Hsp70 mRNA Levels Research Articles

Cerulenin upregulates heat shock protein-70 gene expression in chicken muscle

Lines of evidence suggested that systems involved in the regulation of the stress responses and energy homeostasis are highly integrated. Because cerulenin, the natural antibiotic product of the fungus Cephalosporium ceruleans and a broad-spectrum fatty acid synthesis (FAS) inhibitor, has been shown to affect food intake and energy balance, and because the biomarker of stress Hsp-70 gene was found to interact directly with fatty acids, we hypothesized that cerulenin may regulate Hsp-70 gene expression. Therefore, the present study was undertaken to examine this issue. Cerulenin administration significantly (P < 0.05) decreased food intake and induced Hsp-70 mRNA levels in muscle, but not in liver or hypothalamus of 2-wk-old broiler chickens. These changes were accompanied by an unpregulation of muscle uncoupling protein and carnitine palmitoyltransferase 1 mRNA levels. This result indicated that the regulation of Hsp-70 gene expression in normal chickens, as estimated by oxidative stress indices [TBA reacting substances, ferric reducing/antioxidant power, and ceruloplasmin oxidase activity] levels, is tissue-specific. In attempt to discriminate between the effect of cerulenin and cerulenin-reduced food intake on Hsp-70 gene expression, we also evaluated the effect of food deprivation on the same cellular responses. Food deprivation for 16 h did not affect Hsp-70 gene expression in all tissues examined, indicating that the effect of cerulenin is independent of the inhibition of food intake. To ascertain whether the effect of cerulenin is direct or indirect, we carried out in vitro studies. Cerulenin treatment did not affect Hsp-70 gene expression in Leghorn male hepatoma and quail myoblast cell lines, suggesting that the observed effect in vivo may be mediated through the central nervous system.

IL-17A Mediates Early Post-Transplant Lesions after Heterotopic Trachea Allotransplantation in Mice

Primary graft dysfunction (PGD) and bronchiolitis obliterans (BO) are the leading causes of morbidity and mortality after lung transplantation. Reports from clinical and rodent models suggest the implication of IL-17A in either PGD or BO. We took advantage of the heterotopic trachea transplantation model in mice to study the direct role of IL-17A in post-transplant airway lesions. Across full MHC barrier, early lesions were controlled in IL-17A-/- or anti-IL17 treated recipients. In contrast, IL-17A deficiency did not prevent subsequent obliterative airway disease (OAD). Interestingly, this early protection occurred also in syngeneic grafts and was accompanied by a decrease in cellular stress, as attested by lower HSP70 mRNA levels, suggesting the involvement of IL-17A in ischemia-reperfusion injury (IRI). Furthermore, persistence of multipotent CK14+ epithelial stem cells underlined allograft protection afforded by IL-17A deficiency or neutralisation. Recipient-derived γδ+ and CD4+ T cells were the major source of IL-17A. However, lesions still occurred in the absence of each subset, suggesting a high redundancy between the innate and adaptive IL-17A producing cells. Notably, a double depletion significantly diminished lesions. In conclusion, this work implicated IL-17A as mediator of early post-transplant airway lesions and could be considered as a potential therapeutic target in clinical transplantation.

Cloning and expression analysis of a HSP70 gene from Korean rockfish (Sebastes schlegeli)

The gene encoding HSP70 was isolated from Korean rockfish Sebastes schlegeli by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of HSP70 cDNA was composed of 2259 bp and encoded a polypeptide of 639 amino acids. BLAST analysis showed that HSP70 of S. schlegeli shared high identities with those of the Lates calcarifer, Oreochromis niloticus, Seriola quinqueradiata HSP70s (88–89%). Our current study also revealed that HSP70 of Korean rockfish was expressed in many tissues by RT-PCR under unstressed condition. Quantitative real-time PCR showed that the expression patterns of Korean rockfish HSP70 were developmental stage-dependency. The expression of HSP70 was measured by quantitative real-time PCR after different oxygen treatments. The results showed that expression of HSP70 increased significantly after exposure to hypoxia for 30 min in gill and ovary, and then decreased for 60 min, and the level in spleen and liver gradually increased and reached the highest at 60 min. In addition, in gill, spleen and liver, the HSP70 mRNA level reached the maximum in hypoxia group after one hour different oxygen concentration stress. Increased amounts of serum thyroxine (T4), and triiodothyronine (T3) were also found during 30 min hypoxia treatment and 60 min normoxia group in our study. All of the results provide information to further study the mechanism of physiology and immune function under stress conditions of ovoviviparous teleosts.

