Daily quadratic trend in basal monocyte expressed HSP72 in healthy human subjects

The inducible human stress protein heat shock protein 72 (HSP72) performs vital roles within the body at rest and during periods of stress. Recently it was shown over a 24 h period that basal HSP72 followed a diurnal variation. However, these results and previous literature demonstrate noticeable inter-subject variation in basal HSP72 expression. The notion of intra/inter-day variation in basal HSP72 expression has not been explored in detail. Basal monocyte expressed HSP72 was determined every 3 h, over a 9 h period in 12 healthy male subjects (20.2 ± 1.9 years, 178.7 ± 5.6 cm, 75.1 ± 6.0 kg) within a temperature controlled laboratory. A significant quadratic trend was observed for time (F = 26.0, P = 0.001, partial η² = 0.74), where HSP72 decreased between 0800 and 1100 hours (P < 0.001) and then increased between 1100 and 1400 hours (P = 0.015). The main effect for day (F = 2.6, P = 0.14) and the day × time interaction effect (F = 3.9, P = 0.08) were not significant. There was no correlation between serum and monocyte expressed HSP72, with no significant effect for time (F = 2.0, P = 0.21) in serum HSP72 expression. The results support findings by others that basal monocyte expressed HSP72 follows a diurnal variation which incorporates a quadratic trend, which is not compromised by any significant daily variation and that serum HSP72 expression has no endogenous circadian rhythm. The significant quadratic trend in basal monocyte HSP72 expression shown here highlights the need to tightly control variables, such as timing of sample collection, as it is known basal values influence the magnitude of HSP72 expression post-stressor/intervention.     

HSP72 gene expression progressively increases in human skeletal muscle during prolonged, exhaustive exercise.

To examine the effect of exercise on heat shock protein (HSP) 72 mRNA expression in skeletal muscle, five healthy humans (20 +/- 1 yr; 64 +/- 3 kg; peak O(2) uptake of 2.55 +/- 0.2 l/min) cycled until exhaustion at a workload corresponding to 63% peak O(2) uptake. Muscle was sampled from the vastus lateralis, and muscle temperature was measured at rest (R), 10 min of exercise (Min10), approximately 40 min before fatigue (F-40 = 144 +/- 7 min), and fatigue (F = 186 +/- 15 min). Muscle samples were analyzed for HSP72 mRNA expression, as well as glycogen and lactate concentration. Muscle temperature increased (P < 0.05) during the first 10 min of exercise but then remained constant for the duration of the exercise. Similarly, lactate concentration increased (P < 0.05) when Min10 was compared with R but decreased (P < 0.05) thereafter, such that concentrations at F-40 and F were not different from those at R. In contrast, muscle glycogen concentration fell progressively throughout exercise (486 +/- 74 vs. 25 +/- 7 mmol/kg dry weight for R and F, respectively; P < 0.05). HSP72 mRNA was detected at R but did not increase by Min10. However, HSP72 mRNA increased (P < 0.05) 2.2 +/- 0.5- and 2.6 +/- 0.9-fold, respectively, when F-40 and F were compared with R. These data demonstrate that HSP72 mRNA increases progressively during acute cycling, suggesting that processes that take place throughout concentric exercise are capable of initiating a stress response.     

IL-6 activates HSP72 gene expression in human skeletal muscle

To determine whether the cytokine interleukin (IL)-6 induces heat shock protein (HSP) 72 gene expression in skeletal muscle, 18 healthy, young men had either a high dose of IL-6 (HiIL-6; n=6), low dose IL-6 (LoIL-6; n=6), or saline (CON; n=6) infused into one femoral artery for 3h. Muscle biopsies were obtained from the vastus lateralis of the infusion limb and samples were analyzed for HSP72 mRNA. In addition, blood samples were collected from the femoral vein of the infusion limb and analyzed for plasma IL-6. In CON, femoral vein IL-6 concentration remained at basal levels throughout the experiment but in both HiIL-6 and LoIL-6, femoral vein IL-6 concentrations were markedly elevated (P<0.05). HSP72 gene expression did not increase above resting levels in CON. In contrast, in both HiIL-6 and LoIL-6, HSP72 mRNA increased (P<0.05) 2.5- and 2.3-fold, respectively after 30min of infusion and remained elevated (P<0.05) for 24h following infusion. These data demonstrate that IL-6 can rapidly induce HSP72 gene expression in human skeletal muscle.     

Augmented expression of HSP72 protein in normal human fibroblasts irradiated with ultraviolet light.

