Short Communication: Effect of heat stress on heat-shock protein (Hsp60) mRNA expression in rainbow trout Oncorhynchus mykiss.

The enhanced expression of heat shock proteins (hsps) in organisms can be detected in response to many kinds of stressor. For fish, high temperature is an important stressor, and hsp expression is associated with differences in environmental temperature. In this study, rainbow trout (Oncorhynchus mykiss) that were accustomed to an aquatic temperature of 18°C were exposed to an elevated temperature (25°C), and hsp60 expression in the gill, liver, spleen, heart, and head kidney was quantified using real-time polymerase chain reaction in unstressed and heat-stressed animals. The fish responded to heat stress in a time- and tissue-specific manner. Cardiac hsp60 mRNA levels were largely unchanged, and the greatest induction of hsp60 in heat-stressed animals was recorded in the liver, suggesting that protein damage and the consequent requirement for the Hsp60 protein are probably greater in hepatic tissue. Therefore, fish must be provided with optimal temperature conditions in order to realize their potential growth and maximize fish farm profits.

Temporal variations of Hsp60 and HSF-1 in primary rat myocardial cells in vitro under heat stress.

The mechanisms involved in sudden animal death due to acute heart failure during heat stress are not well understood. We examined the relationship between heat stress-induced variations of protective Hsp60 and expression of its regulatory factor, HSF-1, in heat-stressed primary myocardial cells of neonatal rats in vitro through cardiac enzyme detection, immunoblotting, immunocytochemistry, and qPCR. Increases in cardiac damage-related enzyme levels demonstrated injury to myocardial cells after heat exposure at 42°C. Hsp60 expression levels fluctuated during heat stress; they decreased significantly after 20 min, then increased at 120 min and decreased again at 360 min after initiation of heat stress. The highest levels of Hsp60 were observed at 240 min, while the lowest were at 60 min. Damage to myocardial cells was characterized by increases in cardiac enzyme levels and low levels of Hsp60 due to functional disorder of myocardial cells at early stages of heat stress. However, the significant induction of hsp60 mRNA levels from the beginning up to 240 min of heat stress was not consistent with the classic regulatory mechanisms that link transcription and translation, suggesting that Hsp60 expression is delayed due to loss of Hsp60 during the early stages of heat stress. hsf-1 mRNA levels were significantly increased from 10 min of heat stress; however, HSF-1 protein levels did not simultaneously increase, indicating that HSF-1 is not the sole regulator of Hsp60 expression.

Changes in the expression of Heat Shock Proteins in ovaries from bovines with cystic ovarian disease induced by ACTH.

Cystic ovarian disease (COD), which is considered one of the most important causes of reproductive failure in dairy cattle, induces intraovarian changes in the expression of numerous genes. The purpose of this study was to analyze the changes in the expression of Heat Shock Proteins (HSPs) in ovaries from bovines with cystic ovarian disease induced by ACTH. Immunoreactivity for Heat Shock Proteins (HSPs) in ovaries of cows with induced COD showed differential expression patterns in growing follicles from the control group. The immunopositive area for Hsp27 and Hsp60 in granulosa cells showed significant differences between tertiary follicles from normal cycling animals and those from animals with induced COD. The cysts showed increased Hsp27 immunostaining in theca cells in relation to tertiary follicles from normal cycling cows. Hsp70 immunostaining was more intense in cystic follicles than in other follicular categories from animals with induced COD, in both granulosa and theca cells. In granulosa cells, tertiary follicles from the control group showed higher levels of Hsp90 than cysts. These results demonstrate that there are differences in HSP protein expression when COD is induced. In fact, HSP expression would be part of the functional response to the changes in hormones and neurotransmitters induced by stress, indicating that HSPs can control hormonal functions and vice versa.

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-ß - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice.

Morphological and biochemical changes during formocresol induced cell death in murine peritoneal macrophages: apoptotic and necrotic features.

The present study was conducted to investigate the role of Formocresol (FC)-induced apoptosis and necrotic cell death in murine peritoneal macrophages (pMø). Macrophages were cultured with 1:100 FC for 2 to 24 h. The viability (trypan blue assay), cell morphology (scanning electronic microscope), and apoptotic and necrotic indexes (light and fluorescent microscopy) were determined at different scheduled times. Simultaneously, the expressions of proteins related to stress, survival, and cell death were measured by western blotting. FC-exposed macrophages exhibited maximal apoptosis from 2 to 6 h, coincident with Bax overexpression (P < 0.001). Additionally, Bcl-x(L) showed maximal expression between 12 and 24 h suggesting its survival effect in pMø. The lowest pMø viability and the increment of the necrotic rate from 4 to 12 h were observed in accordance to Fas and Hsp60 overexpressions. In summary, all the experimental data suggest that two different pathways emerge in pMø exposed to FC, one leading Bax-dependent apoptosis (2-6 h) and the other one favoring necrosis (4-18 h), related to Fas-receptor and Hsp60 stress signal.

