Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631bp, consisting of a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 573bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response.     

Changes in cell morphology and actin organization during heat shock in Dictyostelium discoideum: does HSP70 play a role in acquired thermotolerance?

In response to heat shock (34°C, 30 min), cell morphology and actin organization in Dictyostelium discoideum are drastically changed. Loss of pseudopodia and disappearance of F-actin-containing structures were observed by using fluorescence microscopy. These changes were paralleled by a rapid decrease of the F-actin content measured by a TRITC-phalloidin binding assay. The effects of heat shock on cell morphology and actin organization are transient: After heat shock (34°C) or during a long-term heat treatment (30°C), cell morphology, F-actin patterns and F-actin content recovered/adapted to a state which is characteristic for untreated cells. Because F-actin may be stabilized by increased amounts of heat shock proteins, their response and interaction with F-actin was analyzed. After a 1 h heat treatment (34°C), the major heat shock protein of D. discoideum (HSP70) showed maximally increased synthesis rates and levels. During recovery from a 34°C shock or during a continuous heat treatment at 30°C, the HSP70 content first increased and then declined slowly toward normal levels. Pre-treatment of cells with a short heat shock of 30 min at 34°C stabilized the F-actin content when the cells were exposed to a second heat shock. Furthermore, a transient colocalization of HSP70 and actin was observed at the beginning of heat treatment (30°C) using immunological detection of HSP70 in the cytoskeletal actin fraction.     

Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock

Changes in the content of constitutive and inducible proteins of the family of heat shock 70 kDa proteins (HSP70) caused by heat shock in human neutrophils, white blood cells with an atypically short lifespan, which provide a nonspecific defense of the organism against bacterial pathogens, have been studied. An analysis of the intracellular content of the constitutive and inducible HSP70 proteins by flow cytometry revealed a biphasic dynamics of changes in the protein level, which was characterized by an increase in the protein level immediately after heat shock followed by a decrease within 15–30 min after the termination of heat treatment. Because the inhibitor of protein synthesis cycloheximide did not change the dynamics profile, it was assumed that the increase in the HSP70 level is related not to the de novo synthesis of these proteins but to conformational changes of HSP70 molecules and an increased accessibility of some epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or the C-terminal substrate-binding domains of the protein, it was shown by cell immunofluorescence and flow cytometry that the heat shock-associated increase in the intracellular HSP70 level results from an increased efficiency of the binding of antibodies recognizing the substrate-binding domain. It was also demonstrated that the decrease in the intracellular HSP70 level after the heat shock, may be partially due to a release into the extracellular space of both the constitutive and inducible HSP70 proteins, which is regulated with the involvement of ABC-transporters.     

Conditions for a heat shock response during oogenesis and embryogenesis of the amphibian Pleurodeles waltl

The optimal conditions capable of inducing an increase in HSP70 neosynthesis during development of the urodele amphibian Pleurodeles waltl were determined in this study. These conditions depend on temperature, heat shock duration and recovery duration. In oocytes, a heat shock response was repeatedly obtained at 37°C for 15 min followed by 1 h recovery. These results provided evidence for heat shock response at every stage considered. An increase in HSP70 synthesis was noted throughout oogenesis, but it did not lead to an increase in the amount of soluble HSP70, except for stage VI oocytes. Such results suggest that from stage II to stage IV oocytes, an equilibrium occurs between the HSP70 used and the HSP70 neosynthesized. In contrast, in stage VI oocytes, heat shock led to overproduction of HSP70. During early development, the heat shock response was repeatedly obtained only from the gastrula stage with a 37°C shock and a 15min duration of treatment. Surprisingly, during cleavage stage, the soluble HSP70 total amount increased after heat shock at a time when no HSP70 neosynthesis occurred.