Normal human fibroblasts synthesized heat shock protein (HSP) 72 constitutively and its expression was augmented 6 hours after UV irradiation. Maximum induction of HSP72 was obtained at 12 hours and HSP72 showed a punctuated distribution in nucleus. While unscheduled DNA synthesis was almost completed 12 hours after UV irradiation, the S phase fraction decreased immediately and recovered after 6 hours. Thus, HSP72 augmentation was occurred coincidentally with the recovery of S phase, and suggested that HSP72 had some function during the recovery of DNA replication inhibited after UV irradiation.     

Hepoxilin A3 induces heat shock protein (HSP72) expression in human neutrophils

In this paper we show that hepoxilin A3 induces the expression of heat shock protein expression in human neutrophils at a concentration of 100 nM using Western blotting techniques employing the use of a commercial monoclonal antibody to HSP72. No regiospecificity was observed as the 8S enantiomer of HxA3 was as active as the 8R enantiomer of HxA3. Comparison of the effects of HxA3 with 12S-HETE and PGA1 indicated that HxA3 was as effective as 12S-HETE although PGA1 was essentially inactive at the same concentration used for these 12-lipoxygenase products.     

Estrogen attenuates HSP 72 expression in acutely exercised male rodents

Estrogen has been shown to reduce post-exercise skeletal muscle damage. Exercise-induced muscle damage may be a factor in the elevated post-exercise expression of heat-shock proteins (HSPs). Thus, the present investigation was conducted in order to examine the influence of estrogen on post-exercise levels of HSP 72 and heat-shock cognate, HSC 73, in male and female rodents. Prior to an acute bout of treadmill running, male and female Sprague-Dawley rats received daily injections of either 40 microg x kg(-1) of beta-estradiol 3-benzoate or olive oil vehicle for 2 weeks. A two- to fourfold reduction in post-exercise HSP 72 content was observed in the heart, liver, lung and red and white vastus muscles of estradiol-treated males compared with their vehicle-injected counterparts (P < 0.05). Compared to the males, the females had significantly lower post-exercise HSP 72 levels which were not affected by estradiol supplementation. Moreover, estradiol administration in male rodents resulted in a HSP response similar to that of females following exercise. Thus, the results of the present investigation suggest that estrogen is the factor responsible for the observed differences in post-exercise HSP 72 levels between males and females.     

Volatile organic compounds cytotoxicity and expression of HSP72, HSP90 and GRP78 stress proteins in cultured human cells

The aim of this study was to determine whether overexpression of stress proteins (SPs) could be a sensitive biomarker for cell injury due to exposure to low doses of volatile organic compounds (VOCs) such as benzene, ethylbenzene, toluene, xylene, and chlorinated derivatives (ClB). Sublethal and cytotoxic threshold concentrations of the VOCs were determined by studying the growth rate of normal (fibroblasts) or tumor-derived human cell lines (A549, HepG2) exposed for 4 days to VOCs. Changes in SP expression as a function of concentrations were investigated by Western blotting.VOC toxicity was found to be correlated with their degree of chlorination and their hydrophobicity. Cytotoxic threshold concentrations (no-observed effect concentration, NOEC) were found to be similar for the three cell lines. It was observed that using a mixture of VOCs, each of them at concentration below the NOEC, resulted in an actual toxicity to the cells. This finding reveals a synergistic effect and should be taken into account when assessing threshold risk and exposure limit values in the worker's environment when several pollutants may be present. HSP72 and HSP90 expression levels were not affected whereas GRP78 expression was increased by all the VOCs. Taking into account the specific molecular function of GRP78, it suggests that VOC exposure results in misfolded or underglycosylated protein accumulation in the endoplasmic reticulum. GRP78 overexpression was closely related to the magnitude of growth inhibition due to increasing concentrations of each VOC. The overexpression was found to be significant for concentrations 5 to 30 times higher than NOEC, indicating that, under our experimental conditions, GRP78 expression cannot be considered as a sensitive biomarker of exposure to environmental VOCs.     

Expression of HSP72 in the gastric mucosa is regulated by gastric acid in rats-correlation of HSP72 expression with mucosal protection