Human hepatoma cell line (HepG2) cellular response to hypothermic stress with recovery. Induction of Hsp70, Hsp60 and Hsf1 expression.

The cell response of human HepG2 cells exposed to hypothermia with rewarming was analyzed. Ultrastructural findings in hypothermic stressed cells showed swollen mitochondria, dispersed chromatin, vacuoles and ring-shape nucleolar reorganization. These changes were coupled with significative differences in the induction of Hsp60, inducible Hsp70 and monomeric Hsfl in all treated samples, but not in Hsc 70 expression. Cellular response to hypothermia could be associated with the synergistic induction of Hsp expression.

Antibodies against recombinant heat shock proteins of 60 kDa from enterobacteria in the sera and synovial fluid of HLA-B27 positive ankylosing spondylitis patients.

OBJECTIVE: To study the association of HLA-B27 with IgG antibodies to different enterobacterial HSP60s in patients with ankylosing spondylitis (AS). METHODS: IgG antibodies to 60 kDa enterobacterial HSPs were determined by ELISA in paired samples of sera and synovial fluid from 21 HLA-B27+ ankylosing spondylitis (AS) patients; and in sera from 32 HLA-B27+ AS patients, 35 HLA-B27+ healthy relatives of AS patients, and 60 HLA-B27- healthy individuals with no family members with AS. RESULTS: HLA-B27+ patients and healthy individuals showed significantly higher IgG antibody levels to recombinant enterobacterial HSP60s than HLA-B27- healthy controls. The levels of anti-HSP60Sf and anti-HSP60Ec antibodies correlated with disease activity and anti-HSP60Ec antibodies with male gender. No association between enterobacterial HSP60 antibody levels and disease duration was observed. All groups had lower levels of IgG antibodies to rHSP60 from Streptococcus pyogenes (rHSP60 Spy). In paired samples of sera and synovial fluid from B27+ patients, IgG antibodies to enterobacterial HSP60s were detected, but in significantly higher levels in sera than in synovial fluid. The anti-rHSPSpy IgG response in these samples was lower and similar in the three groups. CONCLUSIONS: A correlation was found between HLA-B27 and the response to recombinat enterobacterial HSP60s. This response could be associated with disease activitir and gender in some proteins and the presence eof IgG antibodies to these proteins in synovial fluid could be associated with the inflammatory process and initiation of AS.

Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis.

We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.

Stress protein response and catalase activity in freshwater planarian Dugesia (Girardia) schubarti exposed to copper.

The hsp60 expression pattern and catalase activity in the freshwater planarian Dugesia schubarti exposed to copper under laboratory conditions were investigated. In the hsp60 induction experiments, planarians were exposed to a range of copper concentrations (0-960 microgCu/L) for 4 or 24h, to concentrations of 50 or 100 microgCu/L for 2, 4, 8, and 24h at 19 degrees C, and to heat shock at 27 degrees C for 24h. The concentrations of hsp60 in whole-body homogenates were determined immunochemically by Western blotting. Stress protein induction was detected only after 24h treatment at 27 degrees C. The tissue concentration of hsp60 remained unaltered in Cu-exposed planarians under the experimental conditions used. Catalase activity was significantly induced at concentrations of 40, 80, and 160 microgCu/L after 24h exposure. Our results suggest that catalase levels in planarians could represent biomarkers of interest for the estimation of copper effects in freshwater ecosystems.

Expression of the major heat shock proteins DnaK and GroEL in Streptococcus pyogenes: a comparison to Enterococcus faecalis and Staphylococcus aureus.

One of the outstanding problems in the field of heat shock response has been to elucidate the mechanism underlying the induction of heat shock proteins (HSPs). In this work, we initiate an analysis of the expression of heat shock groEL and dnaK genes and their promoters in S. pyogenes. The synthesis of total cellular proteins was studied upon transfer of a log-phase culture from 37 degrees C to 42 degrees C by performing 5-min pulse-labeling experiments with (35)S-Met. The heat shock responses in the pathogenic Gram-positive cocci, Enterococcus faecalis and Staphylococcus aureus, were also analyzed.

Molecular cloning, purification and immunological responses of recombinants GroEL and DnaK from Streptococcus pyogenes.

To better understand the roles of heat shock proteins in streptococcal diseases, the groEL and dnaK genes from Streptococcus pyogenes were cloned and their products (GroEL and DnaK) and derivatives (F2GroEL, F3GroEL and C1DnaK) purified as His-tagged fusion proteins. Western blot analysis of the purified proteins with sera from individuals with streptococcal diseases demonstrated that 29 out of 36 sera tested were reactive with GroEL and eight recognized DnaK. Rabbit antiserum against myosin recognized both GroEL and DnaK. Antibodies raised against purified F2GroEL and DnaK reacted with myosin in the ELISA but not in a Western immunoblot. These data indicate that the S. pyogenes GroEL and DnaK may be important immunogens during streptococcal infections. Furthermore, we provide evidence of an immunogenic relatedness of the GroEL and DnaK proteins with myosin that could play a role in the pathogenesis of streptococcal non-suppurative sequelae.