Thermal manipulation during chicken embryogenesis results in enhanced Hsp70 gene expression and the acquisition of thermotolerance

The aim of this study was to evaluate the effect of thermal manipulation (TM) during embryogenesis on hatchability, growth performance and thermotolerance acquisition parameters during thermal challenge (TC). Seven-hundred and fifty fertile chicken eggs were divided randomly into three groups (250 eggs each): control group was maintained at 37.8°C and 56% relative humidity (RH), TM1 was subjected to TM at 38.8°C for 6h and 65% RH during embryonic days (ED)10–18 and TM2 was subjected to TM at 38.8°C for 18h and 65% RH during ED10–18. Hatched chicks from each treatment group were then randomly divided into two sub-treatment groups (Naive and TC). Chicks in TC groups were subjected to TC by adjusting room temperature to 41.0°C for 6h on days 3, 7, and 42 of age while naïve chicks were kept under regular conditions (25±1°C and 50–60% RH). Percentage of hatched eggs was recorded and post-hatch chick performance was evaluated by recording chick body weight (BW). Chick’s response to TC was evaluated by determination of body temperature (Tb), plasma T3 and T4 levels, and muscle mRNA levels of Hsp70. There was a significant increase in muscle mRNA levels of Hsp70 during embryogenesis and during TC in post-hatch chicks. While hatchability was not adversely affected, the body weight in TM2 chicks was significantly higher at the end of the study period (42days). Results of this study indicated a long-term enhancement of Hsp70 gene expression associated with improved thermotolerance acquisition in treated chicks without adversely affecting performance.

Assessment of the viability of Cryptosporidium parvum oocysts with the induction ratio of hsp70 mRNA production in manure

Determining the presence of viable Cryptosporidium parvum oocysts in complex environmental matrices in hygiene control can prevent the contamination of water resources and food with this pathogen. This study assessed the induction ratio of hsp70 mRNA production by heat shock in different oocysts as a marker of viability. Using different procedures for (m)RNA extraction directly from manure and reverse transcription real-time qPCR, this study found slightly increased hsp70 mRNA contents in viable oocysts that were heat shock induced at 45°C for 20min compared to not induced oocysts (1.6 fold induction in average). Prolonging the heat shock treatment to 2h did not further increase the copy numbers. Heat shock by consecutive stimuli, such as freezing and then heating, did not yield significantly higher copy numbers than the 45°C treatment. There was a certain background level of hsp70 mRNA in viable oocysts that were not exposed to heat shock, indicating a constitutive production of the transcripts in the oocysts. The production of hsp70 mRNA induced by heat shock in oocysts aged for 9months that exhibited reduced viability was lower than in fresher oocysts (induction ratio<1.2). No production of hsp70 mRNA by heat shock was detected in 12months old oocysts that were not viable in the excystation test. Oocysts inactivated at 75°C for 30min were not able to respond to heat shock, and low amount of copies were occasionally measured only in total RNA extracts, but not in mRNA extracts that were purified directly with an oligo (dT)25 based system. The induction ratio of hsp70 mRNA varied according to the viability of the organisms in a sample. Copy numbers of β-tubulin mRNA in viable oocysts were lower than hsp70 mRNA, therefore the latter is more suitable to detect low numbers of oocysts by RT-qPCR.

