Prokaryotic and eukaryotic organisms respond to various stressors with the production of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is constitutively expressed in most adult mammalian tissues. In the present study, we have examined for the first time the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. The Xenopus hsp110 cDNA encodes an 854 amino acid protein, which shares 74% identity with mice and humans. In Xenopus A6 kidney epithelial cells hsp110 mRNA was detected constitutively and was heat inducible. Enhanced hsp110 mRNA levels were detected within 1 h, and remained elevated for at least 6 h. A similar accumulation of hsp70 mRNA was observed, but only in response to stress. Treatment of A6 cells with sodium arsenite and cadmium chloride also induced hsp110 and hsp70 mRNA accumulation. However, while ethanol treatment resulted in the accumulation of hsp70 mRNA no effect was seen for hsp110. Similarly, HSP110 and HSP70 protein increased after a 2 h heat shock and 12 h sodium arsenite treatment. The elevation in HSP110 and HSP70 protein in response to heat was detectable for up to 6 h. Recent studies with mice suggest an important role for HSP110 during development. Analysis of Xenopus embryos revealed that hsp110 mRNA was present in unfertilized eggs, indicating that it is a maternal mRNA, unlike the hsp70 message which was only detectable in response to heat shock. Heat shock-induced hsp110 mRNA accumulation was developmentally regulated, similar to hsp70, since it was not detectable until after the midblastula stage of development. Enhanced hsp110 mRNA accumulation was evident with heat shock at the blastula stage, and levels continued to increase reaching a maximum at the late tailbud stage. Message for the small heat shock protein, hsp27, was not detectable until the early tailbud stage, indicating that this hsp was not present maternally and was developmentally regulated. In situ hybridization analysis revealed that hsp110 mRNA was present in control embryos in the lens placode, spinal cord and somites, but increased upon heat shock in the anterior and posterior region, the lens placode, as well as in the somites and spinal cord. A similar distribution was observed for the hsp27 message, although it was not detectable until the early tailbud stage in control or heat-shocked embryos. The intracellular localization of HSP110 protein in response to stress was also investigated. HSP110 was detected predominantly in the cytoplasm in either a diffuse pattern or in long spindle-shaped fibres. Additionally, HSP110 was present in the nucleus. In heat shocked Xenopus A6 cells, HSP110 localized in distinct patterns surrounding the nucleus and was enhanced in the nucleus after prolonged heat stress. Sodium arsenite-treated cells displayed a similar pattern in which HSP110 localized on opposite ends of the nucleus. In contrast, in response to stress HSP30 was homogeneously distributed in the cytoplasm, moving into the nucleus only upon intense stress. This study presents, for the first time, a characterization of HSP110 in Xenopus laevis, adding to the growing knowledge of HSPs in this important model organism.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.

Heat shock proteins (HSPs) are stress-inducible and evolutionarily conserved molecular chaperones that are involved in protein binding and translocation. As molecular chaperones, HSPs bind to denatured proteins, inhibit their aggregation, maintain their solubility, and assist in refolding. This process inhibits the formation of protein aggregates which can be lethal to the cell. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most non-native proteins. Furthermore, proteasome inhibition has been shown to induce hsp gene expression. Celastrol, a quinone methide triterpene, was shown to have an inhibitory effect on proteasome function in mammalian cells. The present study determined that celastrol induced the accumulation of ubiquitinated proteins and reduced proteasomal chymotrypsin-like activity in Xenopus laevis A6 kidney epithelial cells. In addition, incubation of A6 cells with celastrol induced the accumulation of HSP30 and HSP70 in a dose- and time-dependent manner with maximal levels of HSP accumulation occurring after 18 h of exposure. In A6 cells recovering from celastrol, the relative levels of HSP30 and HSP70 accumulation remained elevated for 18-24 h after removal of celastrol. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in celastrol-induced hsp gene expression in A6 cells, since the HSF1 inhibitor, KNK437, repressed the accumulation of HSP30 and HSP70. Exposure of A6 cells to simultaneous celastrol and mild heat shock treatment enhanced the accumulation of HSP30 and HSP70 to a greater extent than the sum of both stressors individually. Additionally, concurrent treatment of A6 cells with low concentrations of both celastrol and MG132 produced different patterns of HSP30 and HSP70 accumulation. While combined treatment with celastrol and MG132 acted synergistically on HSP30 accumulation, relative levels of HSP70 were similar to those observed with MG132 alone. Immunocytochemical analysis of celastrol- or MG132-treated A6 cells revealed HSP30 accumulation in a punctate pattern primarily in the cytoplasm with some staining in the nucleus. Also, in some cells treated with celastrol or MG132 large HSP30 staining structures were observed in the cytoplasm. Lastly, exposure of A6 cells to celastrol induced rounder cell morphology, reduced adherence and disorganization of the actin cytoskeleton. In conclusion, this study has shown that celastrol inhibited proteasome activity in amphibian cultured cells and induced HSF1-mediated expression of hsp genes.

