Soy isoflavones reduce heat shock proteins in postmenopausal women

Background: Atherosclerosis and its complications remain the most common cause of death in postmenopausal women. But there are few studies evaluating in hormonal theraphy can affect the autoimmune response involved in atherosclerosis. Objective to evaluate the effects to soy germ isoflavones and hormone replacement theraphy on antibodies against heat shock proteins (HPSP60, HPSP70 and HSC70) in moderately hypertensive hypercholesterolemic postmenopausal women. Methods: Women were treated with soy germ (2g/day) 17'beta'-estradiol(2 mg/day) or 17'beta'-estradiol (2mg/day)+noretisterone acetate (1mg/day), for 3 months after taking placebo for 1 month. The plasma autoantibodies to HSP60, HSP70 and HSC70 were determined by ELISA. Results: Data showed a reduction of autoantibodies against HSC70 after treatment in the 3 studies groups in relation to the placebo. The antibodies reactive to HSP70 were reduced only in women receiving soy germ. No significant differences were found for antibodies against HSP60. Conclusion: The soy germ isoflavones and 17'beta'-estradiol, alone or associated with noretisterone acetate, had similar effects on reduction of antibodies reactive to HSP70 in moderately hypertensive hypercholesterolemic postmenopausal women after 3 months of treatment. Thus, there results indicate that soy isoflavnes and hormone theraphy may modulate some pathways of the immune-inflammatory process in postmenopausal women at high risk for atherosclerosis.

Consequences for the Bovine Embryo of Being Derived from a Spermatozoon Subjected to Post-Ejaculatory Aging and Heat Shock: Development to the Blastocyst Stage and Sex Ratio

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the. embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.

Hormone replacement therapy increases levels of antibodies against heat shock protein 65 and certain species of oxidized low density lipoprotein

Hormone replacement therapy (HRT) reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp) and oxidized low density lipoprotein (LDL) have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11) and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02) (P<0.05 and P<0.001, respectively, ANOVA). The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.

L-NAME Treatment Enhances Exercise-induced Content of Myocardial Heat Shock Protein 72 (Hsp72) in Rats

Background/Aim: Nitric oxide (NO) modulates the expression of the chaperone Hsp72 in the heart, and exercise stimulates both NO production and myocardial Hsp72 expression. The main purpose of the study was to investigate whether NO interferes with an exercise-induced myocardial Hsp72 expression. Methods: Male Wistar rats (70-100 days) were divided into control (C, n= 12), L-NAME-treated (L, n= 12), exercise (E, n= 13) and exercise plus L-NAME-treated (EL, n= 20) groups. L-NAME was given in drinking water (700 mg. L(-1)) and the exercise was performed on a treadmill (15-25 m.min(-1), 40-60 min. day(-1)) for seven days. Left ventricle (LV) protein Hsp content, NOS and phosphorylated-NOS (p-NOS) isoforms were measured using Western blotting. The activity of NOS was assayed in LV homogenates by the conversion of [(3)H] L-arginine to [(3)H] L-citrulline. Results: Hsp72 content was increased significantly (223%; p < 0.05) in the E group compared to the C group, but exercise alone did not alter the NOS content, p-NOS isoforms or NOS activity. Contrary to our expectation, L-NAME enhanced (p < 0.05) the exercise-induced Hsp72 content (EL vs. C, L and E groups = 1019%, 548% and 457%, respectively). Although the EL group had increased stimulatory p-eNOS(Ser1177) (over 200%) and decreased inhibitory p-nNOS(Ser852) (similar to 50%) compared to both the E and L groups (p < 0.05), NOS activity was similar in all groups. Conclusions: Our results suggest that exercise-induced cardiac Hsp72 expression does not depend on NO. Conversely, the in vivo L-NAME treatment enhances exercise-induced Hsp72 production. This effect may be due to an increase in cardiac stress. Copyright (C) 2011 S. Karger AG, Basel

Hepatic concentration of heat shock protein 70 kD (Hsp70) in broilers subjected to different thermal treatments

1. The relationship between repeated thermal treatments and hepatic synthesis of Hsp 70 was studied in broiler chickens.2. Sixty broilers were submitted to 5 different treatments (12 birds each) from day 1 to day 42. Four groups were kept in a thermoneutral environment and subjected to 0, 1, 2 and 3 heat stress episodes at 35 degrees C for 4 h per week (TN-0, TN-1, TN-2 and TN-3, respectively). The last group (HT-35) was reared at a room temperature of 35 degrees C.3. From 39 to 42 old, the birds experienced acute heat stress at 41 degrees C. Resistance to heat stress was evaluated by the time taken for rectal temperature to increase by 3 degrees C above the pre-treatment value. Livers were collected (before and after heat stress) and Hsp70 was determined using Western Blot analysis with monoclonal anti-Hsp70 antibody.4. Resistance to heat stress and concentration of Hsp70 were higher in those birds subjected to more heat stress episodes during the experimental period (TN-3) and HT-35. A positive correlation was observed between Hsp70 concentration and the time taken for a 3 degrees C increase in rectal temperature (r=0.42; P<0.01).5. Exposing birds to episodes of heat stress (35 degrees C) during rearing may improve their resistance to acute heat stress, but the previous thermal history did not seem to influence the hepatocyte Hsp70 content after exposure to more severe heat stress (41 degrees C).

