Protein Synthesis and Breakdown during Heat Shock of Cultured Pear (Pyrus communis L.) Cells

Cultured pear (Pyrus communis L. cv Passe Crassane) cells were subjected to temperatures of 39, 42, and 45[deg]C. Heat-shock protein (hsp) synthesis was greater at 30[deg]C than at temperatures above 40[deg]C and continued for up to 8 h. Both cellular uptake of radiolabeled methionine and total protein synthesis were progressively lower as the temperature was increased. Polysome levels decreased immediately when cells were placed at 39 or 42[deg]C, although at 39[deg]C the levels began to recover after 1 h. In cells from both temperatures, reassembly occurred after transfer of cells to 25[deg]C Four heat-shock-related mRNAs[mdash]hsp17, hsp70, and those of two ubiquitin genes[mdash]all showed greatest abundance at 39[deg]C and decreased at higher temperatures. Protein degradation increased with time at 42 and 45[deg]C, but at 39[deg]C it increased for the first 2 h and then decreased. In the presence of cycloheximide, which prevented hsp synthesis, protein degradation at 39[deg]C was as great as that at 45[deg]C in the absence of cycloheximide. The data suggest that hsps may have a role in protecting proteins from degradation at the permissive temperature of 39[deg]C. At temperatures high enough to inhibit hsp synthesis, protein degradation was enhanced. Although ubiquitin may play a role in specific protein degradation, it does not appear to be involved in increased protein degradation occurring above 40[deg]C.     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.     

Cloning and regulation by glucocorticoid receptor ligands of a rat hsp90

We have isolated a full length cDNA that encodes a heat shock protein, hsp90, from a rat brain library and present the nucleotide sequence and deduced amino acid sequence. Comparison of the entire nucleotide sequence with mouse hsp84 and human hsp90β cDNAs reveal sequence similarities of 92 and 87%, respectively. The coding region of 2172 nucleotides corresponds to a polypeptide chain of 724 amino acids. Comparison with mouse hsp84 and human hsp90β amino acid sequences indicates a similarity of 97%, respectively. Characterization of the constitutive expression of this cDNA both by RNA blot hybridization and immunoblotting, reveals that it is expressed in all rat tissues examined. Hsp90 has been shown to form a transient complex with steroid hormone receptors. In order to further elucidate the role of hsp90 in the endocrine response of cells, we have examined the effects of dexamethasone and RU38486 on the level of hsp90 mRNA in a system in which glucocorticoids down-regulate glucocorticoid receptor mRNA levels. In this system, a subtle but reproducible approx. 2-fold decrease in hsp90 mRNA levels is observed after 48 h treatment with dexamethasone.     

Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts

A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates. These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells.     

Facing warm temperatures during migration: cardiac mRNA responses of two adult Oncorhynchus nerka populations to warming and swimming challenges

The main findings of the current study were that exposing adult sockeye salmon Onchorhynchus nerka to a warm temperature that they regularly encounter during their river migration induced a heat shock response at an mRNA level, and this response was exacerbated with forced swimming. Similar to the heat shock response, increased immune defence‐related responses were also observed after warm temperature treatment and with a swimming challenge in two different populations (Chilko and Nechako), but with some important differences. Microarray analyses revealed that 347 genes were differentially expressed between the cold (12–13° C) and warm (18–19° C) treated fish, with stress response (GO:0006950) and response to fungus (GO:0009620) elevated with warm treatment, while expression for genes involved in oxidative phosphorylation (GO:0006119) and electron transport chain (GO:0022900) elevated for cold‐treated fish. Analysis of single genes with real‐time quantitative PCR revealed that temperature had the most significant effect on mRNA expression levels, with swimming and population having secondary influences. Warm temperature treatment for the Chilko population induced expression of heat shock protein (hsp) 90α, hsp90β and hsp30 as well as interferon‐inducible protein. The Nechako population, which is known to have a narrower thermal tolerance window than the Chilko population, showed even more pronounced stress responses to the warm treatment and there was significant interaction between population and temperature treatment for hsp90β expression. Moreover, significant interactions were noted between temperature treatment and swimming challenge for hsp90α and hsp30, and while swimming challenge alone increased expression of these hsps, the expression levels were significantly elevated in warm‐treated fish swum to exhaustion. In conclusion, it seems that adult O. nerka currently encounter conditions that induce several cellular defence mechanisms during their once‐in‐the‐lifetime migration. As river temperatures continue to increase, it remains to be seen whether or not these cellular defences provide sufficient protection for all O. nerka populations.     