In mammals, the neurogenic inflammatory response can be induced by stimulation or activation on the peripheral sensory C-fibers to release neuropeptides from the peripheral terminals, at the same time their afferent functions are enhanced. There are several neuropeptides stored and released from peripheral terminals of the afferent fibers, such as substance P (SP), neurokinin A, and calcitonin gene related peptide (CGRP). SP is one of the major inflammatory mediators of neurogenic inflammation that can act on neurokinin-1 receptors on smooth muscles and endothelial cells of blood vessels, causing vasodilatation, endothelial gap formation, and local plasma leakage. There are many studies and reports indicate that animals pretreated with a short period non-lethal hyperthermia can induce heat shock response and activate the expression of a group of inducible proteins called heat shock proteins (HSPs), and this stress response reduces the injury by same or other following stresses. In this study, the hyperthermia treatment (HT) was implemented by 42¢J hot water bath and the core body temperature of anesthetized rat was elevated and maintained around 42.0¡Ó0.5¢J for 15 min, and the normothermia control treatment (NT) was implemented by 37¢J warm water bath with the same period. 24 hours after NT or HT, the neurogenic plasma leakage was induced by intravascular injection with capsaicin (90 £gg/kg), SP (3 £gg/kg), or electrical stimulation on the right thoracic vagus nerve. The blood pressures of each animal were continually recorded during the neurogenic inflammation induction or sham operation. The amount of neurogenic inflammation of airway was evaluated by the area density leaky blood vessels. The leaking vessels were labeled with India ink and quantitative analysis by morphometric method. Plasma leakage was also measured by interstitial Evans blue concentration. The results indicated that HT could reduce plasma leakage and hypotension of the neurogenic inflammation that induced by capsaicin, SP or electrical stimulation on vagus nerve. Animals pretreated with aminoguanidine (a selective inhibitor of iNOS) had no significant effect on the neurogenic inflammation by following systemic SP infusion, but that could eliminate the anti-neurogenic inflammatory effect of HT. Animal applied with diphenhydramine (an antagonist of histamine H1 receptor) could attenuate the neurogenic inflammation by following systemic SP infusion, and HT could attenuate the neurogenic inflammation that with or without H1 receptor antagonist. This result indicates that NO synthesis and the activity of iNOS have few effects on neurogenic inflammation of airway, but it plays a critical factor on the initiation of heat shock response. The neurogenic inflammation induced by SP not only direct act on blood vessels but have other indirect effect by the histamine H1 receptor to enhance inflammation. Neonatal rats received high dose capsaicin treatment would induce irreversible sensory C-fiber denervation. The adult rats that were neonatally treated with capsaicin showed a more serious inflammatory response to systemic SP infusion as compared with animals neonatally treated with vehicle. HT still had the anti-inflammatory effects on the neurogenic inflammation that induced by SP. The results indicated that animals with sensory C-fiber denervation might conserve their neurogenic inflammatory responses and were hypersensitive to SP. In conclusion, the HT could attenuate the neurogenic inflammation that induced by different drugs or methods, and the anti-inflammatory effects were correlated with the increase in HSP72 expression. In the neurogenic inflammation induced by SP, the activation of histamine H1 receptors may enhance inflammation, but the activity of endogenous iNOS was less effective.

