Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock

Heat shock proteins (Hsps) are a family of highly conserved cellular proteins present in all organisms, mediating a range of essential housekeeping and cytoprotective functions as well-known molecular chaperons and recently as regulators of the immune response. By subtractive suppression hybridization, three Hsp40 homologues have been identified in the flounder (Paralichthys olivaceus) embryonic cells (FEC) after treatment with UV-inactivated turbot (Scophthalmus maximus L.) rhabdovirus (SMRV), termed PoHsp40A4, PoHsp40B6 and PoHsp40B11, whose encoded proteins all possess the conserved DnaJ domain, a signature motif of the Hsp40 family. Based on different protein structure and phylogenetic analysis, they can be categorized into two subfamilies, PoHsp40A4 for Type I Hsp40, PoHsp40B6 and PoHsp40B11 for Type 11 Hsp40. Further expression analysis revealed two very different types of kinetics in response either to heat shock or to virus infection, with a marked induction for PoHsp4OA4 and a weak one for both PoHsp40B6 and PoHsp40B11. A very distinct tissue distribution of mRNA was also revealed among the three genes, even between PoHsp40B6 and PoHsp40B11. This is the first report on the transcriptional induction of Hsp40 in virally stimulated fish cells, and the differential expressions might reflect their different roles in unstressed and stressed cells. (c) 2005 Elsevier Ltd. All rights reserved.

cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamysfarreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd2+, Pb2+ and Cu2+ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment. (c) 2007 Elsevier Inc. All rights reserved.

Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity and regulation of a subset of cytosolic proteins. In the present study, the cDNA of Argopecten irradians HSP90 (designated AiHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of AiHSP90 was of 2669 bp, including an open reading frame (ORF) of 2175 bp encoding a polypeptide of 724 amino acids with predicted molecular weight of 83.08 kDa and theoretical isoelectric point of 4.81. BLAST analysis revealed that AiHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in AiHSP90, which indicated that AiHSP90 should be a cytosolic member of the HSP90 family. Fluorescent real-time quantitative PCR was employed to examine the expression pattern of AiHSP90 mRNA in haemocytes of scallops challenged by Gram-negative bacteria Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus. In both bacterial challenged groups, the relative expression level of AiHSP90 transcript was up-regulated and reached maximal. level at 9 h after injection, and then dropped progressively to the original level at about 48 h post challenge. The results indicated that AiHSP90 was potentially involved in the immune responses against bacteria challenge in scallop A. irradian. (c) 2007 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Enhanced expression of 70-kilodalton heat shock protein limits cell division in a sepsis-induced model of acute respiratory distress syndrome.

OBJECTIVE: Fibrotic changes are initiated early in acute respiratory distress syndrome. This may involve overproliferation of alveolar type II cells. In an animal model of acute respiratory distress syndrome, we have shown that the administration of an adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) limited pathophysiological changes. We hypothesized that this improvement may be modulated, in part, by an early AdHSP-induced attenuation of alveolar type II cell proliferation. DESIGN: Laboratory investigation. SETTING: Hadassah-Hebrew University and University of Pennsylvania animal laboratories. SUBJECTS: Sprague-Dawley Rats (250 g). INTERVENTIONS: Lung injury was induced in male Sprague-Dawley rats via cecal ligation and double puncture. At the time of cecal ligation and double puncture, we injected phosphate-buffered saline, AdHSP, or AdGFP (an adenoviral vector expressing the marker green fluorescent protein) into the trachea. Rats then received subcutaneous bromodeoxyuridine. In separate experiments, A549 cells were incubated with medium, AdHSP, or AdGFP. Some cells were also stimulated with tumor necrosis factor-alpha. After 48 hrs, cytosolic and nuclear proteins from rat lungs or cell cultures were isolated. These were subjected to immunoblotting, immunoprecipitation, electrophoretic mobility shift assay, fluorescent immunohistochemistry, and Northern blot analysis. MEASUREMENTS AND MAIN RESULTS: Alveolar type I cells were lost within 48 hrs of inducing acute respiratory distress syndrome. This was accompanied by alveolar type II cell proliferation. Treatment with AdHSP preserved alveolar type I cells and limited alveolar type II cell proliferation. Heat shock protein 70 prevented overexuberant cell division, in part, by inhibiting hyperphosphorylation of the regulatory retinoblastoma protein. This prevented retinoblastoma protein ubiquitination and degradation and, thus, stabilized the interaction of retinoblastoma protein with E2F1, a key cell division transcription factor. CONCLUSIONS: : Heat shock protein 70-induced attenuation of cell proliferation may be a useful strategy for limiting lung injury when treating acute respiratory distress syndrome if consistent in later time points.