Physiological responses and HSP70 mRNA expression of GIFT strain of Nile tilapia (Oreochromis niloticus) under cold stress

An investigation on the physiological responses and HSP70 expression under cold stress was conducted on Genetically Improved Farmed Tilapia (GIFT) strain of Nile tilapia (Oreochromis niloticus). Tilapia were acclimated at 28°C as control and then the water temperature was reduced from 28°C to 15°C at a rate of 1°C h−1, and serum biochemical indices, antioxidant enzymes and expression of HSP70 mRNA were analysed on days 0, 0.5, 1, 2, 3 and 5 after exposure to 15°C. The concentration of serum K, Na, Cl and Ca concentration showed instability during cold stress. Glucose rapidly increased on day 0 followed by a declining trend. Triglyceride, cholesterol, low density lipoprotein-cholesterol signifi-cantly decreased and high density lipoprotein-cholesterol showed instability during cold stress. Aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase activities were prominently elevated under cold stress. Superoxide dismutase and catalase activities showed a remarkable rise on day 0.5 under cold stress, and gradually decreased thereafter. HSP70 mRNA level significantly increased both in liver and muscle under cold stress, especially on days 0.5 and 1. These results suggest that cold influences several physiological responses of tilapia, and the cold resistance and cold tolerance of tilapia will benefit from the enhancement of antioxidant enzymes activities and upregulated HSP70 mRNA expression.

Effects of the dinoflagellate Alexandrium minutum and its toxin (saxitoxin) on the functional activity and gene expression of Crassostrea gigas hemocytes

Blood cells or hemocytes are the major component of the cellular immune responses in bivalves. Recent evidences have demonstrated that the immune functions of these cells can be impaired by harmful algae and their toxins. In this study, we compared the in vitro effect of the toxic dinoflagellate Alexandrium minutum and its purified paralytic toxin, saxitoxin (STX) on the oyster Crassostrea gigas hemocytes. Oyster hemocytes were incubated with A. minutum or STX, and their size, complexity, viability, phagocytic activity and reactive oxygen species (ROS) production were analyzed through flow cytometry. Also, expression of eleven defense-related genes was evaluated through real-time quantitative PCR. Cell incubation with both, the noxious microalga and STX did not alter hemocyte viability neither complexity. However, their phagocytic capacity and ROS production were significantly affected by both treatments. On the other hand, only A. minutum exposure resulted in increase of hemocyte size. Transcript levels of the gene encoding the cytokine Interleukin-17 was significantly higher (4.5-fold) after hemocytes were exposed to A. minutum, which contrasted with a decreased (7-fold) number of transcripts, when hemocytes were treated with STX. The purified toxin also led to lower transcript levels of a cytochrome P450 gene, the CYP356A1 (3.5-fold). On the other hand, hemocytes incubated with STX presented higher mRNA levels of antimicrobial peptide defensin-2 (7-fold) and heat shock protein Hsp70 (2-fold) genes. Altogether, these results suggest that the noxious alga A. minutum and STX can indeed negatively affect immunocompetence of C. gigas hemocytes, although in distinct manners. These differences could be due to a lower STX bioavailability in living microalgal cultures or alternatively to the presence of allelopathic compounds produced by living microalgae. This is the first report comparing the transcription of defense-related genes in oysters exposed to a harmful alga and its purified toxin.

Fas receptor-deficient lpr mice are protected against acetaminophen hepatotoxicity due to higher glutathione synthesis and enhanced detoxification of oxidant stress

Acetaminophen (APAP) overdose is a classical model of hepatocellular necrosis; however, the involvement of the Fas receptor in the pathophysiology remains controversial. Fas receptor-deficient (lpr) and C57BL/6 mice were treated with APAP to compare the mechanisms of hepatotoxicity. Lpr mice were partially protected against APAP hepatotoxicity as indicated by reduced plasma ALT and GDH levels and liver necrosis. Hepatic Cyp2e1 protein, adduct formation and hepatic glutathione (GSH) depletion were similar, demonstrating equivalent reactive metabolite generation. There was no difference in cytokine formation or hepatic neutrophil recruitment. Interestingly, hepatic GSH recovered faster in lpr mice than in wild type animals resulting in enhanced detoxification of reactive oxygen species. Driving the increased GSH levels, mRNA induction and protein expression of glutamate-cysteine ligase (gclc) were higher in lpr mice. Inducible nitric oxide synthase (iNOS) mRNA and protein levels at 6h were significantly lower in lpr mice, which correlated with reduced nitrotyrosine staining. Heat shock protein 70 (Hsp70) mRNA levels were substantially higher in lpr mice after APAP. Our data suggest that the faster recovery of hepatic GSH levels during oxidant stress and peroxynitrite formation, reduced iNOS expression and enhanced induction of Hsp70 attenuated the susceptibility to APAP-induced cell death in lpr mice.