Involvement of differential gene expression and mRNA stability in the developmental regulation of the hsp 30 gene family in heat-shocked Xenopus laevis embryos

Four complete hsp 30 genes have been isolated from Xenopus laevis: hsp 30A, hsp 30B (a pseudogene), hsp 30C, and hsp 30D. The hsp 30A and hsp 30C genes are first heat inducible at the early tailbud stage, as determined by RNase protection and RT-PCR assays. In this study, we determined by RT-PCR that the hsp 30D gene was first heat inducible (33^oC for 1 h) at the mid-tailbud stage, approximately 1 day later in development than hsp 30A and hsp 30C. Furthermore, using Northern blot analysis, we detected the presence of very low levels of hsp 30 mRNA at the heat-shocked late blastula stage. The relative levels of these pre-tailbud (PTB) hsp 30 mRNAs increased at the gastrula and neurula stage followed by a dramatic enhancement in heat shocked tail-bud and tadpole stage embryos (50- to 100- fold relative to late blastula). Interestingly, treatment of blastula or gastrula embryos at high temperatures (37^oC for 1 h) or with the protein synthesis inhibitor, cycloheximide, followed by heat shock, led to enhanced accumulation of the pre-tailbud (PTB) hsp 30 mRNAs. hsp 70, hsp 87, and actin messages were not stabilized at high temperatures or by cycloheximide treatment. Finally, hsp 30D mRNA was not detected by RT-PCR analysis of cycloheximidetreated, heat-shocked blastula stage embryos, confirming that it is not a member of the PTB hsp 30 mRNAs. This study indicates that differential gene expression and mRNA stability are involved in the regulation of hsp 30 gene expression during early Xenopus laevis development. © 1995 Wiley-Liss, Inc.     

Identification of two hsp90 genes in carp

Two hsp90 cDNA isoforms (hsp90alpha and hsp90beta) were isolated from the common carp (Cyprinus carpio). Gene-specific probes and primers were selected and used in Northern blot hybridization and RT-PCR reactions to measure the basal hsp90 mRNA levels and to follow the inducer-specific expression of the hsp90 genes in different tissues during in vivo studies. The hsp90beta gene is largely constitutively expressed at a fairly high level in all the examined tissues (brain, liver and kidney) and is slightly inducible by an elevated temperature. Hsp90alpha mRNA is present in the brain, but is hardly detectable in the kidney and liver of unstressed animals. In the brain, this gene is greatly upregulated following thermal stress, whereas in the liver and kidney heat shock has only minor effects on its expression. Hsp90alpha, but not hsp90beta, responds to an elevated level of Cd in a dose-, time- and tissue-dependent manner.     

Expression of heat shock genes during differentiation of mammalian osteoblasts and promyelocytic leukemia cells.

The progressive differentiation of both normal rat osteoblasts and HL-60 promyelocytic leukemia cells involves the sequential expression of specific genes encoding proteins that are characteristic of their respective developing cellular phenotypes. In addition to the selective expression of various phenotype marker genes, several members of the heat shock gene family exhibit differential expression throughout the developmental sequence of these two cell types. As determined by steady state mRNA levels, in both osteoblasts and HL-60 cells expression of hsp27, hsp60, hsp70, hsp89 alpha, and hsp89 beta may be associated with the modifications in gene expression and cellular architecture that occur during differentiation. In both differentiation systems, the expression of hsp27 mRNA shows a 2.5-fold increase with the down-regulation of proliferation while hsp60 mRNA levels are maximal during active proliferation and subsequently decline post-proliferatively. mRNA expression of two members of the hsp90 family decreases with the shutdown of proliferation, with a parallel relationship between hsp89 alpha mRNA levels and proliferation in osteoblasts and a delay in down-regulation of hsp89 alpha mRNA levels in HL-60 cells and of hsp89 beta mRNA in both systems. Hsp70 mRNA rapidly increases, almost twofold, as proliferation decreases in HL-60 cells but during osteoblast growth and differentiation was only minimally detectable and showed no significant changes. Although the presence of the various hsp mRNA species is maintained at some level throughout the developmental sequence of both osteoblasts and HL-60 cells, changes in the extent to which the heat shock genes are expressed occur primarily in association with the decline of proliferative activity. The observed differences in patterns of expression for the various heat shock genes are consistent with involvement in mediating a series of regulatory events functionally related to the control of both cell growth and differentiation.     