BACKGROUND AND AIM: The real mechanism of adaptive cytoprotection in the gastric mucosa is not well established. In the present study, we investigated the effect of acid suppressing agents on a 72-kDa heat shock protein (HSP72) expression, which is known as endogenous cytoprotective factor, in the gastric mucosa. Also, the association of gastric mucosal protective function against HCl-challenge was compared between HSP72-induced and -reduced group. MATERIALS AND METHODS: Expression of HSP72 was measured by Western blotting in the gastric mucosa before and after administration of famotidine or omeprazole. The gastric mucosal protective function against 0.6 N HCl was compared between control group and HSP72-reduced group. Also, the effect of increased expression of gastric HSP72 by additional administration of zinc sulfate or zinc L-carnosine, which is known as HSP72-inducer, on mucosal protective function was studied. RESULTS: HSP72 expression in the gastric mucosa was reduced by acid suppressing agents. The lowest expression level of HSP72 was observed 12 h (famotidine, H2-receptor antagonist) or 48 h (omeprazole, proton pump inhibitor) after administration. The gastric mucosal protective ability against 0.6 N HCl was also reduced when HSP72 expression was decreased by famotidine or omeprazole. This phenomenon was reversed by HSP72 induction by additional administration of zinc derivatives. CONCLUSION: Our results might indicate that the expression of HSP72 in the gastric mucosa is physiologically regulated by gastric acid, and that HSP72 induction could be important in view of mucosal protection especially when HSP72 expression is reduced by administration of acid suppressing agents such as proton pump inhibitor or H2 receptor antagonist.     

Daily variations in platelet aggregation and adhesion in healthy subjects.

Platelet aggregation is known to show a morning rise. The present study was undertaken to examine whether platelet aggregation and adhesion show a peak in the afternoon. Platelet aggregation stimulated by 4 microM of adenosine diphosphate, 1 micrograms/ml of collagen, 4 microM of epinephrine and 0.5 mM of arachidonic acid, and platelet adhesion determined by platelet retention on a glass bead column were measured for a period of 28-hour with an interval of 4 hours in 6 healthy subjects. Platelet aggregation in response to 4 different aggregating agents showed a bimodal daily variation with peaks in the morning and in the afternoon. However platelet adhesion only showed a peak in the morning. Previous studies have demonstrated the increases in the onset of acute myocardial infarction (MI) in the morning and afternoon periods. As enhanced platelet aggregation is involved in the development of acute MI, the present study suggests that the rise in platelet aggregation contributes to the increase in acute MI in the morning and in the afternoon. The present study suggests that the enhancement of platelet adhesion, which might be involved in thromboembolic events, may be another triggering factor for the onset of acute MI.     

Small doses of capsaicin given intragastrically inhibit gastric basal acid secretion in healthy human subjects.

Although the direct inhibitory effect of small dose of capsaicin on gastric secretory responses was proved in animal observations, the role of capsaicin-sensitive afferent nerves (CSAN) and the effect of capsaicin applied in small and high doses on gastric secretion in human has not been clarified yet. In this study we investigated the influence of different small doses (100-800 microg) of capsaicin given intragastrically through an orogastric tube on gastric basal secretory responses in 10 healthy human subjects. Gastric basal secretory responses (volume, H+-concentration, H+-output) were measured from the suctions of gastric juice for a 1-h period. It has been found that: a) capsaicin dose-dependently inhibited the volume and H+-output of gastric juice; b) ID50 was found to be about 400 microg for capsaicin on gastric acid secretion; c) the time interval for capsaicin-induced gastric inhibition existed for about 1 h indifferently from the higher dose (800 microg) of capsaicin given after. It has been concluded that the capsaicin (given in small doses) inhibits the gastric basal acid output via stimulation of the inhibition of capsaicin sensitive afferent nerves.     

Endogenous expression and developmental changes of HSP72 in rat skeletal muscles

The purpose of the present study was to determine whether endogenous factor(s) contributes to the expression of heat shock proteins (HSPs) during the early developmental stages of rat skeletal muscles. HSP72 was expressed in both the soleus and plantaris muscles at embryonic day 22 (E22). On the basis of myosin heavy chain (MHC) immunohistochemistry, HSP72 was specifically expressed in slow type I fibers in both muscles. These slow fibers were observed throughout the entire cross section of the soleus muscle and only in the deep region (close to the bone) of the plantaris muscle. These results indicate that the expression of HSP72 is related to endogenous factors associated with type I fibers, because E22 rats have minimal exogenous influences and the soleus and plantaris muscles of E22 rats have similar metabolic and contractile profiles at this stage of development. We then examined the changes in HSP72 and heat shock cognate (HSC) 73 in the same two muscles from E22 to postnatal day 56 via Western blotting. The level of HSP72 in the soleus muscle gradually increased in parallel with the increment in the type I MHC isoform. Compared with the soleus, only a small amount of HSP72 could be detected in the plantaris muscle throughout the developmental period. For both muscles, HSC73 reached levels observed in adult muscles at postnatal day 3, and these levels were unchanged thereafter. These results indicate that the expression of HSP72, but not HSC73, is influenced by both endogenous and exogenous factors during the embryonic and early developmental periods.     