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 1) or high incubation temperature (Experiment 11). In each experiment, fertile eggs were distributed in three incubators kept at 37.8degreesC. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32degreesC) or heat (40degreesC) 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. (C) 2004 Wiley-Liss, Inc.

Heat shock protein production and immunity and altered fetal development in diabetic pregnant rats

We evaluated associations between the concentrations of heat shock proteins (hsp60 and hsp70) and their respective antibodies, alterations in maternal reproductive performance, and fetal malformations in pregnant rats with hyperglycemia. Mild diabetes (MD) or severe diabetes (SD) was induced in Sprague-Dawley rats prior to mating; non-treated non-diabetic rats (ND) served as controls. On day 21 of pregnancy, maternal blood was analyzed for hsp60 and hsp70 and their antibodies; and fetuses were weighed and analyzed for congenital malformations. Hsp and anti-hsp levels were correlated with blood glucose levels during gestation. There was a positive correlation between hsp60 and hsp70 levels and the total number of malformations (R∈=∈0.5908, P∈=∈0.0024; R∈=∈0.4877, P∈=∈0.0134, respectively) and the number of malformations per fetus (R∈=∈0.6103, P∈=∈0.0015; R∈=∈0.4875, P∈=∈0.0134, respectively). The anti-hsp60 IgG concentration was correlated with the number of malformations per fetus (R∈=∈0.3887, P∈=∈0.0451) and the anti-hsp70 IgG level correlated with the total number of malformations (R∈=∈0.3999, P∈=∈0.0387). Moreover, both hsp and anti-hsp antibodies showed negative correlations with fetal weight. The results suggest that there is a relationship between hsp60 and hsp70 levels and their respective antibodies and alterations in maternal reproductive performance and impaired fetal development and growth in pregnancies associated with diabetes. © 2012 Cell Stress Society International.

Overexpression of inducible 70-kDa heat shock protein in mouse improves structural and functional recovery of skeletal muscles from atrophy

Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7 days and subsequently released from immobilization and evaluated after 7 days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.

A randomized, blinded, multicenter trial of lipid-associated amphotericin B alone versus in combination with an antibody-based inhibitor of heat shock protein 90 in patients with invasive candidiasis

BACKGROUND: Mycograb (NeuTec Pharma) is a human recombinant monoclonal antibody against heat shock protein 90 that, in laboratory studies, was revealed to have synergy with amphotericin B against a broad spectrum of Candida species. METHODS: A double-blind, randomized study was conducted to determine whether lipid-associated amphotericin B plus Mycograb was superior to amphotericin B plus placebo in patients with culture-confirmed invasive candidiasis. Patients received a lipid-associated formulation of amphotericin B plus a 5-day course of Mycograb or placebo, having been stratified on the basis of Candida species (Candida albicans vs. non-albicans species of Candida). Inclusion criteria included clinical evidence of active infection at trial entry plus growth of Candida species on culture of a specimen from a clinically significant site within 3 days after initiation of study treatment. The primary efficacy variable was overall response to treatment (clinical and mycological resolution) by day 10. RESULTS: Of the 139 patients enrolled from Europe and the United States, 117 were included in the modified intention-to-treat population. A complete overall response by day 10 was obtained for 29 (48%) of 61 patients in the amphotericin B group, compared with 47 (84%) of 56 patients in the Mycograb combination therapy group (odds ratio [OR], 5.8; 95% confidence interval [CI], 2.41-13.79; P<.001). The following efficacy criteria were also met: clinical response (52% vs. 86%; OR, 5.4; 95% CI, 2.21-13.39; P<.001), mycological response (54% vs. 89%; OR, 7.1; 95% CI, 2.64-18.94; P<.001), Candida-attributable mortality (18% vs. 4%; OR, 0.2; 95% CI, 0.04-0.80; P = .025), and rate of culture-confirmed clearance of the infection (hazard ratio, 2.3; 95% CI, 1.4-3.8; P = .001). Mycograb was well tolerated. CONCLUSIONS: Mycograb plus lipid-associated amphotericin B produced significant clinical and culture-confirmed improvement in outcome for patients with invasive candidiasis.

