In vitro heat shock of human monocytes results in a proportional increase of inducible Hsp70 expression according to the basal content

Heat shock proteins play an important role as molecular chaperones of the cell. Inducible heat shock protein 70 is rapidly synthesised in response to numerous stressors and monocytes are sensitive to changes in core temperature resulting in a circadian variation of Hsp70 expression. Monocytes were isolated via density centrifugation from nine healthy male volunteers at 5 am, 1 pm and 9 pm, representing the nadir (5 am), peak (9 pm) and intermediate (1 pm) of Hsp70 expression in the 24-h cycle. Analysis of freshly isolated monocytes for Hsp70 expression confirmed Hsp70 levels at the three selected time points. Monocytes were subjected to in vitro heat shock at 40°C (±0.1) for 90 min with a 90 min 37°C (±0.1) exposure acting as a control. A significant increase in Hsp70 was observed at 5 am (p < 0.001) and 1 pm (p = 0.028) at 40°C when compared to 37°C but not at 9 pm (p = 0.19). A significant increase was also observed from the basal levels of Hsp70, measured on freshly isolated monocytes and the levels detected after heat shock at 40°C at 5 am (p < 0.001) and 1 pm (p = 0.001), which was not observed at 9 pm (p = 0.15). Furthermore, a significant correlation was observed in the heat shock response at 40°C and that obtained at 37°C (p < 0.001). In conclusion, the heat shock response in monocytes is directly proportional to the amount of Hsp70 present in the cells and the stress response may be much higher at different times of the day.     

Starvation reduces the heat shock protein responses in white sturgeon larvae

This study investigates the responses of white sturgeon larvae (Acipenser transmontanus) to starvation and thermal stress, through the measurement of nutritional status (i.e. growth performances) and cellular biomarkers: heat shock proteins (Hsp) 70 and 90. White sturgeon larvae (25 day post hatch; initial weight 179.0 ± 5.1 mg) were fed (20% body weight per day) or starved for 24, 48 or 72 hrs. Every 24 hrs, five larvae from each of the starved or fed treatment replicates were exposed to heat shock resulting from an increase in water temperature from 19°C to 26°C, at a rate of 1°C per 15 min, and maintained at 26°C for 4 hrs. No mortality was observed in this study. Starvation significantly (p < 0.05) decreased the body weight and body contents of energy, protein, and lipid of the experimental larvae, compared to the fed larvae. Heat shock induced the expressions of Hsp70 and Hsp90 in both the fed and starved group; however, starvation reduced the induction at all sampling points. The current study demonstrates that poor larval nutritional status, assessed by the aforementioned parameters, reduced heat shock responses to thermal stress, as measured by heat shock protein levels. Furthermore, Hsp70 and 90 are more sensitive to heat shock and starvation, respectively. This may be, in part, a result of the different functioning of the heat shock proteins in cellular stress response and warrants further study.     

The effect of hyperbaric oxygen preconditioning on heat shock protein 72 expression following in vitro stress in human monocytes

Hyperbaric oxygen (HBO) is thought to confer protection to cells via a cellular response to free radicals. This process may involve increased expression of heat shock proteins, in particular the highly inducible heat shock protein 72 (Hsp72). Healthy male volunteers (n = 16) were subjected to HBO for 1 h at 2.8 ATA. Inducible Hsp72 expression was measured by flow cytometry pre-, post- and 4 h-post HBO. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood via density centrifugation pre-, post- and 4 h post-HBO. PBMC were then subjected to an in vitro heat shock at 40°C or hypoxia at 37°C (5% O₂) with a control at 37°C. Cells were then analysed for Hsp72 expression by flow cytometry. Monocytes showed no significant changes in Hsp72 expression following HBO. No detectable Hsp72 was seen in lymphocytes or neutrophils. Following in vitro hypoxic exposure, a significant increase in Hsp72 expression was observed in monocytes isolated immediately post- (p = 0.006) and 4 h post-HBO (p = 0.010) in comparison to control values. HBO does not induce Hsp72 expression in PBMC. The reported benefits of HBO in terms of pre-conditioning are not due to inducement of Hsp72 expression in circulating blood cells, but may involve an enhancement of the stress response.     