Physiological responses and HSP70 mRNA expression in GIFT tilapia juveniles,Oreochromis niloticusunder short-term crowding

Physiological responses and HSP70 mRNA expression to short-term crowding were tested in juveniles genetically improved farmed tilapia (Oreochromis niloticus) (70.43 ± 4.43 g). Fish were kept at control group (5 g L−1) and stress groups (low density 10 g L−1, medium density 40 g L−1 and high density 70 and 100 g L−1) for 48 h. Each density was tested in triplicates. The effects of a short-term exposure on the physiological responses of fish were determined before stress (0 h) and at 6, 12, 24 or 48 h post-crowding. There was a significant increase in serum cortisol, total protein, lysozyme and aspartate aminotransferase (AST) activities in all stressed groups at 24 h post-crowding (P < 0.05). Serum glucose and alanine aminotransferase (ALT) of 100 g L−1 stressed group were lower than that of the control after 48 h of crowding stress (P < 0.05). The levels of serum triglyceride and cholesterol in crowding stress with high density were significantly decreased compared with the control group at 48 h. The mRNA expression data showed that hepatic HSP70 mRNA levels were markedly elevated at all stressed groups. HSP70 mRNA levels of 70 and 100 g L−1 stressed groups decreased at 48 h compared with the 24 h post-crowding. The protective ability of HSP70 was limited.

Thermal manipulation during chicken embryogenesis results in enhanced HSP70 gene expression and the acquisition of thermotolerance

SUMMARYThe aim of this study was to evaluate the effect of thermal manipulation (TM) at 38.5°C for 6 h or 18 h during embryonic days (ED) 10–18 on the muscle mRNA levels of heat shock protein 70 (HSP‐70) and the chick's ability to cope with the thermal challenge (TC; 41°C for 6h) at day 3, 7 and 42 of age. 750 fertile chicken eggs were obtained and divided randomly into 3 different treatment groups: control group was maintained at 37.8°C and 56% relative humidity (RH), TM1 was subjected to TM at 38.8°C for 6 h and 65% RH during ED 10–18 and TM2 was subjected to TM at 38.8°C for 18 h and 65% RH during ED 10–18. After hatching, the chicks from each treatment groups were randomly divided into 2 sub‐treatment groups (Naive and challenged). Chick's response to thermal challenged (41°C fro 6 hrs) was evaluated by determination of body temperature, plasma thyroid hormone levels (T3 and T4), plasma HSP70 and muscle mRNA levels of Hsp70. Our results indicated a long‐term improvement in the gene expression of HSP70 in the thermal manipulated chicks when compared with control, as manifested by a significant increase in the muscle mRNA levels of Hsp70 in thermally manipulated chicks during embryogenesis and during thermal challenge in post‐hatch chickens.

Multiple effects of repetitive transcranial magnetic stimulation on neuropsychiatric disorders

Repetitive transcranial magnetic stimulation (rTMS) is a new tool that has been used for the treatment of patients with neuropsychiatric disorders. However, the mechanisms underlying the effects of rTMS are still unclear. We analyzed the changes in mRNA expression in mouse brain that occurred after rTMS with an Affymetrix GeneChip. Following 20days of rTMS, many genes were differentially expressed in the mouse brain. Downregulation of Period 2 and 3 mRNA expression levels and a subsequent decrease in food and water intake were observed. HSP70 mRNA expression levels were upregulated after transient and chronic rTMS. In N2A 150Q cells, an upregulation of HSP70 mRNA and protein levels and subsequent cell-protective effects were observed after chronic rTMS. In addition, dopamine receptor 2 mRNA expression levels were downregulated, and a subsequent decrease in the binding of [3H]raclopride was observed. These results indicated that the modulation of several genes may be involved in the therapeutic mechanisms of chronic rTMS for patients with neuropsychiatric disorders.

