Hsc70 mRNA Research Articles

Effects of juvenile thermal preconditioning on the heat-shock, immune, and stress responses of rainbow trout upon a secondary thermal challenge

Higher water temperatures and pathogens are both significant factors that negatively affect the welfare of teleost fish. In aquaculture, compared to natural populations, these problems are especially exacerbated, as the animals have relatively limited mobility, and the higher density promotes faster spread of infectious diseases. Because of the potential harm these stressors can inflict, methods that can limit the damage of these stressors are particularly valuable. As a method of interest, early-life thermal preconditioning of animals demonstrated some potential for effective improvements in thermotolerance. However, the potential effects of the method on the immune system via the heat-stress model have not been explored. In this experiment, juvenile-stage thermal preconditioned rainbow trout (Oncorhynchus mykiss) were subjected to a secondary thermal challenge, animals were collected and sampled at the time of lost equilibrium. The effects of preconditioning on the general stress response was assessed by measuring the plasma cortisol levels. In addition, we also examined hsp70 and hsc70 mRNA levels in the spleen and gill tissues, as well as IL-1β, IL-6, TNF-α, IFN-1, β2m, and MH class I transcripts via qRT-PCR. No changes in CTmax were observed between the preconditioned and control cohorts upon the second challenge. IL-1β and IL-6 transcripts were generally upregulated with increased temperature of the secondary thermal challenge, whereas IFN-1 transcripts were upregulated in the spleen, but downregulated in the gills, along with MH class I. The juvenile thermal preconditioning produced a series of changes in transcript levels for IL-1β, TNF-α, IFN-1, and hsp70 but the dynamics of these differences were inconsistent. Finally, analysis of plasma cortisol levels presented significantly lower cortisol levels in the pre-conditioned animals compared to the non-pre-conditioned control cohort.

Expression of Heat Shock Genes in Hepatocytes is Affected by Age and Food Restriction in Rats

The objective of this study was to determine how food restriction altered the age-related changes in the basal and heat-induced expression of heat shock genes (hsc70, grp78, and ubiquitin) by hepatocytes isolated from young (4 to 6 mo old) and old (26 to 28 mo old) male Fischer 344 rats. The basal levels of the mRNA transcripts for hsc70 and ubiquitin were similar for hepatocytes isolated from young and old rats fed with free access to the diet (control) or from rats fed a restricted diet (40% restriction of food intake). However, the induction of the mRNA transcripts for hsc70 and ubiquitin by a heat shock (42.5°C for 30 min) was significantly higher for hepatocytes isolated from old rats fed the restricted diet compared with old rats fed with free access to the diet. The changes in hsc70 mRNA levels were paralleled by similar changes in hsc70 transcription by isolated nuclei; therefore, the greater induction of hsc70 expression by food restriction results from changes in the transcription of hsc70 gene. In contrast with hsc70 and ubiquitin, the basal levels of grp78 were reduced with age and by food restriction. Therefore, the effect of aging and food restriction on the basal and heat-induced expression of heat shock genes varies considerably from gene to gene.

Chaperone Mediated Autophagy Substrates and Components in Cancer.

Chaperone-mediated autophagy (CMA) represents a specific way of lysosomal protein degradation and contrary to macro and microautophagy is independent of vesicles formation. The role of CMA in different physiopathological processes has been studied for several years. In cancer, alterations of the CMA principal components, Hsc70 and Lamp2A protein and mRNA levels, have been described in malignant cells. However, changes in the expression levels of these CMA components are not always associated with changes in CMA activity and their biological significance must be carefully interpreted case by case. The objective of this review is to discuss whether altering the CMA activity, CMA substrates or CMA components is accurate to avoid cancer progression. In particular, this review will discuss about the evidences in which alterations CMA components Lamp2A and Hsc70 are associated or not with changes in CMA activity in different cancer types. This analysis will help to better understand the role of CMA activity in cancer and to elucidate whether CMA can be considered as target for therapeutics. Further, it will help to define whether the attention of the investigation should be focused on Lamp2A and Hsc70 because they can have an independent role in cancer progression beyond of their participation in altered CMA activity.

Physicochemical changes in liver and Hsc70 expression in pikeperch Sander lucioperca under heat stress

The pikeperch Sander lucioperca is an economically important freshwater species that is currently threatened by higher summer temperatures caused by global warming. To clarify the physiological state of pikeperch reared under relatively high temperatures and to acquire valuable biomarkers to monitor heat stress in this species, 100 fish were subjected to five different temperature treatments, ranging from 23 °C (control) to 36 °C. The physiological and biochemical indexes of liver and blood were determined, and heat-shock cognate 70 kDa protein (Hsc70) mRNA expression profiles were analyzed. The results showed that the activities of superoxide dismutase, catalase, and glutathione peroxidase in heat-stressed pikeperch first increased and then decreased, exhibiting peaks at 34 °C, 28 °C, and 28 °C, respectively. The level of thiobarbituric acid-reactive substances (TBARS) in all experimental groups was significantly higher than that of the control. The numbers of red blood cells, the packed-cell volume, and the contents of hemoglobin were significantly higher in the 34 °C and 36 °C treatment groups. Under heat stress, the albumin, cholesterol, and triglycerides contents decreased with increasing temperatures. Real-time fluorescence-based quantitative RT-PCR showed that Hsc70 mRNA levels increased in all eight of the tested tissues under heat stress. Expression reached maximum levels at 34 °C in the muscle, heart and gill tissues, and at 36 °C in the other five tissues. These results demonstrate that several physiological and biochemical phenotypes, such as oxidative stress, antioxidant enzymes and molecular chaperones, could be important biomarkers of heat stress in pikeperch, and are potentially valuable to uncover the mechanisms of heat-stress responses in fish.