Increased circulating heat shock protein 70 levels in pregnant asthmatics

Asthma is one of the most common diseases complicating pregnancy and represents a risk factor for several maternal and perinatal complications. The natural history of asthma is known to change in pregnancy, but very few data are available in the terms of pathomechanism of this change during gestation. Circulating heat shock protein 70 (Hsp70) levels are decreased in healthy pregnancy, which might reflect physiological immunotolerance. The aim of our study was to determine the serum levels of Hsp70 in asthmatic women during gestation. Forty pregnant women with bronchial asthma and 40 healthy pregnant women matched for maternal and gestational age were involved in this case-control study. Serum Hsp70 levels were measured using the ELISA Kit of R&D Systems. Spirometry and oxygen saturation measurements were performed in asthmatic patients. In asthmatic pregnant women, an increase of serum Hsp70 levels was observed compared to healthy pregnant women (median (25–75 percentile): 0.44 ng/ml (0.36–0.53) versus 0.21 ng/ml (0–0.27), p < 0.001). Fetal birth weight of asthmatic mothers was significantly smaller than of healthy controls, but in the normal range (3,230 g (2,690–3,550) versus 3,550 g (3,450–3,775), p < 0.05). A statistically significant negative correlation between maternal age and serum Hsp70 concentrations (Spearman R = −0.48, p = 0.0018) and a significant positive correlation between gestational age and serum Hsp70 levels (Spearman R = 0.83, p < 0.001) were detected in healthy pregnant women. In conclusion, this study proves an elevation of circulating Hsp70 levels during asthmatic pregnancy compared to healthy pregnant women. However, further studies are warranted to determine the role of circulating Hsp70 in the pathogenesis of maternal and perinatal complications of asthma in pregnancy.     

First-trimester plasma extracellular heat shock proteins levels and risk of preeclampsia

Preeclampsia (PE) occurs annually in 8% of pregnancies. Patients without risk factors represent 10% of these. There are currently no first-trimester biochemical markers that accurately predict PE. An increase in serum 60- and 70-KDa extracellular heat shock proteins (eHsp) has been shown in patients who developed PE at 34 weeks. We sought to determine whether there is a relationship between first-trimester eHsp and the development of PE. This was a prospective cohort study performed at a third level hospital in Mexico City from 2019 to 2020. eHsp levels were measured during the first-trimester ultrasound in singleton pregnancies with no comorbidities. First-trimester eHsp levels and biochemical parameters of organ dysfunction were compared between patients who developed preeclampsia and those who did not. All statistical analyses and model of correlation (r) between eHsp and clinical parameter were performed using bootstrapping R-software. p-values <0.05 were considered significant. The final analysis included 41 patients. PE occurred in 11 cases. eHsp-60 and eHsp−70 were significantly higher at 12 weeks in patients who developed PE (p = 0.001), while eHsp-27 was significantly lower (p = 0.004). Significant differences in first-trimester eHsp concentration suggest that these are possible early biomarkers useful for the prediction of PE.     

Influence of Therapy with Metformin on the Concentration of Certain Divalent Cations in Patients with Non-insulin-Dependent Diabetes Mellitus

Research was performed on a group of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM), who never received antidiabetic medication before, and on a group of 17 healthy adults. The patients were administered treatment with metformin, 1,000 mg/day. Plasmatic and urinary concentration of magnesium have been measured, copper and zinc along with the concentrations of glucose, HDL, LDL, cholesterol, tryglicerides, HbA1c, and total erythrocyte magnesium, in advance and after 3 months of treatment. Data showed significant differences in the NIDDM group vs the control group: for plasma magnesium—1.95 ± 0.19 vs 2.20 ± 0.18 mg/dl, p < 0.001; urine magnesium—237.28 ± 34.51 vs 126.25 ± 38.22 mg/24 h, p < 0.001; erythrocyte magnesium—5.09 ± 0.63 vs 6.38 ± 0.75 mg/dl, p < 0.001; plasma zinc—67.56 ± 6.21 vs 98.41 ± 20.47 μg/dl, p < 0.001; urine zinc—1,347.54 ± 158.24 vs 851.65 ± 209.75 μg/24 h, p < 0.001; plasma copper—111.91 ± 20.98 vs 96.33 ± 8.56 μg/dl, p < 0.001; and urine copper—51.70 ± 23.79 vs 36.00 ± 11.70 μg/24 h, p < 0.05. Treatment with metformin for 3 months modified significant erythrocyte magnesium—5.75 ± 0.61 vs 5.09 ± 0.63 mg/dl, p < 0.001 and urine magnesium—198.27 ± 27.07 vs 237.28 ± 34.51 mg/24 h, p < 0.001, whereas it did not modify significant the plasmatic and urinary concentration of the other cations. The erythrocyte magnesium concentration was inversely correlated with HbA1c (r = −0.438, p = 0.015). The plasma level of copper was positively correlated with HbA1c (r = 0.517, p < 0.003), tryglicerides (r = 0.534, p < 0.003), and cholesterol (r = 0.440, p < 0.05), and the plasma level of zinc was inversely correlated with glycemia (r = −0.399, p = 0.029). Our data show a significant action of metformin therapy, by increasing the total intraerythrocyte magnesium concentration and decreasing the urinary magnesium elimination, positively correlated with the decrease of glycemia and HbA1c in NIDDM patients.     