The immunogenicity and protective efficacy of a DNA vaccine encoding a truncated groEL heat shock gene (pcDNA3/groEL) from Burkholderia pseudomallei was evaluated in vaccinated BALB/c mice infected with B. pseudomallei or B. cenocepacia. After vaccination, the levels of anti-GroEL total IgG and IgG2a were increased in mouse sera. The clonal expansion of the spleen cells increased, and the GroEL protein induced IFN-£^ production by spleen cells. The anti-GroEL antibody-mediated opsonic killing effect was not able to eliminate the growth of B. pseudomallei but was able to eliminate the growth of B. cenocepacia. After intravenous challenge of the vaccinated Balb/c mice with B. pseudomallei, the number of bacteria colonizing the in liver and/or spleen was not reduced. Over 50% of vaccinated mice infected with B. pseudomallei died within 7 days post-infection. By contrast, the bacterial loads in organs were significantly reduced if the vaccinated mice were infected with B. cenocepacia. All of vaccinated mice were alive 7 days post-infection. Liver damage, as assessed by histological observation, and abnormalities in the levels of liver enzymes rapidly resolved in vaccinated mice. We suggest that B. pseudomallei groEL plasmid DNA immunization of Balb/c mice induces a Th1-type immune response and provides cross-protection against B. cenocepacia but not against B. pseudomallei infection.

Many proteins can easily attain a non-native fold and be of no use or even a detriment to the host. The host cell has a myriad of molecules dedicated to assisting nascent and existing proteins in folding properly and maintaining the native fold. Of these molecular chaperones, the small Heat Shock Proteins (sHSP’s) are an important group and worthy of study. The sHSP’s are a diverse group of proteins that have in common an a-crystallin domain and generally display a chaperone activity. A sHSP (HSP20) isolated from the cattle parasite Babesia bovis has similar activities, and limited sequence homology to other a-crystallins. The gene encoding HSP20 was cloned into an expression system where the gene product was induced and purified for study. It was shown that HSP20 inhibits thermally induced aggregation of alcohol dehydrogenase at equimolar ratios. HSP20 was also used to significantly reduce amyloid formation of the b-Amyloid (1-40) Peptide in vitro at the sub-stoichiometric ratio of 1:10. A study of the oligomeric forms of HSP20 using size exclusion chromatography and gel electrophoresis revealed a broad range of multimers present in solution. The distribution of oligomers was affected by altering the solution conditions and concentration of the protein. The domains responsible for multimerization of HSP20 were mapped via sequence homology with known a-crystallins. These regions correspond to 12 carboxy-terminal amino acids and 50 amino-terminal amino acids. Truncated versions of HSP20 lacking these proposed oligomerization domains were created using PCR of the original gene and cloning into an expression vector as before. Using size exclusion chromatography, gel electrophoresis and analytical centrifugation, we show that the deleted domains alter the multimeric population of the protein in solution. The carboxy-terminal domain has a slight effect on multimerization while the amino-terminal deletion results in a drastic reduction in any multimers above a dimer under the conditions tested. Despite this drastic change in the multimerization of HSP20, there were no changes in the activities observed when compared to the full-length form. From this we conclude that the regions responsible for multimerization play little role in the observed activities of HSP20.