Effects of alprazolam and clonidine on carbon dioxide-induced increases in anxiety ratings in healthy human subjects

In order to investigate possible neurobiologic mechanisms underlying carbon dioxide-induced anxiety, the effects of oral alprazolam 0.75 mg and intravenous clonidine 2 mcg/kg on CO2-induced increases in ratings of subjective anxiety, pulse rate, and ventilation were measured in healthy human subjects. Pretreatment with alprazolam but not with clonidine significantly reduced the CO2-induced increase in ratings of anxiety. Neither drug altered CO2-induced increases in pulse rate or ventilatory responses. Clonidine did produce potent sedative and hypotensive effects. The behavioral data suggest that the mechanisms through which CO2 induces anxiety-like effects involve neural systems regulated by benzodiazepine receptors and, secondly, that they appear not to require normal functioning of noradrenergic systems. Carbon dioxide may provide a useful model system for identification of new drugs with anxiolytic properties.     

Sprint interval running increases insulin sensitivity in young healthy subjects

High intensity cycling training increases oxidative capacity in skeletal muscles and improves insulin sensitivity. The present study compared the effect of eight weeks of sprint interval running (SIT) and continuous running at moderate intensity (CT) on insulin sensitivity and cholesterol profile in young healthy subjects (age 25.2 ± 0.7; VO(2max) 49.3 ± 1.2 ml·kg(-1)·min(-1)). SIT and CT increased maximal oxygen uptake by 5.3 ± 1.8 and 3.8 ± 1.6%, respectively (p < 0.05 for both). Oral glucose tolerance test (OGTT) was performed before and 60 h after the last training session. SIT, but not CT, reduced glucose area under curve and improved HOMA β-cell index (p < 0.05). Insulin area under curve did not decrease significantly in any group. SIT, but not CT, reduced LDL and total cholesterol. In conclusion, sprint interval running improves insulin sensitivity and cholesterol profile in healthy subjects, and sprint interval running may be more effective to improve insulin sensitivity than continuous running at moderate intensity.     

Prostaglandin E2 signalling pathway in human T lymphocytes from healthy and conjunctiva basal cell carcinoma-bearing subjects

Prostaglandin E-induced signal transduction pathways in human T cells from healthy and uveal melanoma-bearing subjects were studied. Transfection experiments showed that PGE2 was able to phosphorylate and activate the fusion trans-activator of the cAMP responsive element-binding protein (CREB). Phosphorylation was at least partially mediated by protein kinase A, as evidenced by the effects of specific kinase inhibitors. Western blotting experiments, which were performed to identify the CREB/ATF2 family members involved in the response to PGE2, revealed a modulation of proteins CREB1, CREB2 and ATF2 and phosphorylation of the 43 kDa form of CREB. Experiments of immunoprecipitation with CREB-binding protein (CBP) demonstrated that, after PGE2 treatment, all of the CREB/ATF isoforms studied, as well as the phosphorylated form of CREB (p-CREB), interacted with CBP. In basal conditions, T cells from patients with conjunctiva basal cell carcinoma showed the presence of p-CREB, which coimmunoprecipitated with CBP. CREB phosphorylation did not modify after PGE2 treatment whereas the p-CREB fraction bound to CBP increased in a delayed manner compared to normal subjects.     

Asymptomatic Helicobacter pylori infection increases asymmetric dimethylarginine levels in healthy subjects

BACKGROUND: Chronic infections have been demonstrated to be early factors of atherosclerosis and cardiovascular diseases, and their relevance increases when they are caused by agents with extremely broad spectrum of disease outcome such as Helicobacter pylori. The consequent endothelial impairment leads to a reduced bioavailability of nitric oxide. Increasing evidences have pointed out that the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine, defined as a risk factor for cardiovascular disease, may increase in infections and plays an important role impairing the vascular functions of the endothelium. Starting from these findings, we aim to investigate whether H. pylori may affect asymmetric dimethylarginine levels. MATERIALS AND METHODS: The study was carried out on a group of 186 subjects (age 46.2 +/- 14.9 years). We evaluated asymmetric dimethylarginine, symmetric dimethylarginine, L-arginine, presence of H. pylori by 13C-urea breath test, and the main parameters of glyco and lipo metabolic balance. RESULTS: Increased levels of asymmetric dimethylarginine were found in H. pylori-positive subjects with respect to H. pylori-negative subjects (0.46 x/ / 1.13 versus 0.42 x/ / 1.23 mol/l, p < .001, respectively). No differences were detected in L-arginine levels between the two groups. Multiple regression analysis performed in H. pylori-positive subjects and H. pylori-negative subjects showed profound differences in the variables related to asymmetric dimethylarginine (R2 = 66.9%, p < .01 versus 34.3%, p < .01, respectively) and symmetric dimethylarginine (R2 = 39.2%, p < .01 versus 20.6%, p = .09, respectively) levels. CONCLUSIONS: Our data clearly demonstrate that H. pylori infection increases asymmetric dimethylarginine levels. Moreover, this infection causes a profound metabolic modification that alters the role of the known determinants of asymmetric dimethylarginine levels. We conclude that H. pylori infection must be taken into account as a cause of increased asymmetric dimethylarginine levels and that the eradication of H. pylori may therefore lead to a decrease in asymmetric dimethylarginine levels, which is a further reason for the reduction of the risk for cardiovascular disease in this large portion of population.     

