Heat Shock Protein Content Research Articles

Effects of Quercetin on the Severity of Chemically Induced Convulsions and 70-kDal Heat Shock Protein Content in Brain Structures in Rats

Behavioral methods were used to study the effects of the 70-kDal heat shock protein (Hsp70) expression inhibitor quercetin on convulsions and motor disorders evoked by N-methyl-D-aspartic acid (N-methyl- D-aspartate (NMDA)) or corazol in adult Wistar rats. These experiments showed that intraperitoneal administration of quercetin 4 h before microinjection of NMDA into the third ventricle of the brain increased the duration of the toxic component of convulsive seizures and the severity of convulsions and ataxic symptoms. The same dose and route of quercetin increased the duration of clonic and tonic convulsions but did not alter the severity of convulsive seizures or ataxia symptoms induced by intraperitoneal injection of corazol. Immunoblotting studies showed that administration of quercetin decreased the content of the inducible form of Hsp70 in the hippocampus, thalamus, and corpus callosum. These data provide evidence that the Hsp70 expression inhibitor quercetin has anticonvulsant properties. It is suggested that Hsp70 has a role in the central mechanisms regulating behavioral convulsions and motor disorders induced by NMDA and corazol in rats.

Comparative studies on temperature threshold for heat shock protein 70 induction in young and adult Murrah buffaloes

To know the temperature threshold for heat shock protein 70 (HSP70) induction in lymphocytes and to assess physiological changes, if any, in relation to HSP70 induction in young and adult Murrah buffaloes, this study was divided into two parts: I. In vivo study: where assay of HSP70 was performed in blood samples collected from acutely exposed young and adult Murrah buffaloes (n = 6) inside a climatic chamber at 40, 42 and 45 °C for 4 h and thermoneutral temperature (22 °C). Physiological parameters viz., rectal temperature, respiratory rate, pulse rate and skin temperature of different body parts were monitored to assess magnitude of stress in the animals owing to thermal exposure II. For in vitro study, equal numbers of lymphocyte cells were separated from blood collected from young and adult buffaloes and were subjected to four temperature treatments (38, 40, 42 and 45 °C) for 4 h. A significant increase (p < 0.05) in all the physiological parameters in both young and adult buffaloes was observed after exposure to 40, 42 and 45 °C for 4 h as compared to 38 °C. The average plasma HSP70 concentrations (ng/ml) were significantly higher (p < 0.05) at 40, 42 and 45 °C as compared to 38 °C in both young and adult and were higher in young than adult buffaloes at 38 and 45 °C. Heat shock protein 70 level in lymphocyte lysate showed highest concentration after 3-h exposure to all temperatures (40, 42 and 45 °C) in both young and adult buffaloes. The intensity of changes of all physiological parameters was more in young animals than in the adults indicating the greater susceptibility of younger animals to heat stress and was found to be changed at around 40 °C when animals were exposed to different temperatures, indicating the possibility that HSP70 production may be initiated at this temperature which is 2 or 3 °C higher than core body temperature.

Identification and validation of rice reference proteins for western blotting

Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen–antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.

Relationship between the content of heat shock protein 70 and the level of sepsis patients

Objective To investigate the relationships between the level of heat shock protein (HSPs) 70 and severity of sepsis in order to find its effects on the status of sepsis and the prognosis of the patients. Methods In total, 120 patients diagnosed to be suffering from sepsis from the emergency department were selected as studying subjects. According to SAPS Ⅱ score, all of these patients were divided into mild, moderate and severe groups (n =40 in each group) , and at the same time, 40 healthy volunteers were studied as controls. The levels of intracellular HSPs70 in peripheral blood neutrophil cells in the mild, moderate and severe groups were determined by using flow cytometry, while serum levels of procalcitonin (PCT) and C-reactive protein ( CRP) were measured as well. Results ① The levels of heat shock proteins HSPs70 in neutrophil cells and PCT in patients with sepsis escalated gradually with the increase in SAPS Ⅱ score, and there were significant different among the mild, moderate, and severe sepsis groups(P<0.05). ② In all septic patients, the death happened only in the severe group. Compared other markers in the survivals , the level of heat shock protein HSPs70 was significantly higher in fatal patients. ③ In curve plot analysis in the patients with sepsis done by the curve of receiver operating characteristic (ROC) , the area of HSPs70 in neutrophil cells under the curve was significantly larger as compared with that of PCT and CRP. Conclusions Heat shock protein HSPs70 has a certain clinical value in determining the severity of disease and prognosis of patients suffering from sepsis. Key words: Heat shock protein 70; Sepsis; Procalcitonin; C-reactive protein; SAPS Ⅱ score; Prognosis ; Value of diagnosis; Assessment