¡iAbstract¡j Neurogenic inflammation can be initiated by activation of sensory nerves to release neuropeptides, including tachykinins and calcitonin gene-related peptide. Capsaicin stimulation induces the release of substance P, the most important tachykinin and other neurotransmitters from sensory nerves to cause an increase of plasma leakage via the binding of substance P to NK1 receptors on endothelial cells. It has been proven that hyperthermic pretreatment decreases microvascular protein leakage and attenuates hypotension in anaphylactic shock in rats. Heat shock proteins¡]HSPs¡^are families of phylogenetically conserved molecules that have a protective role in all living cells under stress . Heat shock proteins are induced by whole-body hyperthermia and persist for 6 days. To establish the relationship between heat shock and neurogenic inflammation, the present study investigated whether whole-body hyperthermia pretreatment, at 42 ¢J for 15 min in rats 1 day earlier, could suppress inflammatory response in the lower airways and esophagus evoked by capsaicin (90 µg/ml/kg) or substance P (3 µg/ml/kg ). The magnitude of neurogenic inflammation in the trachea and bronchi was expressed by the area density of India ink-labeled leaky blood vessels in the airway mucosa. One day after heat shock pretreatment, capsaicin-evoked inflammation was reduced by one half to two thirds, and reduced substance P-evoked inflammation by one third. Six days after exposure to heat shock, neurogenic inflammation was not inhibited. HSPs appeared overexpressed in trachea and esophagus tissue in the rats one day after hyperthermia, but was less expressed 6 days after hyperthermia. It is suggested that exposure of the rats to whole-body hyperthermia caused an increased production of HSPs that might influence the affinity of the binding of substance P to NK1 receptors on venule endothelial cells, and reduce the amount of neurogenic plasma leakage.

YDL100Cp is the ArsA homologue protein found in S. cerevisiae. In bacteria, ArsA protein is involved in As3+detoxification but the function of YDL100Cp is still unknown. Previous studies show that deletion of YDL100C in S. cerevisiae was not lethal and had no effect on As3+ sensitivity or growth at 30¢J. However, when grown at 40¢J, growth of YDL100C disrupted strain (JSY1) was inhibited. To study the role of YDL100C in response to lethal heat shock, wild type (W303-1B) and JSY1 cells were exposed to 50¢J for 15 min. The survival rate of JSY1 cells was half of W303-1B cells and the difference in survival rate was complemented by introduction of plasmid carrying YDL100C. It suggests that YDL100Cp plays a role in acquisition of thermotolerance to lethal heat shock. It is believed that there are two factors involved in heat-induced cell death: the heat damage and the oxidative damage. Determinations of heat-damage related defense system in S. cerevisiae, including trehalose (a thermoprotectant) content, Hsp70 expression and Hsp104 expression, demonstrate that heat damage should not be the major cause of JSY1 cell death during heat shock. For the oxidative damage, the measurement of in vivo reactive oxygen species reveal the lower protein damage caused by reactive oxygen species (ROS) in JSY-1 after 50¢J 15 min heat shock, this might reflect the difference in viability of three strains under lethal heat shock. And with the intra cellular content of glutathione, it revels that the YDL100C deficient caused cell got more serious oxidative damage under 50¢J heat shock. But the observation of thermotolerance related ROS scavenger system (including the catalase, and superoxide dismutase) expression with reverse transcription polymerase chain reaction suggested that YDL100C deficient had no effect on triggering these system. As the result, it is suggested that the function of YDL100Cp in S. cerevisiae might be an oxidative damage repair system, such as the glutathione peroxidase. It might react with the oxidative damage substance and function as a deoxidizer.

Cancer has become the first among the 10 major death causing factors in Taiwan. Glioblastoma multiforme (GBM) is the most common malignant tumor in adult human brain tumors. Previously, heat shock or all trans retinoic acid (ATRA) treatment has been shown to be effective in inducing cell apoptosis and cell cycle arrest in several cancer cell lines. In this study, human brain tumor cell line GBM8401 was exposed to 43¢J for 30 min followed by incubation with ATRA. The treatment resulted in up to 50% inhibition of cell growth rate and 50% reduction of cell survival rate . Analysis of cell apoptosis related gene expression and protein expression with RT-PCR and Western blot has showen that p21, p27, pro-caspase 3, phospho-JNK and phospho-p38 were overexpressed after treatment of tumor cells with 43¢J for 30 min followed by addition of ATRA for 15 min to 8 hr. The immunocytochemistry assay revealed that overexpression of phospho-p53 in the nuclei after tumor cells were treated with 43¢J for 30 min followed by addition of ATRA for 8 hr. Results from this study show that treating tumor cells with heat shock before incubation with ATRA will enhance cell apoptosis and inhibit cell growth.

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