Cloning of heat shock protein genes (hsp70, hsc70 and hsp90) and their expression in response to larval diapause and thermal stress in the wheat blossom midge, Sitodiplosis mosellana

Sitodiplosis mosellana Géhin, one of the most important pests of wheat, undergoes obligatory diapause as a larva to survive unfavorable temperature extremes during hot summers and cold winters. To explore the potential roles of heat shock proteins (hsp) in this process, we cloned full-length cDNAs of hsp70, hsc70 and hsp90 from S. mosellana larvae, and examined their expression in response to diapause and short-term temperature stresses. Three hsps included all signature sequences of corresponding protein family and EEVD motifs. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known classification of insects. Expression of hsp70 and hsp90 were highly induced by diapause, particularly pronounced during summer and winter. Interestingly, hsp70 was more strongly expressed in summer than in winter whereas hsp90 displayed the opposite pattern. Abundance of hsc70 mRNA was comparable prior to and during diapauses and was highly up-regulated when insects began to enter the stage of post-diapause quiescence. Heat-stressed over-summering larvae (⩾30°C) or cold-stressed over-wintering larvae (⩽0°C) could further elevate expression of these three genes, but temperature extremes i.e. as high as 45°C or as low as −15°C failed to trigger such expression patterns. Notably, hsp70 was most sensitive to heat stress and hsp90 was most sensitive to cold stress. These results suggested that hsp70 and hsp90 play key roles in diapause maintenance and thermal stress; the former may be more prominent contributor to heat tolerance and the latter for cold tolerance. In contrast, hsc70 most likely is involved in developmental transition from diapause to post-diapause quiescence, and thus may serve as a molecular marker to predict diapause termination.

Potential role of heat-shock protein 70 and interleukin-15 in the pathogenesis of threatened spontaneous abortions.

The role of HSP70 and both its constitutive (Hsc) and inducible (Hsp) forms in the pathogenesis of threatened spontaneous abortions was investigated. Immunohistology and/or immunofluorescence was used to analyze paraffin-embedded tissue sections, and reverse transcriptase-quantitative polymerase chain reaction and flow cytometry were used for analyses of decidual mononuclear cells (DMCs) and confocal microscopy for the detection of perforin, granulysin, and lysosome-associated membrane protein-1 (LAMP-1) in decidual lymphocytes (DLs). The percentage of single Hsp70(+) , Hsc70(+) , and IL-15(+) cells and mRNA levels of HSP70, CD91, and TLR4 were lower in the decidua basalis in cases of threatened miscarriages compared to that in cases of normal pregnancy. In a suspension of normal DMCs, IL-15 significantly decreased the HSP70 members and TLR4 in dendritic cells, T cells, and NK cells while increasing CD91 in NK cells alone. Downregulation of Hsc70, Hsp70, and IL-15 expression at gene and/or protein levels might support the retention of fertilization products in cases of missed abortion and blighted ovum.

Rotenone down-regulates HSPA8/hsc70 chaperone protein in vitro: A new possible toxic mechanism contributing to Parkinson’s disease

HSPA8/hsc70 (70-kDa heat shock cognate) chaperone protein exerts multiple protective roles. Beside its ability to confer to the cells a generic resistance against several metabolic stresses, it is also involved in at least two critical processes whose activity is essential in preventing Parkinson’s disease (PD) pathology. Actually, hsc70 protein acts as the main carrier of chaperone-mediated autophagy (CMA), a selective catabolic pathway for alpha-synuclein, the main pathogenic protein that accumulates in degenerating dopaminergic neurons in PD. Furthermore, hsc70 efficiently fragments alpha-synuclein fibrils in vitro and promotes depolymerization into non-toxic alpha-synuclein monomers.Considering that the mitochondrial complex I inhibitor rotenone, used to generate PD animal models, induces alpha-synuclein aggregation, this study was designed in order to verify whether rotenone exposure leads to hsc70 alteration possibly contributing to alpha-synuclein aggregation. To this aim, human SH-SY5Y neuroblastoma cells were treated with rotenone and hsc70 mRNA and protein expression were assessed; the effect of rotenone on hsc70 was compared with that exerted by hydrogen peroxide, a generic oxidative stress donor with no inhibitory activity on mitochondrial complex I. Furthermore, the effect of rotenone on hsc70 was verified in primary mouse cortical neurons. The possible contribution of macroautophagy to rotenone-induced hsc70 modulation was explored and the influence of hsc70 gene silencing on neurotoxicity was assessed. We demonstrated that rotenone, but not hydrogen peroxide, induced a significant reduction of hsc70 mRNA and protein expression. We also observed that the toxic effect of rotenone on alpha-synuclein levels was amplified when macroautophagy was inhibited, although rotenone-induced hsc70 reduction was independent from macroautophagy. Finally, we demonstrated that hsc70 gene silencing up-regulated alpha-synuclein mRNA and protein levels without affecting cell viability and without altering rotenone- and hydrogen peroxide-induced cytotoxicity.These findings demonstrate the existence of a novel mechanism of rotenone toxicity mediated by hsc70 and indicate that dysfunction of both CMA and macroautophagy can synergistically exacerbate alpha-synuclein toxicity, suggesting that hsc70 up-regulation may represent a valuable therapeutic strategy for PD.