Effect of combined drought and heat stress on heat shock proteins in wheat varieties

Introduction: The re sponse of crop plants ex posed on drought or heat shock is related to de crease in the synthesis of normal proteins, accompanied by increased translation of heat shock proteins (HSPs). Though drought and heat stress have been studied individually, little is known about their combined effect on plants. Methods: The wheat (Triticum aestivum L.) varieties (Katya-tolerant, Sadovo or Mladka-susceptible) were potted in soil. Eight-day-old plants were ex posed to with drawing water for seven days. Heat shock was realized in growth chamber at 40 °C for 6h. A combination of drought and heat shock was per formed by subjecting drought-stressed plants to heat shock treatment. Expression of HSPs in the first leaf of wheat varieties was analyzed by SDS electrophoresis and immunoblotting. Polyclonal antibodies against HSP20, HSP60, HSP110 and mononclonal antibodies against HSP70 were used to distinguish the mentioned HSPs. Results: The leaf relative water content (RWC), which indicated the level of plant dehydration decreased significantly (34 %) under drought stressed conditions The electrolyte leakage of ions (EL), representing the level of the cell membrane stability in creased mark edly (68 %), especially under combination of drought and heat. Maximum EL was ob served in drought susceptible varieties Sadovo and Mladka. Drought and heat shock combination in the wheat plants resulted in the induction of specific HSPs. Conclusions: Our results demonstrate that the response of the wheat plants to a combination of drought and heat stress is different from the response of plants to each of these stresses applied separately. Induction of synergetic effect on HSP expression in case of combination between drought and heat was discussed in the case of two contrasting wheat varieties.

Functional and physiological consequences of genetic variation at phosphoglucose isomerase: Heat shock protein expression is related to enzyme genotype in a montane beetle

Allele frequency variation at the phosphoglucose isomerase (PGI) locus in Californian populations of the beetle Chrysomela aeneicollis suggests that PGI may be undergoing natural selection. We quantified (i) apparent Michaelis-Menten constant (Km) of fructose 6-phosphate at different temperatures and (ii) thermal stability for three common PGI genotypes (1–1, 1–4, and 4–4). We also measured air temperature (Ta) and beetle body temperature (Tb) in three montane drainages in the Sierra Nevada, California. Finally, we measured 70-kDa heat shock protein (Hsp70) expression in field-collected and laboratory-acclimated beetles. We found that PGI allele 1 predominated in the northernmost drainage, Rock Creek (RC), which was also significantly cooler than the southernmost drainage, Big Pine Creek (BPC), where PGI allele 4 predominated. Allele frequencies and air temperatures were intermediate in the middle drainage, Bishop Creek (BC). Differences among genotypes in Km (1–1 > 1–4 > 4–4) and thermal stability (4–4 > 1–4 > 1–1) followed a pattern consistent with temperature adaptation. In nature, Tb was closely related to Ta. Hsp70 expression in adult beetles decreased with elevation and differed among drainages (BPC > BC > RC). After laboratory acclimation (8 days, 20°C day, 4°C night) and heat shock (4 h, 28–36°C), Hsp70 expression was greater for RC than BPC beetles. In RC, field-collected beetles homozygous for PGI 1–1 had higher Hsp70 levels than heterozygotes or a 4–4 homozygote. These results reveal functional and physiological differences among PGI genotypes, which suggest that montane populations of this beetle are locally adapted to temperature.

The Effect Of Celastrol, A Triterpene With Antitumorigenic Activity, On Conformational And Functional Aspects Of The Human 90kda Heat Shock Protein Hsp90α, A Chaperone Implicated In The Stabilization Of The Tumor Phenotype.

Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.

Photochemical heat-shock response in common bean leaves as affected by previous water deficit

The heat sensitivity of photochemical processes was evaluated in the common bean (Phaseolus vulgaris) cultivars A222, A320, and Carioca grown under well-watered conditions during the entire plant cycle (control treatment) or subjected to a temporal moderate water deficit at the preflowering stage (PWD). The responses of chlorophyll fluorescence to temperature were evaluated in leaf discs excised from control and PWD plants seven days after the complete recovery of plant shoot hydration. Heat treatment was done in the dark (5 min) at the ambient CO2 concentration. Chlorophyll fluorescence was assessed under both dark and light conditions at 25, 35, and 45 degrees C. In the dark, a decline of the potential quantum efficiency of photosystem II (PSII) and an increase in minimum chlorophyll fluorescence were observed in all genotypes at 45 degrees C, but these responses were affected by PWD. In the light, the apparent electron transport rate and the effective quantum efficiency of PSII were reduced by heat stress (45 degrees C), but no change due to PWD was demonstrated. Interestingly, only the A222 cultivar subjected to PWD showed a significant increase in nonphotochemical fluorescence quenching at 45 degrees C. The common bean cultivars had different photochemical sensitivities to heat stress altered by a previous water deficit period. Increased thermal tolerance due to PWD was genotype-dependent and associated with an increase in potential quantum efficiency of PSII at high temperature. Under such conditions, the genotype responsive to PWD treatment enhanced its protective capacity against excessive light energy via increased nonphotochemical quenching.

Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42 degreesC for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37 degreesC, those exposed to heat stress (42 degreesC for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate. hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Polydnavirus particle proteins with similarities to molecular chaperones, heat-shock protein 70 and calreticulin

Multipartite nucleic acid-containing virus-like particles, known as polydnaviruses, are special structures produced by female parasitoid wasps to deliver wasp components into the body of their host at oviposition. The particles confer protection for the developing parasitoid by passive and active means. Although several genes expressed from the circular DNA of these particles have been identified from various host-parasitoid systems, there is not much known about the structural proteins of these particles. Here we report on two genes encoding Cotesia rubecula particle proteins with similarities to molecular chaperones, calreticulin and heat-shock protein 70.