An examination of transcriptional and translational regulation of the 70kDa heat shock proteins following amputation of the tail and forelimb in the newt Notophthalmus viridescens

Several stresses to tissues including hyperthermia, ischemia, mechanical trauma and heavy metals have been demonstrated to affect the regulation of a subset of the family of heat shock proteins of70kOa (hsp70). In several organisms following some of these traumas, the levels of hsp70 mRNA and proteins are dramatically upregulated. However, the effects of the stress on limb and tail amputation in the newt Notophthalmus viridescens, involving mechanical tissue damage, have not adequately been examined. In the present study, three techniques were utilized to quantitate the levels of hsp70 mRNA and protein in the tissues of the forelimbs and tails of newts during the early post-traumatic events following surgical resection of these:: appendages. These included quantitative Western blotting of proteins separated by both one and twodimensional SDS-polyacrylamide gel electrophoresis and quantitative Northern blot analysis of total RNA. In tissues of both the limb and tail one hour after amputation, there were no significant differences in the levels of hsp70 protein measured by one-dimensional SOSPAGE followed by Western blotting, when compared to the levels measured in the unamputated limb. A 30 minute heat shock at 35°C failed to elicit an increase in the levels of hsp70 protein in these tissues. Further analysis using the more sensitive 20 PAGE separation of stump tissue proteins revealed that at least some of the five hsp70 isoforms of the newt may be differentially regulated in limbs and tails in response to trauma. It appears also that amputation of the tail and limb tissues leads to slight 3 elevation in the levels of HSP70 mRNA when compared to those of their respective unstressed tissues.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Stimulation of multiple mitogen-activated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular myocytes.

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAPKs), namely the stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs), the extracellularly responsive kinases (ERKs) and p38-MAPK, by oxidative stress as exemplified by H2O2 in primary cultures of neonatal rat ventricular myocytes. The 46 and 54 kDa species of SAPKs/JNKs were activated 5- and 10-fold, respectively, by 0.1 mM H2O2 (the maximally effective concentration). Maximal activation occurred at 15-30 min, but was still detectable after 2 h. Both ERK1 and ERK2 were activated 16-fold by 0.1 mM H2O2 with a similar time course to the SAPKs/JNKs, and this was comparable with their activation by 1 microM PMA, the most powerful activator of ERKs that we have so far identified in these cells. The activation of ERKs by H2O2 was inhibited by PD98059, which inhibits the activation of MAPK (or ERK) kinases, and by the protein kinase C (PKC) inhibitor, GF109203X. ERK activation was also inhibited by down-regulation of PMA-sensitive PKC isoforms. p38-MAPK was activated by 0.1 mM H2O2 as shown by an increase in its phosphorylation. However, maximal phosphorylation (activation) was more rapid (<5 min) than for the SAPKs/JNKs or the ERKs. We studied the downstream consequences of p38-MAPK activation by examining activation of MAPK-activated protein kinase 2 (MAPKAPK2) and phosphorylation of the MAPKAPK2 substrate, the small heat shock protein HSP25/27. As with p38-MAPK, MAPKAPK2 was rapidly activated (maximal within 5 min) by 0.1 mM H2O2. This activation was abolished by 10 microM SB203580, a selective inhibitor of certain p38-MAPK isoforms. The phosphorylation of HSP25/27 rapidly followed activation of MAPKAPK2 and was also inhibited by SB203580. Phosphorylation of HSP25/27 was associated with a decrease in its aggregation state. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in neonatal rat ventricular myocytes. Activation of all three MAPKs has been associated with the development of the hypertrophic phenotype. However, stimulation of p38-MAPK and the consequent phosphorylation of HSP25/27 may also be important in cardioprotection.

Lack of glucose and hsp70 responses in haddock Melanogrammus aeglefinus (L.) subjected to handling and heat shock

Juvenile haddock Melanogrammus aeglefinus (c. 39 g) were exposed to either a handling stressor (1 min out of water) or heat shock (increase from 10 to 15° C for 1 h), and plasma cortisol, plasma glucose and gill hsp70 levels were determined before, and at 1, 3, 6, 12, 24 and 48 h post-stress. The pattern of cortisol increase was similar following both stressors, with levels increasing by 25-fold at 1 h post-stress, but returning to pre-stress levels (2–5 ng ml⁻¹) by 3 h. In contrast, neither handling nor heat shock caused an increase in plasma glucose levels. Although gill hsp70 was detected, presumably constitutive levels, in both control and heat shocked groups, there were not significant changes in gill hsp70 levels after exposure to heat shock. The lack of glucose and hsp70 responses to these typical stressors is consistent with previous studies on Atlantic cod Gadus morhua, and suggests that the stress physiology of Gadidae differs from the ‘typical’ teleost.