Effects of Selenium-Enriched Probiotics on Heat Shock Protein mRNA Levels in Piglet under Heat Stress Conditions

The effects of selenium-enriched probiotics (SP) on tissue selenium (Se) deposition, glutathione peroxidase-1 (GPx1) activity and mRNA level, and heat shock protein (Hsp) mRNA levels of piglets under heat stress conditions were investigated. A total of 48 crossbred ([Landrace × Yorkshire] × Duroc) piglets were randomly divided into 4 groups and fed a basal diet (Con, 0.16 mg Se/kg) or basal diets with added probiotics (P, 0.16 mg Se/kg), sodium selenite (SS, 0.46 mg Se/kg), or SP (0.46 mg Se/kg), respectively, for 42 days. Three piglets were randomly selected from each group for blood sample collection at days 0, 14, 28, and 42 and for liver, kidney, and spleen sample collection at day 42. The results showed that P, SS, and SP could significantly down-regulate the average mRNA levels of Hsp70 (17.3, 23.7, and 40.1%) and Hsp27 (22.4, 24.4, and 44.7%) of the tissues, respectively (P < 0.05), whereas SS and SP could significantly elevate Se concentration, GPx1 activity and mRNA level (P < 0.05). The maximal effects of these parameters were observed in SP. It was concluded that SP is a feasible dietary supplementation of piglets under heat stress conditions during the summer season.

Abstract A84: Altered monocyte-derived dendritic cell differentiation in the presence of tumor supernatant: Possible involvement the p38MAPK pathway.

Abstract Introduction: Dendritic cells (DC) are the most efficient antigen-presenting cells in the immune system. In cancer patients, however, these cells present functional and phenotypic disturbances that could be related to a stressful environment induced by the tumor. Heat Shock Proteins (HSP) and the p38MAPK pathway are involved in cell responses against stress and, interestingly, we noted that mature monocyte-derived dendritic cells (Mo-DC) from breast cancer patients present, indeed, higher levels of HSP27 mRNA. Furthermore, HSP27 is a phosphorylation target of the p38 pathway and high HSP27 levels have been associated with impaired Mo-DC differentiation. To investigate these phenomena we exposed monocytes from healthy donors to a tumor cell line supernatant and evaluated their differentiation into DCs and the activation of HSP27 and p38 in the differentiating cells. Methods and Results: Monocytes from healthy donors were negatively selected by magnetic beads and induced to differentiate into DCs (by treatment with GM-CSF and IL-4) either in the presence of 20% conditioned medium (CM) from the breast adenocarcinoma cell line SK-BR-3 (tDC) or not (cDC). The surface phenotype, the presence of total p38, total HSP27, phospho-p38, phospho-HSP27 and the levels of IL-1beta, IL-6, IL-8, IL-10, TNF-alpha and IL-12p70 were evaluated at different time points of the culture, by flow cytometry. At 24h tDC cells expressed less HLA-DR, CD11c and CD86 and showed higher levels of phospho-p38, but less phospho-HSP27. At 72h of culture tDC contained a higher frequency of PD-L1+ cells. Furthermore, during differentiation, tDCs expressed higher levels of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha, but were produced less IL-12p70 in response to lipopolysaccharide (LPS) in comparison with cDCs. Conclusions: CM from SK-BR-3 cells affected the differentiation of Mo-DCs, inducing a phenotype with lesser expression of molecules associated with the induction of immune responses (HLA-DR, CD11c and CD86) but higher expression of PD-L1, a molecule associated with the induction of immune suppression. Dendritic cells exposed to CM also produced high levels of cytokines since the first day of differentiation. This cytokine production exposes the cells simultaneously to signals of differentiation (GM-CSF and IL-4, added to the culture) and maturation (TNF-alpha and IL-1beta produced by the cells), what might impair their differentiation. This was concomitant to an early increase in p38 phosphorylation, but, surprisingly, lower levels of phospho-HSP27 in the same cells. Though preliminary, these data suggest that the p38MAPK pathway may be involved, indeed, in the negative effects of tumors upon the differentiation of Mo-DCs into effective inducers of anti-tumor immune responses. Financial Support: FAPESP. Citation Format: Bruna Zelante, José Alexandre Marzagão Barbuto. Altered monocyte-derived dendritic cell differentiation in the presence of tumor supernatant: Possible involvement the p38MAPK pathway. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A84.