The role of heat shock proteins in inflammatory injury induced by cold stress in chicken hearts

The aim of this study was to investigate the effects of cold stress on the expression levels of heat shock proteins (Hsps90, 70, 60, 40, and 27) and inflammatory factors (iNOS, COX-2, NF-κB, TNF-α, and PTGEs) and oxidative indexes in hearts of chickens. Two hundred forty 15-day-old male chickens were randomly divided into 12 groups and kept at the temperature of 12 ± 1 °C for acute and chronic cold stress. There were one control group and five treatment groups for acute cold stress, three control groups, and three treatment groups for chronic cold stress. After cold stress, malondialdehyde level increased in chicken heart; the activity of superoxide dismutase and glutathione peroxidase in the heart first increased and then decreased. The inflammatory factors mRNA levels were increased in cold stress groups relative to control groups. The histopathological analysis showed that heart tissues were seriously injured in the cold stress group. Additionally, the mRNA levels of Hsps (70, 60, 40, and 27) increased significantly (P < 0.05) in the cold stress groups relative to the corresponding control group. Meanwhile, the mRNA level and protein expression of Hsp90 decreased significantly (P < 0.05) in the stress group, and showed a gradually decreasing tendency. These results suggested that the levels of inflammatory factors and Hsps expression levels in heart tissues can be influenced by cold stress. Hsps commonly played an important role in the protection of the heart after cold stress.     

Upregulation of temperature susceptibility in Bemisia tabaci upon acquisition of Tomato yellow leaf curl virus (TYLCV)

Acquisition of plant viruses has various effects on physiological mechanisms in vector insects. Bemisia tabaci is the only known vector of Tomato yellow leaf curl virus (TYLCV), which is a serious virus affecting tomato cultivars. In this study, the lifespan of Q1 biotype was compared between non-viruliferous (NV) and TYLCV-viruliferous (V) whiteflies. Total lifespan from egg to adult death of NV whiteflies was 62.54days but 10.64days shorter in V whiteflies. We investigated the temperature susceptibility of B. tabaci by comparing mortalities as well as heat shock protein (hsp) mRNA levels between NV and V whiteflies. For this, NV and V whiteflies were exposed for either 1 or 3h at 4, 25, and 35°C. The mortality of V whiteflies was higher than NV ones following exposure at either 4 or 35°C, but there was no significant difference at 25°C. Analysis of the expression level of heat shock protein (hsp) genes using quantitative real-time PCR showed that both cold and heat shock treatments stimulated higher expression of hsps (hsp40, hsp70, and hsp90) at various rates in V whiteflies than NV ones, but there was no difference at 25°C. All together, our results show that TYLCV acquisition accelerated the developmental rate and increased susceptibility to thermal stress in B. tabaci. Therefore, this modification may result in reduced vector longevity due to increased metabolic energy utilization. Our results provide insights into the complex interaction between vector fitness and thermal stress in relation to the acquisition and transmission of plant viruses.     

The Role of the 90-kDa Heat Shock Protein in Cell Cycle Control and Differentiation of the Monoblastoid Cell Line U937

The human monoblastoid cell line, U937, has been widely used to study proliferation and differentiation in the monocyte–macrophage lineage. Recent evidence from other cell systems suggests that heat shock proteins (hsps) may participate in these processes. Therefore, we have examined expression of hsp and the effect of either increased or decreased expression of the hsp90 in U937 cells. Parental U937 cells express high levels of hsp90, hsp73, and hsp65, but little hsp72. Heat shock at 42°C for 30 min increased hsp72 levels but caused no change in hsp90. U937 cells transfected with the expression vector pBA.4 containing either an anti-sense or a sense hsp90 cDNA insert showed constitutive decrease, or increase, in expression of hsp90. Decreased hsp90 levels slowed the rate of cell division and levels of hsp90 correlated both with the responses to phorbol esters and with phenotypic changes: anti-sense-transfected cells expressed less CD50. Sense-transfected cells showed no change in cell cycle, but expressed less CD14 than controls. Thus, hsp90 plays a role in the monocyte–macrophage lineage, participating in proliferation and cell cycle control and in the acquisition of functional heterogeneity of the mature macrophage phenotype, with potential effects on the role of the macrophage in innate immunity.     

