtle Trachemys scripta elegans. Immunoblotting was used to analyze levels of active and inactive forms of the heat shock transcription factor 1 (HSF1), nuclear translocation of HSF1, and the levels of six heat shock proteins (HSPs). PCR was also used to retrieve the turtle HSF1 nucleotide sequence; its deduced amino acid sequence showed 97% identity with chicken HSF1. White skeletal muscle showed a strong fivefold increase in the amount of active HSF1 under anoxic conditions as well as an 80% increase in nuclear localization. This was accompanied by upregulation of five HSPs by 1.8- to 2.9-fold: Hsp25, Hsp40, Hsp70, Hsc70, and Hsp90, the latter two remained elevated after 5 h of aerobic recovery. Kidney and liver showed little change in active HSF1 content during anoxia and recovery, but a significant increase in the nuclear localization of HSF1 during anoxia. This supported enhanced expression of three HSPs in kidney (Hsp40, Hsc70, and Hsp90) and four in liver (Hsp40, Hsp60, Hsp70, Hsc70). Heart displayed a strong increase in active HSF1 during anoxia and recovery (6.6- to 6.8-fold higher than control) and increased nuclear localization but heart HSP levels did not rise. The data demonstrate organ-specific regulation of HSPs during anoxia exposure and aerobic recovery in T. s. elegans and suggest that the heat shock response is an important aspect of cytoprotection during facultative anaerobiosis, particularly with regard to underwater hibernation of turtles in cold water.

The signal transduction pathways of heat shock protein 27 phosphorylation in vascular smooth muscle cells

The objective of this study is to investigate the signal transduction pathways that regulate heat shock protein 27 (HSP27) phosphorylation and migration of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) induced by angiotensin II (AngII) and platelet derived growth factor-BB (PDGF-BB). The activity of HSP27 was evaluated by Western blot with specific phospho-HSP27 antibody. F-actin polymerization was detected by FITC-Phalloidine staining using confocal microscopy. Modified Boyden chamber technique was employed for VSMCs migration assessment. Within a given concentration, the phosphorylation of HSP27 induced by AngII and PDGF-BB was blocked by the specific P38MAPK inhibitor SB202190, the specific PI3K inhibitor LY294002 and the specific ERK1/2 inhibitor U0126 in a concentration-dependent manner, with a peak inhibition rate at 87.2%, 78.4% and 37.3%, respectively, induced by AngII (P < 0.01), with a peak inhibition rate at 85.0%, 55.3% and 41.0%, respectively, induced by PDGF-BB (P < 0.01).The migration of VSMCs induced by AngII and PDGF-BB was inhibited by 100 micromol/l SB202190, 30 micromol/l LY294002, and 30 micromol/l U0126, with a inhibition rate at 60.1%, 71.7% and 47.3%, respectively, provoked by AngII (P < 0.01), with a inhibition rate at 55.3%, 55.6% and 38.1%, respectively, provoked by PDGF-BB (P < 0.01). P38MAPK and PI3 K/Akt are important pathways that contribute to the phosphorylation of HSP27 and migration of VSMCs in response to AngII and PDGF-BB. ERK1/2 might be involved in HSP27 phosphorylation and migration of VSMCs provoked by AngII and PDGF-BB.

Exploring temporal transcription regulation structure of Aspergillus fumigatus in heat shock by state space model