Restoring HSP70 deficiencies improves glucose tolerance in diabetic monkeys

We evaluated heat shock protein 70 (HSP70) changes in diabetes mellitus (DM) in a nonhuman primate model. To this end, two studies were conducted in DM vervet monkeys. 1) Normal control and streptozotocin-induced DM monkeys (Stz-DM) that were differentiated into moderately or poorly controlled DM by judicious insulin administration were evaluated. Liver was collected at 4, 8, 12, 16, and 20 wk after streptozotocin, exposed to ex vivo heat shock at 42°C, and immunoblotted for heat shock factor 1 (HSF1), HSP70, and phosphorylated HSF1. 2) Spontaneous DM monkeys that were not pharmacologically induced were included in a crossover study of the HSP70-inducing drug geranylgeranylacetone (GGA). GGA at 20 mg/kg was given for 14 days with a 6-wk washout period. Glucose tolerance testing and plasma and muscle HSP70 were the primary outcome measurements. In Stz-DM, hyperglycemia reduced hepatic HSP70 in a dose-dependent fashion. HSF1 was increased in livers of monkeys with Stz-DM, but responses to ex vivo heat shock were impaired vs. normal monkeys. Activation of HSF1 appears to be important, because the phosphorylation change with heat stress was nearly perfectly correlated with HSP70 increases. Impaired HSF1 activation was also seen in Stz-DM after chronic hyperglycemia (>12 wk). In naturally occurring DM, increased circulating HSP70 resulted in significantly improved glucose tolerance and significant, positive trends in other measurements of insulin resistance. No change in muscle HSP70 content was observed. We conclude that increasing HSP70, potentially through targeting hyperglycemia-related deficits in HSF1 induction and activation in the liver, is a potent and viable strategy to improve glucose tolerance.

Effects of propofol and isoflurane anesthesia on serum heat shock protein 70 of perioperative patients with traumatic brain injury

Objective To investigate the effect of propofol and isoflurane anesthesia on serum heat shock protein 70 ( HSP70 ) of perioperative patients with brain injury and compare the anesthetic effect of the two narcotic drugs. Methods 30 patients with medium or severe traumatic brain injury,treated by hematoma evacuation operation,were randomly divided into propofol ( n = 15 ) and isoflurane group ( n = 15 ). After anesthesia induction,propofol (target effect concentration of 3 ～ 4 μg/ml ) was intravenously injected and 1.2% isoflurane was inhalated into the patients of the 2 groups,respectively. In addition,vecuronium and fentany were intermittent intravenously injected into patients to maintain the depth of anesthesia. HSP70 in blood of patients were detected before anesthesia induction, 1 h after the dura was opened and 24 h after the operation by ELISA method. Recovery and tracheal extubation time of patients were recorded. Results HSP70 levels in blood of patients in the 2 groups were significantly increased compared with the former time point(P＜0.05 ). HSP70 content in propofol group was significantlyhigher at 1 h after the dura was opened( ( 2.00 ± 0. 24 ) ng/ml, ( 2.19 ± 0.26 ) ng/ml, t = 2. 080, P = 0.047 ) and 24 h after the operation( t=2.086, P=0.046) ,and recovery and tracheal extubation time was shorter compared with the isoflurane group(P＜0. 05). Conclusion Perioperative moderate stress,and early postoperative recovery and extubation can be maintained in patients with traumatic brain injury with profol anesthesia. Key words: Traumatic brain injury; Propofol; Heat shock protein 70