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.

Inducing extra copies of the Hsp70 gene in Drosophila melanogaster increases energetic demand

BackgroundMutations that increase gene expression are predicted to increase energy allocation to transcription, translation and protein function. Despite an appreciation that energetic tradeoffs may constrain adaptation, the energetic costs of increased gene expression are challenging to quantify and thus easily ignored when modeling the evolution of gene expression, particularly for multicellular organisms. Here we use the well-characterized, inducible heat-shock response to test whether expressing additional copies of the Hsp70 gene increases energetic demand in Drosophila melanogaster.ResultsWe measured metabolic rates of larvae with different copy numbers of the Hsp70 gene to quantify energy expenditure before, during, and after exposure to 36°C, a temperature known to induce robust expression of Hsp70. We observed a rise in metabolic rate within the first 30 minutes of 36°C exposure above and beyond the increase in routine metabolic rate at 36°C. The magnitude of this increase in metabolic rate was positively correlated with Hsp70 gene copy number and reflected an increase as great as 35% of the 22°C metabolic rate. Gene copy number also affected Hsp70 mRNA levels as early as 15 minutes after larvae were placed at 36°C, demonstrating that gene copy number affects transcript abundance on the same timescale as the metabolic effects that we observed. Inducing Hsp70 also had lasting physiological costs, as larvae had significantly depressed metabolic rate when returned to 22°C after induction.ConclusionsOur results demonstrate both immediate and persistent energetic consequences of gene copy number in a multicellular organism. We discuss these consequences in the context of existing literature on the pleiotropic effects of variation in Hsp70 copy number, and argue that the increased energetic demand of expressing extra copies of Hsp70 may contribute to known tradeoffs in physiological performance of extra-copy larvae. Physiological costs of mutations that greatly increase gene expression, such as these, may constrain their utility for adaptive evolution.

Effects of dietary nucleotides on acute stress response and cannabinoid receptor 1 mRNAs in sole, Solea solea

The objective of the present study was to evaluate the modulation of acute stress response by dietary nucleotides (NT) in sole, Solea solea. A basal diet was supplemented with levels of 0 (normal diet), or 0.4g NT/kg dry diet for 8weeks. At the end of feeding trial, fish fed the normal and NT-supplemented diet were subjected to a standardized protocol of disturbance and sampled over a 24h recovery after the stressor exposure. Modulatory effects of NT on acute stress response (cortisol and glucose), proopiomelanocortin (POMC) and cannabinoid receptor 1 splice variants (CB1A and CB1B) mRNA levels were studied. Both plasma cortisol and glucose levels of fish fed NT-supplemented diet were significantly lower than fish fed the control diet at 1 and 4h post-stress time-points. There are no significant effects of dietary NT on POMC and HSP70 mRNA levels. In our study, both CB1A and CB1B trascript levels were induced in fish fed the normal diet at 1 and 4h post-stress intervals. Collectively, the results obtained suggest that dietary NT modulates the CB1-like receptor mRNA expressions leading to attenuation in stressor-induced plasma cortisol level in sole.

Expression profiles of heat shock protein gene families in the monogonont rotifer Brachionus koreanus — exposed to copper and cadmium

Heat shock proteins play an important role in protein homeostasis under stress conditions, and act as molecular chaperons in living organisms. Here, we investigate acute toxicity of heavy metals (Cu and Cd) and the transcriptional changes of the heat shock proteins (hsps) family genes (hsp20, hsp27, hsp70, hsp90α-1, and hsp90β) in Brachionus koreanus-exposed to heavy metals. As a result, 48-h LC50 values were 0.956 mg/L (95%-cl. 0.678–1.348) for Cu and 57.563 mg/L (95%-cl. 47.691-69.477) for Cd. The expressions of hsp90α-1 and hsp70 mRNA level at all concentrations were significantly upregulated 24 h after exposure to Cu and Cd. In particular, a significant increase in hsp27 and hsp70 mRNA level indicated higher sensitivity to Cd. This finding indicates that HSPs play protective roles under heavy metal stress in this species. In addition, the expression profiles of hsp27, hsp70, and hsp90α-1 may be useful as molecular marker for monitoring heavy metal toxicity in the aquatic environment.