BackgroundThe thermotolerance of Aspergillus fumigatus plays a critical role in mammalian and avian infections. Thus, the identification of its adaptation mechanism to higher temperature is very important for an efficient anti-fungal drug development as well as fundamental understanding of its pathogenesis. We explored the temporal transcription regulation structure of this pathogenic fungus under heat shock conditions using the time series microarray data reported by Nierman et al. (Nature 2005, 438:1151-1156).ResultsThe estimated transcription regulation structure of A. fumigatus shows that the heat shock proteins are strongly negatively associated with central metabolic pathway genes such as the tricarboxylic acid cycle (TCA cycle) and carbohydrate metabolism. It was 60 min and 120 min, respectively, after the growth temperature changes from 30°C (corresponding to environments of tropical soil) to 37°C and 48°C (corresponding to temperatures in the human body and compost, respectively) that some of genes in TCA cycle were started to be upregulated. In these points, most of heat shock proteins showed lowest expression level after heat shocks. Among the heat shock proteins, the HSP30 (AFU6G06470), a single integral plasma membrane heat shock protein, presented most active role in transcription regulation structure in both heat shock conditions of 37°C and 48°C. The metabolic genes associated with multiple genes in the gene regulation network showed a tendency to have opposite expression patterns of heat shock proteins. The role of those metabolic genes was second regulator in the coherent feed-forward loop type of regulation structure having heat shock protein as its first regulator. This type of regulation structure might be very advantageous for the thermal adaptation of A. fumigatus under heat shock because a small amount of heat shock proteins can rapidly magnify their regulation effect on target genes. However, the coherent feed-forward loop type of regulation of heat shock proteins with metabolic genes became less frequent with increasing temperature. This might be the reason for dramatic increase in the expression of heat shock proteins and the number of heat shock response genes at heat shock of 48°C.ConclusionWe systemically analysed the thermal adaption mechanism of A. fumigatus by state space model with times series microarray data in terms of transcription regulation structure. We suggest for the first time that heat shock proteins might efficiently regulate metabolic genes using the coherent feed-forward loop type of regulation structure. This type of regulation structure would also be efficient for adjustment to the other stresses requiring rapid change of metabolic mode as well as thermal adaptation.

Quantification of coral heat shock proteins from individual coral polyps

The induction and regulation of heat shock proteins (hsps) is a significant defense mechanism that can preserve metabolic function and foster recovery from short-term stress events. Present coral sampling methodologies that involve hsp analysis often require the harvesting of large samples of live coral colonies that may already be stressed or in poor health. In the present study, 3 novel protocols were developed to: (1) extract single coral polyps, minimizing colony trauma; (2) purify protein from single coral polyps (approximately 12 mm 3 ); and (3) develop a more sensitive protein quantification method. The preliminary testing of 5 separate protein preparation methods resulted in a range of total protein yields from 47 to 77 µg coral polyp -1 . The optimized methods were able to recover, on average, 44 ± 12 µg protein polyp -1 (n = 20). Subsequent SDS-PAGE and immunoblotting analysis of single coral polyps resolved as little as 87 pg hsp70 coral polyp -1 . This minimally invasive sampling protocol reduces coral damage and, thus, reduces stress and diseases caused by sampling.

Differences in heat shock protein 70 expression during larval and early spat development in the Eastern oyster, Crassostrea virginica (Gmelin, 1791)

For a variety of species, changes in the expression of heat shock proteins (HSP) have been linked to key developmental changes, i.e., gametogenesis, embryogenesis, and metamorphosis. Many marine invertebrates are known to have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis as they transition to the benthic life stage. A series of experiments were run to examine the expression of heat shock protein 70 (HSP 70) during larval and early spat (initial benthic phase) development in the Eastern oyster, Crassostrea virginica. In addition, the impact of thermal stress on HSP 70 expression during these early stages was studied. C. virginica larvae and spat expressed three HSP 70 isoforms, two constitutive, HSC 77 and HSC 72, and one inducible, HSP 69. We found differences in the expression of both the constitutive and inducible forms of HSP 70 among larval and early juvenile stages and in response to thermal stress. Low expression of HSP 69 during early larval and spat development may be associated with the susceptibility of these stages to environmental stress. Although developmental regulation of HSP 70 expression has been widely recognized, changes in its expression during settlement and metamorphosis of marine invertebrates are still unknown. The results of the current study demonstrated a reduction of HSP 70 expression during settlement and metamorphosis in the Eastern oyster, C. virginica.