Morphofunctional Characteristics of Syncytiotrophoblast and Content of Heat Shock Protein 70 in It during Exacerbation of Herpesvirus Infection in Pregnant Women

Exacerbation of herpesvirus infection during pregnancy is associated with damage to syncytiotrophoblast inflicted by herpesvirus either directly or via TNF-α (due to contact of NK lymphocytes with villus surface). A sharp decrease in the content of heat shock protein with a molecular weight of 70 Da and activation of caspase-3 were noted in placenta homogenate. This leads to disturbances in syncytiotrophoblast cytosol structure and increase in the relative content of apoptotic nuclei.

Heat-shock protein 70 expression in the seminal plasma of patients with chronic bacterial prostatitis and chronic prostatitis/chronic pelvic pain syndrome

The purpose of this study was to assess the diagnostic value and potentially protective capacity of heat-shock protein 70 (HSP70) in chronic bacterial prostatitis and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). In this study, seminal plasma levels of cytokines (tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β)) and HSP70 were evaluated by enzyme-linked immunosorbent assay in 80 men: 15 healthy controls, 16 men with chronic bacterial prostatitis, 23 men with CP/CPPS IIIA and 26 with CP/CPPS IIIB. The HSP70 levels in chronic bacterial prostatitis and CP/CPPS patients were correlated with chronic prostatitis symptom index (CPSI). Significantly increased levels of cytokines (TNF-α and IL-1β) and HSP70 were observed in seminal plasmas from patients with chronic bacterial prostatitis compared with CP/CPPS patients and controls. However, only IL-1β was significantly elevated compared with CP/CPPS IIIB and controls in patients with CP/CPPS IIIA. HSP70 levels in CP/CPPS patients were significantly lower than that in controls. HSP70 concentration in seminal plasma was negatively correlated with CPSI in chronic bacterial prostatitis. The results indicated that HSP70 and IL-1β appear to be the most reliable and predictive surrogate markers to diagnose chronic bacterial prostatitis and CP/CPPS, respectively. HSP70 has an important protective role in the regulation of cell functions in chronic bacterial prostatitis. CP/CPPS would probably be detrimental to the ability of T cells and consequently suppress the expression of HSP70.

Ethanol increases HSP70 concentrations in honeybee ( Apis mellifera L.) brain tissue

Previous research on the honeybee ethanol model established how acute ethanol exposure altered function at different levels of organization: behavior and learning, ecology, and physiology. The purpose of this study was to evaluate whether ethanol doses that affect honeybee behavior also induce a significant stress response, measured by heat shock protein 70 (HSP70) concentrations, in honeybee brain tissues. Experiment 1 examined how pretreatment handling influenced brain HSP70 concentrations in three pretreatment groups of bees; immediately after being collected, after being harnessed and fed, and after 22–24 h in a harness. HSP70 concentrations did not differ among pretreatment groups within replicates, although we observed significantly different HSP70 concentrations between the two replicates. Experiment 2 investigated the relationship between ethanol dose and brain HSP70 concentrations. Bees were placed in seven experimental groups, the three pretreatment groups as in Experiment 1 and four ethanol-fed groups. Bees in ethanol treatments were fed 1.5 M sucrose (control) and 1.5 M sucrose–ethanol solutions containing 2.5, 5, and 10% ethanol, allowed to sit for 4 h, and dissected brains were assayed for HSP70. We observed ethanol-induced increases in honeybee brain HSP70 concentrations from the control group through the 5% ethanol group. Only bees in the 5% ethanol group had HSP70 concentrations significantly higher than the control group. The inverted U-shaped ethanol dose–HSP70 concentration response curve indicated that ingestion of 2.5% ethanol and 5% ethanol stimulated the stress response, whereas ingestion of 10% ethanol inhibited the stress response. Doses that show maximum HSP70 concentration (5% ethanol) or HSP70 inhibition (10% ethanol) correspond to those (≥5% ethanol) that also impaired honeybees in previous studies. We conclude that acute ethanol intoxication by solutions containing ≥5% ethanol causes significant ethanol-induced stress in brain tissue that impairs honeybee behavior and associative learning.

