Synthesis of heat shock proteins (HSP70) in blood leukocytes as a criterion of the resistance to stress injury

The resistance of August rats to ulceration of the gastric mucosa induced by acute emotional stress was higher than in Wistar rats. August rats exhibited not only more potent activation of the protective nitric oxide system and mobilization of the immune system, but also increased synthesis of cytoprotective heat shock proteins HSP70 in blood leukocytes under stress conditions. Our results indicate that HSP70 protein synthesis in blood leukocytes during stress reflects organism’s resistance to stress and, probably, to other adverse factors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 12, pp. 614–617, December, 2006     

Effect of Recombinant Heat Shock Protein 70 of Mycobacterial Origin on Cytotoxic Activity and Immunophenotype of Human Peripheral Blood Mononuclear Leukocytes

Recombinant heat shock protein of mycobacterial origin with a molecular weight 70 kDa in concentration 0.1 μg/ml in vitro activates cytotoxic activity of mononuclear lymphocytes from peripheral blood of healthy donors against K-562 human erythroblastic leukemia cells sensitive to natural killers. In other concentrations (1.0 μg/ml and 10 μg/ml) this heat shock protein did not signifi cantly affect functional activity of mononuclear leukocytes. Expression of CD4, CD25, CD16 and CD56 on membrane of mononuclear leukocytes was also studied.     

Inulinase Immobilization on Macroporous Anion-Exchange Resins by Different Methods

For evaluation of the stress-protective influence of delta-sleep inducing peptide we studied its effects on the system of heat-shock proteins in immune cells using the method of flow cytometry. The peptide affected the expression of heat-shock protein 70 kDa in cultured human myeloleukemia K562 cells. Delta-sleep-inducing peptide reduces accumulation of intracellular heat shock proteins 70 kDa in cells cultured under conditions of high density. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 1, pp. 40-42, January, 2009     

Comparative analysis of polypeptides in the heat shock proteins synthesized in the myocardium of Wistar and August rats

The use of two-dimensional electrophoresis to study the polypeptide composition of heat shock proteins 70 synthesized in the myocardium of Wistar and August rats in response to thermal stress and repetitive immobilization stress revealed interstrain differences in the composition of stimulated polypeptides in these proteins. The differences are mainly due to differential expression of the heat shock protein gene(s) encoding polypeptides with pI 6.2–6.0 in the two strains. The drastically reduced levels of the synthesis and/or accumulation of some heat shock proteins 70 in the hearts of August rats are associated with their failure to develop cardioprotective phenomena in response to repetitive immobilization stress. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 7, pp. 30–32, July, 1995 Presented by R. S. Karpov, Member of the Russian Academy of Medical Sciences     

Long-term exercises increase the concentration of HspBP1, a co-chaperone of 70-KDa heat shock protein

Extracellular concentration of heat shock protein (Hsp) with a molecular weight of 70 kDa (Hsp70) rapidly increases in the serum in response to stress and returns to the basal level during recovery. Further regulation of its blood concentration is unclear. A possible regulator is HspBP1, a protein binding Hsp70. Binding to ATPase domain of Hsp70, HspBP1 inactivates it, thus acting as a factor of nucleotide exchange. Blood sera from athletes were examined at the beginning and end of the last mesocycle of the training period by two-staged immunoaffinity test system. The concentration of HspBP1 increased with decreasing Hsp70 concentration under conditions of long-term training. Presumably, the dynamics of Hsp70 and HspBP1 concentrations can serve as the test for evaluating the adaptation potential.     