136: Heat shock proteins and inflammation-induced preterm birth

Heat shock proteins (HSP) have been suggested to be critical players in inflammatory disease states by binding to Toll-like receptor-4(TLR-4) as endogenous ligands. HSPs are released from cells in response to stress and function as regulators of the innate immune response. Limited obstetrical studies suggest that HSPs may have a mechanistic role in preterm birth (PTB) or may be promising biomarkers. These studies sought to determine 1) the regulation of HSP during pregnancy and in the setting of inflammation-induced PTB and 2) the validity of HSP as a potential non-invasive biomarker using a mouse model of PTB. 2 sets of experiments were performed. 1) Cervical (CX) and uterine (UT) tissue was harvested from CD-1 non-pregnant (NP) and pregnant mice on E15, E17, E19 (n=3-6 mice/group). 2) On E15, dams were randomized to intrauterine infusion of saline or lipopolysaccharide (LPS)(N=5-6/group). 6 hrs later, maternal serum (MS), CX and UT were collected for QPCR and ELISA for evaluatoin of HSP 70. HSP 70 levels were undetectable in saline-exposed dams in maternal serum. While HSP 70 was increased in LPS-exposed, variability between dams was pronounced. SEE TABLETabled 1E15 / NPE15 / NPE15 LPS / E15E15 LPS / E15CervixUterusCervixUterusHSP60−9.4−1.5−1.61.6HSP70−10.22.1−2.51.1HSP90−5.91.7−1.61.3 Open table in a new tab HSPs do not appear to be critically involved in the pathogenesis of PTB. Down-regulation of HSPs in the CX during normal pregnancy could be a protective mechanism against preterm cervical ripening by preventing endogenous ligands from activating TLRs.

Regulation of heat shock proteins in the apple maggot Rhagoletis pomonella during hot summer days and overwintering diapause

Abstract Developing larvae of the apple maggot Rhagoletis pomonella are frequently exposed to summertime apple temperatures that exceed 40 °C and, during their overwintering diapause, pupae are exposed to sub‐zero soil temperatures for prolonged periods. To investigate the potential involvement of heat shock proteins (Hsps) in response to these environmental extremes, the genes encoding Hsp70 and Hsp90 in R. pomonella are cloned and expression monitored during larval feeding within the apple and during overwintering pupal diapause. Larvae reared in the laboratory at constant temperatures of 25, 28 or 35 °C express Hsp90 but very little Hsp70. Larvae do not survive rearing at 40 °C. The temperature cycles to which larvae were exposed inside apples in the field, ranging 16–46.9 °C over a 24‐h period, elicit strong Hsp70 and Hsp90 expression, which begins at mid‐day and reaches a peak in late afternoon, coinciding with peak air and apple temperatures. Heat shock proteins are also expressed strongly by pupae during their overwintering diapause. Hsp70 is not expressed in nondiapausing pupae but is highly expressed throughout diapause. Hsp90 is constitutively expressed in both diapausing and nondiapausing pupae. Rhagoletis pomonella thus strongly expresses its Hsps during pupal diapause, presumably as a protection against low temperature injury, and during larval development to cope with natural temperature cycles prevailing in late summer.

TherpoEoperon regulates heat stress response inBurkholderia pseudomallei

Burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis, one of the important lethal diseases in tropical regions. In this article, we demonstrate the crucial role of the B. pseudomallei rpoE locus in the response to heat stress. The rpoE operon knockout mutant exhibited growth retardation and reduced survival when exposed to a high temperature. Expression analysis using rpoH promoter-lacZ fusion revealed that heat stress induction of rpoH, which encodes heat shock sigma factor (sigma(H)), was abolished in the B. pseudomallei rpoE mutant. Analysis of the rpoH promoter region revealed sequences sharing high homology to the consensus sequence of sigma(E)-dependent promoters. Moreover, the putative heat-induced sigma(H)-regulated heat shock proteins (i.e. GroEL and HtpG) were also absent in the rpoE operon mutant. Altogether, our data suggest that the rpoE operon regulates B. pseudomallei heat stress response through the function of rpoH.