The Heat Shock Protein Profile in Children with Chronic Kidney Disease

Chronic kidney disease (CKD) due to inflammation, lipid disorders, and endothelial dysfunction predisposes to accelerated atherosclerosis. Elevated levels of heat shock proteins (HSPs) and antibodies against them have been described in adults with atherosclerotic lesions and cardiovascular events. However, there are no investigations of these variables in children with CKD treated conservatively or on peritoneal dialysis. Therefore, we decided to evaluate the profile of HSPs and their potential role as markers of atherosclerosis in these groups of patients. The study group consisted of 37 children with CKD treated conservatively and 19 children and young adults on automated peritoneal dialysis (APD). The control group comprised 15 age-matched subjects with normal kidney function. HSP-60, HSP-70, HSP-90alpha, anti-HSP-60, anti-HSP-70, sE-selectin, and interleukin (IL)-4 serum concentrations were assessed by ELISA; high-sensitivity C-reactive protein (hs-CRP) serum levels were assessed by nephelometry. Serum lipid profiles (total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides) were also estimated. HSP-90alpha, anti-HSP-60, and sE-selectin concentrations in the CKD and APD patients were higher than in the controls and were lower in the predialysis subjects than in the children on dialysis. Median values of anti-HSP-70 were higher in the CKD patients than in the control group. Levels of IL-4 were increased in all patients versus controls. Median values of HSP-60 were decreased in the CKD and APD children versus controls. HSP-70 and hs-CRP concentrations were comparable in all groups. The altered HSP and anti-HSP concentrations may imply that the response to stress conditions in the course of CKD is disturbed in children; APD does not aggravate that dysfunction in a significant way. Relationships between HSPs, lipid profile, and markers of inflammation suggest a possible role of the selected HSPs as markers of atherosclerosis in children with CKD.

A Dynamical Model for Heat Shock Protein (HSP) Transcription and its Correlation with HSP70 in vitro Experiments

Heat-shock proteins (HSPs) are well know for their protective mechanism when cells undergo stress conditions such as heat by enhancing protein folding and mitigating apoptosis Heat-shock causes the denaturing of proteins to increase, which leads to the aggregation of denatured and functional proteins. On the other hand, cells develop thermotolerance to protect themselves from later exposure to heat shock or other stresses. Once a cell has been exposed to the heat-shock, the fate of the cell depends on the amount of protein denaturation and aggregation. The cell's ability to become tolerant of heat after the heat-shock is one of the most widely studied activating responses experimentally. In the paper, we develop three mechanistic models in a hierarchical fashion that follow a biological pathway from the initial heat-shock to the HSP70:HSF complex that dissociates to HSP70 and HSF, and aim to predict the HSP concentration in a cell. These models allow investigators to simulate the major/important mechanisms in the upregulation of HSP70. This study was performed to create a depiction of the cellular and nuclear events of the HSP70 response to the heat-shock. The data generated from the model were correlated to favorably experimental data under the assumption of time delay.