Intracellular and extracellular NO differentially modulates the stress response and apoptosis in macrophages exposed to the influence of biological or physical factors

We studied the role of extracellular and intracellular NO in the regulation of the stress response and apoptosis in macrophages of proinflammatory and antiinflammatory phenotypes under the influence of S. aureus and heat shock. Blockade of extracellular nitric oxide synthesis in cells with antiinflammatory phenotype inhibited the stress response induced by S. aureus and heat shock. The decrease in extracellular nitric oxide concentration around antiinflammatory macrophages potentiated the stress response induced by S. aureus, but had no effect on the stress response induced by heat shock. Hence, intracellular NO mediates the stress response induced by S. aureus and heat shock, while extracellular NO inhibits the stress response induced by S. aureus, but has no effect on the stress response induced by heat shock. In cells with antiinflammatory phenotype, intracellular NO plays an antiapoptotic role. S. aureus and heat shock did not cause apoptosis in macrophages with proinflammatory phenotype, while intracellular NO did not play a role in antiapoptotic activity of the proinflammatory phenotype. Extracellular NO synthesized by macrophages protects these cells from apoptosis induced by S. aureus and heat shock. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 6, pp. 614–618, June, 2007     

Extracellular heat shock proteins (eHSP70) in exercise: Possible targets outside the immune system and their role for neurodegenerative disorders treatment

The intracellular heat shock protein 70kDa (iHSP70) is a universal marker of stress protein whose expression is induced by different cell stressors, such as heat, metabolite deprivation, redox imbalances and also during physical exercise. The activation of the iHSP70 is sine qua non for the promotion of tissue repair, since the expression of this chaperone confers cytoprotection and also exerts anti-inflammatory effects. On the other hand, exercise also induces the appearance of HSP70 in the extracellular medium (eHSP70) but, so far, the eHSP70 function has been mainly attributed to the activation of the immune system, seeming to perform an opposite function from the iHSP70. Since a moderate intensity exercise bout induces a general anti-inflammatory response even in the presence of an elevated eHSP70, this protein could carry out other functions rather than immune activation. Because exercise generates heat and metabolic challenges (especially on glucose metabolism) we suggests that the motoneurons, a very active (possibly one of the most stressed cells during exercise) and also very sensitive cells to heat and glucose metabolism imbalances, could be the major sites for the eHSP70 function. Due to the importance of the iHSP70 for repair and stress adaptation, this protein must be present in abundance on the site of stress and, because of its intrinsic inability response to stress [low heat shock factor 1 (HSF-1) activation] and the structure of the motoneurons (very long cells), the iHSP70, produced on the very far nucleus, is not appropriately transported through the axon to the axon terminal, were it is required. Then, during the exercise, the released eHSP70 can be internalized by the motoneurons and act as intracellular chaperons, protecting this cell against oxidative damage, protein denaturation and many others. Since a decreased iHSP70 expression capacity is associated with neurodegeneration diseases (such as Parkinson, polyglutamine, Amyotrophic lateral sclerosis, Alzheimer's, Huntington's and many others), the understanding of the physiological function of the extracellular HSP70 could be helpful on the treatment of neurodegenerative and other neuronal diseases. Besides that, it could explain some of the beneficial effects of the pharmacological HSP70 activators and also the beneficial effects of the exercise among neuronal cells during neurodegenerative-inducing diseases.     

Sub‐lethal heat shock induces plasma membrane translocation of 70‐kDa heat shock protein in viable,but not in apoptotic,U‐937 leukaemia cells

Lasunskaia EB, Fridlianskaia I, Arnholdt AV, Kanashiro M, Guzhova I, Margulis B. Sub‐lethal heat shock induces plasma membrane translocation of 70‐kDa heat shock protein in viable, but not in apoptotic, U‐937 leukaemia cells. APMIS 2010; 118: 179–87. Heat shock protein 70 kDa, Hsp70, is an important intracellular factor that protects cells from stress. Unusual plasma membrane expression of Hsp70, observed in some cancer cells, contributes to the cell’s recognition and elimination by the immune system. Induction of apoptosis in cancer cells was demonstrated to increase Hsp70 translocation to the surface membrane, enhancing immunogenic effects through the stimulation of dendritic cells. As hyperthermia is proposed as a method of choice for anti‐cancer therapy, we examined whether apoptosis induction by heat shock enhances Hsp70 membrane translocation in U‐937 leukaemia cells. Cells were exposed to sub‐lethal heat shock, and intracellular and membrane‐bound Hsp70 expression was evaluated in apoptotic and viable cell sub‐populations, employing flow cytometry and immunofluorescence. Heat shock induced Hsp70 membrane translocation in the viable cells that were able to enhance Hsp70 production upon heating, but not in the cells undergoing apoptosis that continued to express low basal levels of the intracellular protein. Data suggest that the protein translocation was associated with the increasing Hsp70 content rather than the apoptotic process. Apoptosis does not contribute to externalization of Hsp70, at least in the cells with low levels of this protein.     