In Planta Evidence for the Involvement of a Ubiquitin Conjugating Enzyme (UBC E2 clade) in Negative Regulation of Disease Resistance

One of the three main components of the ubiquitylation system is an ubiquitin-conjugating enzyme (UBC, E2) that attaches ubiquitin (Ub) to a substrate. A strong link has been discovered between ubiquitylation and regulation of plant defense responses against plant pathogens. In this study, the role of UBC in response to pathogen attack is investigated in Arabidopsis thaliana as it has three Ubc gene homologs (Ubc1-1, Ubc2-1, and Ubc3-1). Analysis of single mutants of these three Ubc genes revealed enhanced disease resistance. Moreover, virus-induced gene silencing (VIGS) analysis revealed an enhanced level of resistance of Nicotiana benthamiana following inoculation with avirulent Pto DC3000 and virulent Pto DC3000 hopQ1-1 strains. In addition, RAR1, SGT1, and HSP90 mRNA levels are up-regulated following Ubc2 silencing. This study shows that UBC2 negatively regulates disease resistance via SGT1 level. Sub-cellular localization assay of UBC2 protein indicates its nuclear and cytoplasmic localization in planta.

Heat Shock and Caloric Restriction Have a Synergistic Effect on the Heat Shock Response in a sir2.1-dependent Manner in Caenorhabditis elegans

The heat shock response (HSR) is responsible for maintaining cellular and organismal health through the regulation of proteostasis. Recent data demonstrating that the mammalian HSR is regulated by SIRT1 suggest that this response may be under metabolic control. To test this hypothesis, we have determined the effect of caloric restriction in Caenorhabditis elegans on activation of the HSR and have found a synergistic effect on the induction of hsp70 gene expression. The homolog of mammalian SIRT1 in C. elegans is Sir2.1. Using a mutated C. elegans strain with a sir2.1 deletion, we show that heat shock and caloric restriction cooperate to promote increased survivability and fitness in a sir2.1-dependent manner. Finally, we show that caloric restriction increases the ability of heat shock to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1.

Genotoxicity assessment and detoxification induction in Dreissena polymorpha exposed to benzo[a]pyrene

The use of DNA adduct analysis has previously focused on the use of marine organisms for biomonitoring, whereas similar investigations in freshwater organisms are sparse. In that context, we have investigated the relevance of DNA adducts as biomarkers of genotoxicity in the freshwater mussels Dreissena polymorpha. The objective of the present study is to determine the stability of DNA adducts induced by benzo[a]pyrene (B[a]P) in zebra mussels. Mussels were exposed to dissolved B[a]P (10-100 µg/l) for 4 days. Afterwards, mussels were kept in clean water for 28 days and DNA adduct levels were subsequently measured in two different organs, the digestive glands and the gills, using the (32)P-postlabelling technique. In parallel, the expression of genes involved in the detoxification system was assessed by qPCR (catalase, superoxide dismutase, glutathione S transferase, HSP70, aryl hydrocarbon receptor, P glycoprotein). We observed a higher level of DNA adducts in the digestive glands compared to the gills. Moreover, in gills, the level of DNA adduct was dependent on the B[a]P concentration. The levels of adducts tended to decrease in both organs after 28 days in clean water. In addition, an early induction of HSP70, PgP, AHR and SOD mRNA levels was noticed in the gills compared to the digestive glands. CAT and GST gene expression increased from 12h exposure in both organs. A higher gene expression level of those genes was observed in the gills, except for AHR and CAT genes. Data converge towards the fact that DNA adducts hence represent a very promising biomarker of B[a]P contamination and potentially of exposure to polycyclic aromatic hydrocarbons. In addition, for the first time in this study, B[a]P detoxification system was characterised in D. polymorpha.