Glutamine attenuates acute lung injury by inhibition of high mobility group box protein-1 expression during sepsis

Heat shock protein 70 (HSP70) is reported as the main factor responsible for the beneficial effects of glutamine (GLN) and as a negative regulator of high mobility group box protein-1 (HMGB-1) expression. Our aim was to determine whether GLN attenuates acute lung injury (ALI) by the inhibition of HMGB-1 expression during sepsis. Male Sprague-Dawley rats were subjected to caecal ligation and puncture (CLP) to induce sepsis. GLN or saline was administered through tail vein 1 h after CLP. Then, quercetin (Q), an inhibitor of HSP70, was utilised to assess the role of the enhanced HSP70. We observed the survival of the subjects. At 24 h post-CLP, we measured lung HSP70, phosphorylated heat shock factor-1 (HSF-1-p) and HMGB-1 expressions, NF-kappaB DNA-binding activity and ALI occurrence. We also measured serum HSP70, IL-6 and HMGB-1 concentrations. GLN improved survival during sepsis. In GLN-treated rats, lung HSP70 and HSF-1-p expressions were enhanced, lung HMGB-1 expression and NF-kappaB DNA-binding activity were suppressed, and ALI was attenuated. Furthermore, in GLN-administered rats, serum HSP70 concentration was higher, and serum IL-6 and HMGB-1 concentrations were lower than those in non-treated rats. Q inhibited the enhancement of HSP70 and HSF-1-p expressions and abrogated the GLN-mediated benefits. In conclusion, GLN attenuated ALI and improved survival by the inhibition of HMGB-1 expression during sepsis in rats. These benefits were associated with the enhancement of HSP70 expression by GLN.

Heat shock proteins in children and young adults on chronic hemodialysis

Chronic inflammation, lipid and autoimmune disorders are hallmarks of atherogenesis, and hemodialysis per se may be an additional factor predisposing to accelerated atherosclerosis. Elevated levels of heat shock proteins (HSP) and antibodies against these HSP have been described in adults with atherosclerotic lesions and cardiovascular events, but to date there has been a scarcity of investigations on these parameters in adult and pediatric patients on hemodialysis (HD). We have investigated the HSP profile in hemodialyzed children and the impact of a single HD session on those proteins and their correlations with known risk factors for atherosclerosis. The study group consisted of 17 children and young adults undergoing HD with polysulfone membranes. The control group comprised 15 age-matched subjects with normal kidney function. The serum concentrations of Hsp60, Hsp90alpha, anti-Hsp60, anti-Hsp70, and sE-selectin were assessed by an enzyme-linked immunosorbent assay, and serum concentration of high-sensitivity-C-reactive protein was assayed by nephelometry. The serum lipid profile [total cholesterol (CHOL), high-density lipoprotein-CHOL, low-density lipoprotein-CHOL, triglycerides] was also estimated. Compared to the control values, the median values of Hsp60 before the HD session were lower, whereas those of Hsp90alpha and anti-Hsp60 were higher. A single HD session raised the median values of Hsp60 and Hsp90alpha and decreased the concentrations of anti-Hsp60 and anti-Hsp70. In addition, the concentrations of HSPs and the antibodies against them correlated with the lipid markers both before and after HD. The altered HSP and anti-HSP concentrations in HD children, which correlated with the lipid profile and the endothelial markers, suggest a dysfunctional HSP system in this population and the possibility of HSPs being classified as new markers of atherosclerosis.

Heat shock protein 70 is secreted from endothelial cells by a non-classical pathway involving exosomes

Emerging evidence suggests that a high level of circulating heat shock protein 70 (HSP70) correlates with a lower risk of vascular disease; however, the biological significance of this inverse relationship has not been explored. Herein, we report that oxidative low density lipoprotein (Ox-LDL) and homocysteine (Hcy) induce HSP70 release from endothelial cells. In rat endothelial cells, Ox-LDL and Hcy induced robust release of HSP70, independent of the classical route of endoplasmic reticulum/Golgi protein trafficking or the formation of lipid rafts. In contrast, Ox-LDL and Hcy significantly enhanced the exosomal secretory rate and increased the HSP70 content of exosomes. Exogenous HSP70 had no impact on LPS-, Ox-LDL- and Hcy-induced activation of endothelial cells, whereas HSP70 did activate monocytes alone, resulting in monocyte adhesion to endothelial cells. These results indicate that exosome-dependent secretion of HSP70 from endothelial cells provides a novel paracrine mechanism to regulate vascular endothelial functional integrity.