Interaction of serum 70-kDa heat shock protein levels and <Emphasis Type="Italic">HspA1B</Emphasis> (+1267) gene polymorphism with disease severity in patients with chronic heart failure

Background Circulating heat shock protein 70 (Hsp70) is present in the circulation of healthy individuals and in patients with various disorders, including chronic heart failure (CHF). However, the source and routes of release of Hsp70 is only partially characterised in clinical samples. Aims The purpose of this study was to study the clinical and biological correlates of Hsp70 in a CHF population and, for the first time, to investigate the association of HspA1B (also known as Hsp70-2) +1267 alleles with serum Hsp70 levels. Methods A total of 167 patients (123 men, 44 women) with <45% left ventricular ejection fraction (LVEF) were enrolled; serum Hsp70 level was determined by enzyme-linked immunosorbent assay and HspA1B +1267 polymorphism by polymerase chain reaction–restriction fragment length polymorphism. Results Increased Hsp70 levels were present in patients with severe CHF (NYHA III–IV) as compared to the group of NYHA I–II (p = 0.003). Hsp70 levels correlated with LVEF, NT-proBNP, serum bilirubin, aspartate aminotransferase, alanine aminotransferase, γGT (p < 0.05) concentrations in patients with severe CHF, although no correlation was observed between Hsp70 and CRP, TNF-α, or IL-6. HspA1B allele G was associated with higher Hsp70 levels (p = 0.001) in patients in NYHA IV class as compared to carriers of allele A. Conclusions Serum Hsp70 levels were associated with disease severity in heart failure patients. An interaction with the presence of HspA1B +1267 allele G was observed for Hsp70 concentrations. Hsp70 correlates with markers of heart function and hepatic injury, but not with signs of inflammation.     

The effect of 15 consecutive days of heat–exercise acclimation on heat shock protein 70

The purpose of this study was to investigate the alterations in serum heat shock protein (Hsp) 70 levels during a 15-consecutive-day intermittent heat–exercise protocol in a 29-year-old male ultra marathon runner. Heat acclimation, for the purpose of physical activities in elevated ambient temperatures, has numerous physiological benefits including mechanisms such as improved cardiac output, increased plasma volume and a decreased core temperature (T _c). In addition to the central adaptations, the role of Hsp during heat acclimation has received an increasing amount of attention. The acclimation protocol applied was designed to correspond with the athlete’s tapering period for the 2007 Marathon Des Sables. The subject (VO₂max = 50.7 ml·kg⁻¹·min⁻¹, peak power output [PPO] = 376 W) cycled daily for 90 min at a workload corresponding to 50% of VO₂max in a temperature-controlled room (average WBGT = 31.9 ± 0.9°C). Venous blood was sampled before and after each session for measurement of serum osmolality and serum Hsp70. In addition, T _c, heart rate (HR) and power output (PO) was measured throughout the 90 min to ensure that heat acclimation was achieved during the 15-day period. The results show that the subject was successfully heat acclimated as seen by the lowered HR at rest and during exercise, decreased resting and exercising T _c and an increased PO. The heat exercise resulted in an initial increase in Hsp70 concentrations, known as thermotolerance, and the increase in Hsp70 after exercise was inversely correlated to the resting values of Hsp70 (Spearman’s rank correlation = −0.81, p < 0.01). Furthermore, the 15-day heat–exercise protocol also increased the basal levels of Hsp70, a response different from that of thermotolerance. This is, as far as we are aware, the first report showing Hsp70 levels during consecutive days of intermittent heat exposure giving rise to heat acclimation. In conclusion, a relatively longer heat acclimation protocol is suggested to obtain maximum benefit of heat acclimation inclusive of both cellular and systemic adaptations.     