Gastric Autoantigenic Proteins in Helicobacter Pylori Infection

Purpose

This study tried to identify novel gastric autoimmune antigens that might be involved in aggravating the atrophic gastritis among patients with Helicobacter pylori infection using two-dimensional immunoblotting analysis.

Materials and Methods

Proteins from gastric mucosal antrectomy specimens and AGS cells (gastric adenocarcinoma cell lines derived from a Caucasian patient who had received no prior therapy) were 2-dimensionally immunoblotted separately with a pool of 300 sera from H. pylroi-infected patients at Gyeongsang National University Hospital.

Results

Thirty-eight autoantigenic proteins including alcohol dehydrogenase [NADP+], alpha enolase, gastrokine-1, gastric triacylglycerol lipase, heat shock 70 kDa protein 1, and peroxiredoxin-2 were identified in the gastric mucosal tissue. Fourteen autoantigenic proteins including programmed cell death 6-interacting protein, serum albumin and T-complex protein 1 subunit gamma were identified in the AGS cells. Albumin, alpha-enolase, annexin A3, cytoplasmic actin 1, heat shock cognate 71 kDa protein and leukocyte elastase inhibitor were commonly observed autoantigenic proteins in both gastric mucosal tissue and AGS cells. Alpha-enolase, glutathione S-transferase P, heat shock cognate 71 kDa protein, heat shock 70 kDa protein 1, human mitochondrial adenosine triphosphate synthase (ATP) subunit beta, mitochondrial 60 kDa heat shock protein, peroxiredoxin-2, 78 kDa glucose-regulated protein precursor, tyrosine-protein phosphatase non-receptor type 11 and Tryptophan-Aspartic acid (WD) repeat-containing protein 1 showed 60% or higher amino acid positivity.

Conclusion

These newly identified gastric autoimmune antigens might be useful in the control and prevention of gastroduodenal disorders, and might be valuable in breaking the vicious circle that exists in gastroduodenal disorders if their pathophysiological roles could be understood in the progress of chronic atrophic gastritis, gastroduodenal ulcers, intestinal metaplasia, and gastric carcinogenesis.     

CP2 gene as a useful viability marker for Cryptosporidium parvum

The validity of the CP2 gene of Cryptosporidium parvum as a viability marker was evaluated using absolute quantitative real-time polymerase chain reaction (qPCR) assays. Total ribonucleic acid (RNA) was isolated from live and heat-killed C. parvum oocysts, and complementary deoxyribonucleic acid was synthesized and used as a template. The most accurate number of viable C. parvum oocysts was predicted when the CP2 gene was used as a target gene. The lower detection limit of the CP2 gene was ten oocysts, which was the most sensitive among examined target genes. With heat shock induction, only hsp70 messenger RNA (mRNA) was induced, and the predicted viable oocyst number was increased by heat shock for this marker. The CP2, hsp70, Cryptosporidium oocyst wall protein, and β-tubulin mRNAs were not detected in heat-killed oocysts, but the 18S ribosomal ribonucleic acid (rRNA) showed heat stability until 48 h after heat killing. Although the 18S rRNA demonstrated the fastest response in crossing point (CP) value among the examined primer sets in qPCR, overestimation of viable oocysts was noted in the analysis with this gene. In conclusion, the CP2 gene was identified as the most sensitive, reliable, and accurate candidate of a viability marker of C. parvum by qPCR evaluation.     