Chlamydia trachomatis Infection Is Related to Poor Semen Quality in Young Prostatitis Patients

BackgroundThe impact of chronic prostatitis resulting from Chlamydia trachomatis infection on male fertility is controversial. ObjectiveTo investigate the correlation between C. trachomatis infection and semen quality in young male patients affected by chronic prostatitis resulting from C. trachomatis infection and to evaluate the correlation between anti–C. trachomatis immunoglobulin (Ig) A against heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), and semen parameters. Design, setting, and participantsAll patients with clinical and instrumental diagnosis of chronic prostatitis underwent microbiological cultures for common bacteria, DNA extraction, mucosal and serum antibody evaluation for C. trachomatis, and semen parameter analysis. Western blot analysis of mucosal anti–C. trachomatis IgA was performed. InterventionsSubjects were split into two groups: Group A consisted of patients with chronic prostatitis resulting from common bacteria (uropathogens), and group B consisted of patients with chronic prostatitis resulting from C. trachomatis infection. MeasurementsThe relationship between C. trachomatis infection and semen parameters as well as the correlation among IgA levels, IgA characterisation, and semen analysis were determined. Results and limitationsWe enrolled 1161 patients (mean age: 36.5 yr). Of these, 707 patients were placed in group A, and 454 were placed in group B. Significant statistical differences were reported between groups in terms of sperm concentration (p<0.001), percentage of motile sperm (p<0.001), and normal morphologic forms (p<0.001). Strong correlations between mucosal anti–C. trachomatis IgA and sperm concentration (p<0.001) and normal morphologic forms (p<0.001) were reported. Correlations among positivity to HSP60, HSP70, and sperm concentration (p<0.003) and normal morphologic forms (p<0.001) were also reported. ConclusionsThis study demonstrated the role of chronic prostatitis resulting from C. trachomatis in male fertility decrease, highlighting probable immunomediated damage to germinal cells because of C. trachomatis infections.

Nest Site Selection Influences Mortality and Stress Responses in Developmental Stages ofMegachile apicalisSpinola (Hymenoptera: Megachilidae)

We examined stress responses and survival in developmental stages of the invasive solitary bee Megachile apicalis Spinola during two nesting seasons in the Central Valley of California to consider whether abiotic stress tolerance of its offspring contributes to this species' successful colonization of the western United States. In 2001 and 2003, artificial nesting cavities were affixed to vertical plywood boards oriented to maximize nest cavity temperature and humidity differences: one side faced south (exposed to direct sun) and the other one faced north (shaded). After several weeks of nesting activity, we measured heat shock protein 70 (HSP70) concentrations in adults and offspring on 1 d in both years and offspring survival and mortality sources in 2003. In 2001, M. apicalis showed higher HSP70 concentrations in exposed nests than in shaded nests during all developmental stages, adults and their offspring. In 2003, overall survivorship was not significantly different between treatments because exposed nests experienced high offspring mortality caused by heat stress, whereas shaded nests suffered similarly high offspring mortality because of parasitoids. In both years of our study, females preferred shaded nests over exposed nests. M. apicalis successfully reproduces in grasslands of the Central Valley of California where offspring survive hot, dry nest sites and parasitoids in sufficient numbers to inoculate new grassland habitats, unpopulated by tolerance-limited native solitary bees, with incipient populations of this bee, M. apicalis.