Intramuscular heat shock protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes: evidence that insulin resistance is associated with a disturbed antioxidant defense mechanism

To examine whether genes associated with cellular defense against oxidative stress are associated with insulin sensitivity, patients with type 2 diabetes (n = 7) and age-matched (n = 5) and young (n = 9) control subjects underwent a euglycemic-hyperinsulinemic clamp for 120 min. Muscle samples were obtained before and after the clamp and analyzed for heat shock protein (HSP)72 and heme oxygenase (HO)-1 mRNA, intramuscular triglyceride content, and the maximal activities of β-hyroxyacyl-CoA dehydrogenase (β-HAD) and citrate synthase (CS). Basal expression of both HSP72 and HO-1 mRNA were lower (P < 0.05) by 33 and 55%, respectively, when comparing diabetic patients with age-matched and young control subjects, with no differences between the latter groups. Both basal HSP72 (r = 0.75, P < 0.001) and HO-1 (r = 0.50, P < 0.05) mRNA expression correlated with the glucose infusion rate during the clamp. Significant correlations were also observed between HSP72 mRNA and both β-HAD (r = 0.61, P < 0.01) and CS (r = 0.65, P < 0.01). HSP72 mRNA was induced (P < 0.05) by the clamp in all groups. Although HO-1 mRNA was unaffected by the clamp in both the young and age-matched control subjects, it was increased (P < 0.05) ∼70-fold in the diabetic patients after the clamp. These data demonstrate that genes involved in providing cellular protection against oxidative stress are defective in patients with type 2 diabetes and correlate with insulin-stimulated glucose disposal and markers of muscle oxidative capacity. The data provide new evidence that the pathogenesis of type 2 diabetes involves perturbations to the antioxidant defense mechanism within skeletal muscle.

Hepatic concentration of heat shock protein 70 kD (Hsp70) in broilers subjected to different thermal treatments

1. The relationship between repeated thermal treatments and hepatic synthesis of Hsp 70 was studied in broiler chickens.2. Sixty broilers were submitted to 5 different treatments (12 birds each) from day 1 to day 42. Four groups were kept in a thermoneutral environment and subjected to 0, 1, 2 and 3 heat stress episodes at 35 degrees C for 4 h per week (TN-0, TN-1, TN-2 and TN-3, respectively). The last group (HT-35) was reared at a room temperature of 35 degrees C.3. From 39 to 42 old, the birds experienced acute heat stress at 41 degrees C. Resistance to heat stress was evaluated by the time taken for rectal temperature to increase by 3 degrees C above the pre-treatment value. Livers were collected (before and after heat stress) and Hsp70 was determined using Western Blot analysis with monoclonal anti-Hsp70 antibody.4. Resistance to heat stress and concentration of Hsp70 were higher in those birds subjected to more heat stress episodes during the experimental period (TN-3) and HT-35. A positive correlation was observed between Hsp70 concentration and the time taken for a 3 degrees C increase in rectal temperature (r=0.42; P<0.01).5. Exposing birds to episodes of heat stress (35 degrees C) during rearing may improve their resistance to acute heat stress, but the previous thermal history did not seem to influence the hepatocyte Hsp70 content after exposure to more severe heat stress (41 degrees C).

Gastroprotective mechanisms of Citrus lemon (Rutaceae) essential oil and its majority compounds limonene and beta-pinene: Involvement of heat-shock protein-70, vasoactive intestinal peptide, glutathione, sulfhydryl compounds, nitric oxide and prostaglandin E-2

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pre- and post-transplant anti-myosin and anti-heat shock protein antibodies and cardiac transplant outcome

Background: the purpose this study was to investigate the relationship of anti-myosin and anti-heat shock protein immunoglobulin G (IgG) serum antibodies to the original heart disease of cardiac transplant recipients, and also to rejection and patient survival after cardiac transplantation.Methods: Anti-myosin and anti-heat shock protein (anti-hsp) IgG antibodies were evaluated in pre-transplant sera from 41 adult cardiac allograft recipients and in sequential post-transplant serum samples from 11 recipients, collected at the time of routine endomyocardial biopsies during the first 6 months after transplantation. In addition, the levels of these antibodies were determined from the sera of 28 healthy blood donors.Results: Higher anti-myosin antibody levels were observed in pre-transplant sera than in sera from normal controls. Moreover, patients with chronic Chagas heart disease showed higher anti-myosin levels than patients with ischemic heart disease, and also higher levels, although not statistically significant, than patients with dilated cardiomyopathy. Higher anti-hsp levels were also observed in patients compared with healthy controls, but no significant differences were detected among,the different types of heart diseases. Higher pre-transplant anti-myosin, but not anti-hsp, levels were associated with lower 2-year post-transplant survival. In the post-transplant period, higher anti-myosin IgG levels were detected in sera collected during acute rejection than in sera collected during the rejection-free period, whereas anti-hsp IgG levels showed no difference between these periods.Conclusions: the present findings are of interest for post-transplant management and, in addition, suggest a pathogenic role for anti-myosin antibodies in cardiac transplant rejection, as has been proposed in experimental models of cardiac transplantation.