Hsp70 expression in human skeletal muscle after exercise

Prolonged exercise of a sufficiently high intensity is thought to create physiological stress and to disturb cellular homeostasis, ultimately inducing cellular adaptations which enable the organism to better deal with any future exercise challenge. Heat shock proteins (hsp) are expressed when cells are exposed to different types of stress. In this study, we have investigated whether the expression of the heat inducible form of hsp70 is increased in human skeletal muscle cells after a single bout of exercise. Five untrained subjects performed an exercise bout at their individual anaerobic threshold for 30 min on a treadmill. Hsp70 mRNA concentration was significantly increased by a factor of four at 4 min post-exercise. Similarly high levels were also observed 30 min and 3 h after the end of exercise. Hsp70 protein concentration, on the contrary, did not change within 3 h after cessation of exercise. Thus, a single exercise bout in humans is able to increase the steady state concentration of hsp70 mRNA, but is probably not sufficient to have an effect on the already high basal level of its protein. The analysis of hsp70 mRNA is potentially useful as a method to detect stress in tissues with a high basal level of heat shock proteins.     

Stress proteins in the human endometrium and decidua

Protein synthesis in response to heat shock was induced in theproliferative and secretory human endometrium as well as inhuman decidua during a 2-h incubation period at 41°C. Amajor 70 K stress protein and two minor stress proteins of 88and 94 K were detected after [³⁵S]methionlne incorporation followedby SDS polyacrylamide gel electrophoretic separation and autoradiographyof dried gels. Two dimensional isoelectric focusing followedby horography showed the major 70 K stress protein to consistof at least six polypeptides of pH 4.6 to 5.5, the 88 K to consistof at least four polypeptides, of pH 5.5 to 6.0, and the 94K to consist of at least three polypeptides of pH 4.6 to 5.2.Stress proteins in the human endometrium and decidua may proveto be of physiological significance in repductive events.     

Heat shock- and alkaline pH-induced proteins of Campylobacter jejuni: characterization and immunological properties.

The protein response to physiological stress was characterized in Campylobacter jejuni 81176 after exposure to heat and pH shock and following periods of recovery. Immunoreactivities of major stress-related proteins were determined with anti-Campylobacter immune rabbit serum and intestinal lavage fluid. Distinct proteins with molecular masses ranging from 10 to 120 kDa were induced and/or released by selective heat or pH treatments. The most notable responses were those of two proteins with apparent molecular masses of 45 and 64 kDa that were induced and two other proteins of 10 and 12 kDa that were released by selective heat shock, alkaline pH treatment, or both. On the basis of N-terminal sequence analysis and immunological cross-reactivity data, the 64- and 10-kDa proteins were the C. jejuni homologs of Escherichia coli GroEL and GroES proteins, respectively. Enhanced chemiluminescence Western blotting (immunoblotting) revealed that all four proteins were among the major protein antigens recognized by anti-Campylobacter rabbit serum immunoglobulin G (IgG) and immune rabbit intestinal lavage IgA (secretory IgA). The results of this investigation suggest that the C. jejuni 10-, 12-, 45-, and 64-kDa proteins and a number of minor stress-related proteins deserve further evaluation of their respective roles in Campylobacter pathogenesis and immunity.     

Distribution of 70kDa heat shock protein in rabbit brains after heat stress and heat stroke

OBJECTIVE: Evaluation of the relationship between the induction of 70kDa heat shock protein in rabbit brains and heat stress. METHODS: HSP70 was detected using monoclonal antibody by ABC method in rabbit hypothalamus, hippocampus and cerberal cortex. RESULTS: Intense HSP70 staining was displayed in rabbit brains of the heat stroke group (rectal temperature 43 degrees C to death). Positive cells were distributed mainly in the CA1, CA2 regions of the hippocampus; granular cell layer I and pyramidal layer (II) of the cerebral cortex; and the periventricular area of hypothalamus. HSP70-psoitive substances were localized in the cytoplasm and neuronal processes, a few neurons exhibited dark staining nucle. Hosever, the rabbit brains of the general heat stress group (rectal temperature 42.0 degrees C, 30 minutes) had much weaker staining. CONCLUSION: Hyperthermia causes neuronal expression of HSP70, particularly under strong heat stress, and may be sustained till death.