Expression of Heat Shock Proteins after Ultrasound Exposure in HL-60 Cells

One of the important cellular defense mechanisms against stress is the induction of heat shock proteins (HSPs). We have recently demonstrated that a low frequency electromagnetic field is unable to induce the heat shock response (HSR). In the present study, we expanded our investigations to the induction of HSPs, particularly Hsp72, by ultrasound (US). Human promyelocytic leukemia HL-60 cells were exposed in suspension to US at 1, 3 and 10 MHz, as well as combinations of two of these frequencies. The ability of US to induce Hsp72 was tested for different frequencies, intensities and exposure times. In addition, the water bath temperature was varied from 30 to 36°C. The Hsp72 protein expression was determined 4 and 24 h after treatment. We found that the amount of Hsp72 increased with increasing US frequency, reaching its highest level of about 1800%, induced by 10 MHz. After increasing the temperature of the water bath, the amount of Hsp72 in the treated cells was also increased, whereas no induction was observed at 30°C. For all treatment conditions, ultrasound of 1 MHz was unable to significantly induce Hsp72. At 10 MHz, the exposure time was varied from 0 to 20 min. We found that the induction of Hsp72 took place after 5 min of exposure. For a fixed level of absorbed US energy, the continuous regime, as well as a pulsation of 1:2 (5 ms on and 5 ms off) induced the same Hsp72 level. Pulsation of 1:5 (2 ms on and 8 ms off) and 1:10 (1 ms on and 9 ms off) did not show any effect. A single sonication of 20 min, as well as a fractionated sonication of two 10 min exposures induced the same level of Hsp72, whereas four exposures of 5 min reduced the Hsp72 level. At the optimum exposure conditions (10 MHz, 10 min), the concentration of other HSPs was also determined. Hsp27 showed no effect but Hsp32, Hsp40 and Hsp72 were induced. Taken together, these results suggest a synergistic interaction between heat and US. (E-mail: werner.sontag@ibg.fzk.de)

Reduced carbohydrate availability does not modulate training-induced heat shock protein adaptations but does upregulate oxidative enzyme activity in human skeletal muscle

The primary aim of the present study was to test the hypothesis that training with reduced carbohydrate availability from both endogenous and exogenous sources provides an enhanced stimulus for training-induced heat shock protein (HSP) adaptations of skeletal muscle. A secondary aim was to investigate the influence of reduced carbohydrate availability on oxidative adaptations and exercise performance. Three groups of recreationally active men performed 6 wk of high-intensity intermittent running occurring four times per week. Group 1 (n = 8; Low + Glu) and 2 (n = 7; Low + Pla) trained twice per day, 2 days/wk, and consumed a 6.4% glucose or placebo solution, respectively, immediately before every second training session and at regular intervals throughout exercise. Group 3 (n = 8; Norm) trained once per day, 4 days/wk, and consumed no beverage throughout training. Training induced significant improvements in maximal oxygen uptake (Vo(2max)) (P = 0.001) and distance covered on Yo-Yo Intermittent Recovery Test 2 (P = 0.001) in all groups, with no difference between conditions. Similarly, training resulted in significant increases in HSP70, HSP60, and alphaB-crystallin in the gastrocnemius (P = 0.03, 0.02, and 0.01, respectively) and vastus lateralis (P = 0.01, 0.02, and 0.003, respectively) muscles in all groups, with no difference between conditions. In contrast, training resulted in significant increases in succinate dehydrogenase (SDH) activity of the gastrocnemeius (Low + Glu, Low + Pla, and Norm: 27, 76, and 53% increases, respectively; P = 0.001) and vastus lateralis muscles (Low + Glu, Low + Pla, and Norm: 17, 70, and 19% increases, respectively; P = 0.001) where the magnitude of increase in SDH activity was significantly larger for both muscles (P = 0.03 and 0.04 for gastrocnemius and vastus lateralis, respectively) for subjects training in the Low + Pla condition. Data provide the first evidence that in whole body exercise conditions, carbohydrate availability appears to have no modulating effect on training-induced increases of the HSP content of skeletal muscle. In contrast, training under conditions of reduced carbohydrate availability from both endogenous and exogenous sources provides an enhanced stimulus for inducing oxidative enzyme adaptations of skeletal muscle although this does not translate to improved performance during high-intensity exercise.