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.     

Cell-free synthesis of rat glucocorticoid receptor in rabbit reticulocyte lysate. In vitro synthesis of receptor in Mr 90,000 heat shock protein-depleted lysate

The glucocorticoid receptor is present in the cytosol of cell extracts as a large nonactivated (i.e. non-DNA-binding) approximately 9 S (Mr 300,000) complex. Experimental evidence indicates that the purified nonactivated glucocorticoid receptor contains a single steroid-binding protein and two approximately 90-kDa nonsteroid-binding subunits identified as heat shock protein (hsp) 90. Translation of the glucocorticoid receptor mRNA in vitro in reticulocyte lysates produces a large nonactivated glucocorticoid receptor complex similar to that found in cytosols. The cell-free synthesized glucocorticoid receptor is able to bind steroid and can be activated further to the DNA-binding form. To test the hypothesis of an active role played by hsp90 in the stabilization of a competent steroid-binding conformation of the glucocorticoid receptor, we have synthesized the receptor in a reticulocyte lysate that has been depleted of hsp90 by immunoadsorption with AC88 anti-hsp90. Although the translation capacity of the reticulocyte system was reduced considerably upon hsp90 removal, the glucocorticoid receptor was synthesized, and a significant number of molecules were found to bind [3H]triamcinolone acetonide. Chromatography on DEAE-cellulose showed that most of the receptor molecules synthesized in hsp90-depleted lysate had lost the capacity to form an oligomeric receptor complex. Addition of purified rat liver hsp90 to the hsp90-depleted lysate before translation did not increase steroid binding nor did it restore formation of the heteromeric receptor complex. Analysis of [35S] methionine-labeled glucocorticoid receptor molecules synthesized in the hsp90-depleted lysate showed the production of polypeptides differing from the expected chromatographic pattern on DEAE-cellulose. Upon addition of purified hsp90 to the hsp90-depleted lysate, before translation, the 35S-labeled synthesized receptor fractionated on DEAE-cellulose as an intermediate peak between activated and nonactivated receptor forms. The data suggest that hsp90 alone may not be sufficient for the formation of the nonactivated steroid receptor complex.     

Differential Expression of the Two Subunits of Tomato Polygalacturonase Isoenzyme 1 in Wild-Type and rin Tomato Fruit

The [beta] subunit of tomato (Lycopersicon esculentum Mill.) fruit polygalacturonase 1 is a cell wall glycoprotein that binds to and apparently regulates the catalytic PG2 polypeptide in vivo. [beta] Subunit and polygalacturonase 2 (PG2) expression have been investigated in both wild-type and ripening inhibitor (rin) mutant fruit. During fruit development and ripening, [beta] subunit expression was unrelated to expression of the catalytic PG2 protein. In wild-type fruit, [beta] subunit mRNA and protein were first detected early in development and increased to maximal levels before PG2 mRNA and protein were detected. At the onset of ripening [beta] subunit mRNA decreased dramatically, but [beta] subunit protein levels remained stable. In rin fruit, which fail to ripen, [beta] subunit expression was similar to that in wild type, although PG2 mRNA and protein were not detected. These data suggest that [beta] subunit expression is ethylene independent and regulated primarily by developmental cues. This conclusion is supported by results from ethylene-treated immature (20 days after pollination) wild-type and rin fruit in which no significant differences were observed in [beta] subunit expression patterns in response to ethylene treatment. Surprisingly, RNA blot analysis indicated that catalytic PG2 mRNA was induced in immature rin fruit after 3 d of exogenous ethylene treatment. In addition, [beta] subunit mRNA and protein were also detected at lower levels in root, leaf, and flower tissues of both genotypes, suggesting a broader functional role for the protein.