Cognitive training increases platelet PLA2 activity in healthy elderly subjects

Phospholipases A(2) (PLA(2)) are ubiquitous enzymes involved in membrane fatty acid metabolism and intracellular signalling. Recent studies have shown that PLA(2) subtypes are implicated in the modulation of pathways related to memory acquisition and retrieval. We investigated the effects of cognitive training on platelet PLA(2) activity in healthy elderly individuals. Twenty-three cognitively unimpaired older adults were randomly assigned to receive memory training or standard outpatient care only. Both groups were cognitively assessed by the same protocol, and the experimental group (EG) underwent a four-session memory training intervention. Pre- and post-test measures included prose and list recall, WAIS-III digit symbol, strategy use measures and platelet PLA(2) group activity. After cognitive training, patients in the EG group had significant increase in cytosolic, calcium-dependent PLA(2) (cPLA(2)), extracellular (or secreted), calcium-dependent PLA(2) (sPLA(2)), total platelet PLA(2) activity, and significant decrease in platelet calcium-independent PLA(2) (iPLA(2)) activity. Our results suggest that memory training may have a modulating effect in PLA(2)-mediated biological systems associated with cognitive functions and neurodegenerative diseases.     

Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.     

Effects of oral vitamin C on monocyte: endothelial cell adhesion in healthy subjects

Monocyte recruitment and retention in the vasculature is influenced by oxidative stress and is involved in cardiovascular disease (CVD). Individuals with low plasma ascorbate are at elevated risk of CVD. It is unknown whether vitamin C supplementation affects monocyte adhesion to endothelial cells (ECs) in healthy non-smokers. In a randomised double-blind crossover study the effect of vitamin C supplementation (six weeks, 250 mg/day) was determined in subjects with normal (HIC) and below average (LOC) plasma vitamin C concentration at baseline (mean=67 microM, n=20, mean=32 microM, n=20, respectively). LOC subjects showed 30% greater monocyte adhesion to ECs. This was significantly reduced by 37% (P<0.02) following vitamin C supplementation to levels of HIC monocyte adhesion. No differences in plasma malondialdehyde concentrations were observed between groups or after supplementation. In conclusion, vitamin C supplementation normalises monocyte adhesion in subjects with low plasma vitamin C (LOC). This process may be related to a direct effect on monocytes, independent of lipid peroxidation.     

An increased carbon dioxide production in transient hypoxia in healthy subjects at rest

Indices of pulmonary gas exchange and heart rate (HR) have been measured in 24 healthy subjects not adapted to hypoxia after hypoxic aerial mixture (HAM) (17, 15, 13 vol % of oxygen) respiration for 15 min. Using group data analysis, it has been shown that hypoxia under the conditions of inhalation of 17 and 15 vol % of O₂ caused no significant changes. Hypoxia under the conditions of 13 vol % of O₂ inhalation is a threshold one, when ventilation (SpO₂) drops below 85%. A significant increase in the lung ventilation (Ve) (10–14%, p < 0.05) and HR (11–15%, p < 0.05) have been observed in this case. Hyperpnea was accompanied by an increase in the oxygen uptake rate by 10% and carbon dioxide release rate (10–18%, p < 0.05). On the contrary, individual data analysis showed changes in the pulmonary gas exchange indices in 90% of subjects in the case of inhalation of 17 vol % of O₂ HAM. Four response types have been found: ventilation (increase in lung ventilation), hypoxic hypometabolism (decrease in oxygen consumption rate), and mobilization response (increase in oxygen utilization in the lungs), and anaerobic response, which is expressed in an increase in the carbon dioxide release rate along with an increase in the respiratory quotient. All these responses are of an individual type, but the ventilation response is developed in response to hypoxia caused by inhalation of 13 vol % of O₂ HAM and a decrease in SpO₂ below 85% in more than 60% of cases.