Peripheral venous occlusion causing cardiac hypertrophy and changes in biological parameters in rats

Although patients with peripheral vascular disease (PVD) show a high prevalence of coronary artery disease and myocardial hypertrophy, influences of PVD alone on cardiac hypertrophy, and its subcellular mechanism remain unclear. This study investigates the influences of PVD alone on cardiac muscles and the changes in biological parameters that potentially affect cardiac hypertrophy and cardioprotection. Several veins from rat hind limb were occluded. Two weeks later, the weight of heart relative to body weight in the occluded group increased by approximately 10% compared to that in the sham-operated group. A significant increase was observed in insulin-like growth factor-1 expression, and a significant decrease was observed in myostatin and mechano growth factor in the occluded group compared to the sham-operated group. Tissue concentration of nitric oxide, and content of heat shock protein (HSP)-72, HSP-90, and heat shock cognate protein-70 significantly decreased in the hypertrophied myocardium. These results suggest that peripheral occlusion alone causes cardiac hypertrophy associated with changes in biological parameters.

250 EFFECT OF LOW OXYGEN TENSION ATMOSPHERE AND MATURATION MEDIA ON IN VITRO-MATURED SWINE OOCYTES

The aim of this study was to evaluate the efficiency of a low oxygen tension atmosphere (5% CO2, 5% O2, and 90% N2) on swine oocyte maturation in chemically defined media or when supplemented with porcine follicular fluid (PFF). Briefly, oocytes were in vitro matured for 44 h in TCM-199 with 10% PFF or 0.1% PVA added, under a low oxygen tension atmosphere or a normal oxygen tension atmosphere (5% CO2 in air, approximate 20% O2). At 0 and 44 h of maturation, cumulus oophorus cells were removed. To evaluate the migration of cortical granules, oocytes were fixed, permeabilized, and incubated in 100 μg of FITC-PNA mL–1 for 30 min. Oocytes were then incubated in 10 μg mL–1 of Rnase for 30 min and in 10 μg mL–1 of propidium iodide for 10 min to verify nuclear maturation by confocal microscopy (Zeiss LSM 510 Meta). Heat shock protein 70 (HSP70) content was assessed as described in Kawarsky and King (2001 Zygote 9(3), 39–50) to verify oxidative stress. Data were analyzed by the SAS System for Windows (2000). The nonparametric ANOVA NPAR1WAY procedure was applied to evaluate nuclear maturation rate by comparing groups in pairs. Migration of cortical granules and HSP70 content were analyzed using PROC GLM (LSD test of means). The effects of treatment and manipulation were verified in all analyses. The significance level was 5%, and data were presented as means ± SEM. Results indicated that the percentage of metaphase II oocytes did not differ among groups after 44 h of maturation [PFF 5% O2 (89.16 ± 3.73a), PFF 20% O2 (86.59 ± 6a), PVA 5% O2 (79.62 ± 8.22a), and PVA 20% O2 (93 ± 5.17a)]. However, these groups were different from the 0-h group (0 ± 0b). Results for the percentage of cortical granule migration showed that 0-h oocytes (38.92 ± 2.75a) had lower migration rates compared with other groups. After 44 h of maturation, migration of the cortical granule rate of the PFF-supplemented group under a 5% O2 atmosphere (61.66 ± 3.21b) was different when compared with the PVA 20% O2 group (50.97 ± 3.48c). The other groups showed intermediate results, but without statistical differences [PFF 20% O2 (58.51 ± 2.5bc) and PVA 5% O2 (53.75 ± 3.14bc)] for the migration of cortical granules. Moreover, no difference at pixel quantification of HSP70 was observed among groups after 44 h of maturation [PFF 5% O2 (116.45 ± 40.94a), PFF 20% O2 (44.44 ± 12.66a), PVA 5% O2 (29.95 ± 7.95a), and PVA 20% O2 (58.49 ± 22.2a)], although these groups were different from the 0-h group (247.41 ± 38.59b). Although the HSP content decreased throughout in vitro maturation of swine oocytes under the low and high oxygen tension atmospheres, it can be concluded that a low oxygen tension atmosphere did not affect nuclear maturation and rates of cortical granule migration regardless of maturation media supplementation. Financial support: FAPESP (grant no. 05/01420-7).