Inducible and constitutive heat shock gene expression responds to modification of Hsp70 copy number in Drosophila melanogaster but does not compensate for loss of thermotolerance in Hsp70 null flies

Background  

The heat shock protein Hsp70 promotes inducible thermotolerance in nearly every organism examined to date. Hsp70 interacts with a network of other stress-response proteins, and dissecting the relative roles of these interactions in causing thermotolerance remains difficult. Here we examine the effect of Hsp70 gene copy number modification on thermotolerance and the expression of multiple stress-response genes in Drosophila melanogaster, to determine which genes may represent mechanisms of stress tolerance independent of Hsp70.     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

The effects of the new guidance ‘take your sleeping pills 7 h before your wake-up time’: a pilot study

The aim of this study is to investigate the effectiveness and practicality of the newly proposed guidance to take hypnotics 7 h before one’s usual wake-up time, compared to the conventional guidance to do so 30 min before bedtime. Subjects with primary insomnia who were not satisfied with their hypnotics were included between November 2014 and October 2015. Participants were instructed to take their own sleeping pills 7 h before their usual wake-up times, and sleep-related time variables and symptom questionnaires were assessed at baseline and after 2 weeks. Among 32 subjects, 23 patients were successfully followed up. Adhering to the said 7-h instruction, 73.9 % (n = 17) were satisfied with their sleeping pills. Mean hypnotics administration time was significantly delayed from 9:32 p.m. ± 0:58 to 10:55 p.m. ± 0:46 (p < 0.001), duration from pills to wake-up time (PTW) was shortened from 9.0 ± 1.1 to 7.1 ± 0.8 h (p < 0.001), and sleep latency (p = 0.023) was significantly shortened. Scores of ISI and PSQI significantly improved (p < 0.001), and the improvements of ISI and PSQI were positively correlated with the shortened sleep latency (r = 0.49, p < 0.05) and PTW (r = 0.54, p < 0.05), respectively. Advising patients to take hypnotics about 7 h before their usual wake-up time could increase the level of satisfaction with their original medication as is. In incorporating concepts of cognitive-behavioral therapy, this recommendation may serve as a simple but considerably useful guidance on the proper timing for taking prescribed sleeping pills.

     

Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treatment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and massive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42°C for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and nontreated cells (67.69% ± 8.35% versus 58.79% ± 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% ± 8.22% versus 28.27% ± 6.32%, P < 0.01) and more than threefold at 120 h (26.33% ± 5.54% versus 8.79% ± 2.51%, P < 0.01). Histological analysis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock pretreatment of myoblast in vitro is a simple and effective way to enhance cell survival after transplantation in pig. It might represent a potential method to overcome the limitations of MT treatment.     

Increased serum HSP70 levels are associated with the duration of diabetes

The evolutionary conserved family of heat shock proteins (HSP) is responsible for protecting cells against different types of stress, including oxidative stress. Although the levels of HSPs can be readily measured in blood serum, the levels of HSP70 in patients with different durations of diabetes have not been studied before. We quantified serum HSP70 levels in a healthy control group (n = 36) and two groups of type 2 diabetic patients, defined as newly diagnosed diabetes (n = 36) and patients with diabetes duration of more than 5 years (n = 37). The clinical characteristics and biochemical parameters were evaluated in the studied population. We found that serum HSP70 levels were significantly higher in patients with diabetes when compared with controls (p < 0.001) and it was higher in patients with disease for more than 5 years than in newly diagnosed patients (p < 0.001). Serum HSP70 was inversely correlated with fasting blood sugar in patients with diabetes for more than 5 years (r = −0.500, p = 0.002), positively correlated with the history of hypertension in newly diagnosed patients (p < 0.001), and positively correlated with age in patients with diabetes (r = 0.531, p = 0.001). Serum level of HSP70 is significantly higher in patients with diabetes and correlates with the duration of disease. Higher HSP70 in prolonged diabetes versus newly diagnosed diabetes may be an indicator of metabolic derangement in the course of diabetes.     

Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal

We studied various aspects of heat‐shock response with special emphasis on the expression of heat‐shock protein 70 (hsp70) genes at various levels in two congener species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal which show striking differences in their vertical distribution and thermal tolerance. Although both the species studied demonstrate high constitutive levels of Hsp70, the thermotolerant E. cyaneus exhibited a 5‐fold higher basal level of Hsp70 proteins under normal physiological conditions (7 °C) and significantly lower induction of Hsp70 after temperature elevation compared with the more thermosensitive E. verrucosus. We isolated the hsp70 genes from both species and analysed their sequences. Two isoforms of the cytosolic Hsp70/Hsc70 proteins were detected in both species under normal physiological conditions and encoded by two distinct hsp/hsc70 family members. While both Hsp70 isoforms were synthesized without heat shock, only one of them was induced by temperature elevation. The observed differences in the Hsp70 expression patterns, including the dynamics of Hsp70 synthesis and threshold of induction, suggest that the increased thermotolerance in E. cyaneus (compared with E. verrucosus) is associated with a complex structural and functional rearrangement of the hsp70 gene family and favoured the involvement of Hsp70 in adaptation to fluctuating thermal conditions. This study provides insights into the molecular mechanisms underlying the thermal adaptation of Baikal amphipods and represents the first report describing the structure and function of the hsp70 genes of endemic Baikal species dwelling in thermally contrasting habitats.     

Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treat-ment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and mas-sive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42℃ for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and non- treated cells (67.69% ± 8.35% versus 58.79% ± 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% ± 8.22% versus 28.27% ± 6.32%, P < 0.01) and more than threefold at 120 h (26.33% ± 5.54% versus 8.79% ± 2.51%, P < 0.01). Histological analy-sis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock pretreatment of myoblast in vitro is a simple and effective way to enhance cell survival after transplantation in pig. It might represent a potential method to overcome the limitations of MT treat-ment.     

COVID-19: comparative clinical features and outcome in 114 patients with or without pneumonia (Nord Franche-Comte Hospital,France)

COVID-19 patients (n = 114) were included (55 patients with pneumonia (group P) and 59 without pneumonia (group NP). Patients in group P were older (69 (±17) years vs 46 (±16); p < 0.001) with a male predominance (58.2% vs 27.1%; p < 0.001). The symptoms which were statistically more frequents in patients with pneumonia were fever ≥ 38 °C (93% vs 70%; p = 0.002) and dyspnea (73% vs 22%; p < 0.001). Symptoms such as facial headache (42% vs 15%; p = 0.001), sore throat (39% vs 16%; p = 0.007), dysgeusia (61% vs 33%; p = 0.003), anosmia (63% vs 31%; p = 0.001) were statistically more frequents in patients without pneumonia.     

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.     

Effect of allozyme heterozygosity on basal and induced levels of heat shock protein (Hsp70), in juvenile Concholepas concholepas (Mollusca)

Organisms cope physiologically with extreme temperature by producing heat shock proteins (HSPs). Expression of Hsp70 enhances thermal tolerance and represents a key strategy for ectotherms to tolerate elevated temperature in nature. Synthesis of these proteins, together with other physiological responses to elevated temperatures, increases energy demands. A positive association between multiple and single locus heterozygosity (MLH and SLH, respectively) and individual fitness has been widely demonstrated. In molluscs, MLH can decrease routine metabolic rates and improve energetic status. Juvenile Concholepas concholepas live in the intertidal zone and are constantly exposed to temperature fluctuations. Thus, these young individuals are exposed both to thermal risks and the large metabolic costs required to cope with thermal stress. We evaluated the effects of allozyme MLH and SLH on basal (control animals) and induced (stressed animals) levels of the Hsp70 in juveniles C. concholepas. Juveniles (n = 400) were acclimated at 16 °C for 2 weeks; then 100 animals were exposed to 24 °C (stress) and 100 were kept at 16 °C (control) for 2 and 7 days. The variability of 20 loci was analyzed by starch gel electrophoresis. For SLH effects we used 7 polymorphic loci. We quantified expression of Hsp70 by Western blot analyses. Hsp70 expression increased markedly (~ 90%) with temperature. We found a positive association between MLH and basal and induced levels of Hsp70 in the 2-day exposure experiment. Regardless of temperature, Hsp70 levels increased with MLH (r² = 0.7 and 0.9, for basal and induced levels, respectively) reaching maximal levels in juveniles with intermediate and high MLH levels (2 and 3 loci), and decreasing slightly (but not significantly) in juveniles with highest MLH (≥ 4 heterozygous loci). However, after 7 days of exposure to thermal stress, less heterozygous juveniles attained the same levels of Hsp70 than more heterozygous juveniles. Given the faster increment of Hsp70 in C. concholepas juveniles with intermediate-high levels of MLH, these individuals could be less affected by thermal stress in the intertidal zone. We found an association between specific loci genotype and higher Hsp70 levels (basal or induced). In comparison to homozygous juveniles, heterozygous juveniles for several loci showed higher Hsp70. However, these associations were not for the same loci in juveniles exposed to high temperature for 2 and 7 days. This suggests genotypic variation at some allozyme loci could be more important in the period of initial response to high temperature and others can be more important in the response to the chronic temperature stress.