Study on the immune response to recombinant Hsp70 protein from Megalobrama amblycephala

The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1β mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance.

Effect of chaperone-mediated autophagy in MPP+-induced SH-SY5Y cells and interventional effect of puerarin

To study the protective effect of puerarin on MPP(+) -induced SH-SY5Y cells by chaperone-mediated autophagy (CMA). The Parkinson's disease cell model was established by injuring SH-SY5Y cells with 1 mmol x L(-1) MPP+. The CCK-8 staining was adopted to detect the effect the puerarin of different concentrations on the survival rate of MPP(+)-induced SH-SYSY cells. The autophagosome formation was observed under transmission electron microscope. The AO staining showed the changes in the lysosome activity. RT-PCR was used to detect the changes in Lamp2a and Hsc70 mRNA expressions. The western blotting was adopted to test the expressions of Lamp2a, Hsc70 and alpha-synuclein protein in cells. Within the concentration range of 12. 5-50.0 micromol x L(-1), the pretreatment with puerain for 30 minutes could protect the injury of MPP+ in SH-SY5Y cells, and showed a certain dose-effect relationship. The AO staining and electron microscope showed the effect of puerain within the concentration range of 12.5-50.0 micromol x L(-1) on 1 mmol x L(-1) MPP(+)-induced SH-SY5Y cells; autophagosomes emerged in cells, and increased along with the rise in the puerarin dose. The results of the flow cytometry revealed that 50.0 micromol x L(-1) of puerarin could protect against the increase of the ROS level in 1 mmol x L(-1) MPP(+) -induced SH-SY5Y cells and prevent the oxidative injury. The results of RT-PCR and western blotting indicated that puerain within the concentration range of 12.5-50.0 micromol x L(-1) alleviated the MPP(+)-induced SH-SY5Y cell injury, and inhibited the accumulation of alpha-synuclein proteins in MPP(+) -induced SH-SY5Y cells by up-regulating Hsc70, Lamp2a mRNA and protein level. Puerarin could protect against the MPP(+) -induced cell injury, whose protective mechanism may be related to the chaperone-mediated autophagy pathway of interventional molecules.

Anti-stress properties and two HSP70s mRNA expressions of blunt snout bream (Megalobrama amblycephala) fed with all-plant-based diet

The influence of all-plant-based diet on fingerling blunt snout breams (Megalobrama amblycephala) was tested by examining growth performance, anti-stress properties and related gene expression. Healthy fish were randomly divided into triplicate groups per dietary treatment and fed with different formulated diets. The results showed that both weight gain, specific growth rate and protein efficiency ratio of all-plant-based diet group were significant higher than those of the control (p<0.05). In contrast, FCR of all-plant-based diet group was significantly lower than that of the control (p<0.05). Therefore, all-plant-based diets could not affect the growth performance of blunt snout breams. Compared to the control group, the lysozyme levels in serum and mucus, and glutamic-oxaloacetic transaminase activities in serum and liver decreased significantly (p<0.05). In contrast, the glutamic-pyruvic transaminase activities in serum and liver increased significantly (p<0.05). For blunt snout breams fed with all-plant-based diets, the superoxide dismutase activities in mucus, serum and liver as well as catalase activity in serum and liver were decreased significantly (p<0.05) comparing with that of the control group. But malondialdehyde contents were higher (p<0.05) in serum and liver than that of control group. The expression of HSC70 mRNA increased significantly (p<0.05) in blunt snout breams fed with all-plant-based diet, whereas the HSP70 mRNA expression decreased significantly (p<0.05) when compared with control group. In conclusion, all these results indicated that the application of all-plant-based diet could decrease the anti-stress properties (non-specific immunity, stress resistance and antioxidant ability) and HSP70 mRNA expression in blunt snout breams fingerling. Although all-plant-based diets could not affect the growth performance of blunt snout breams, the application of all-plant-based diet should be discreet in the production practice.

Chaperone‐mediated autophagy components are upregulated in sporadic inclusion‐body myositis muscle fibres

Sporadic inclusion-body myositis (s-IBM) is an age-associated degenerative muscle disease. Characteristic features are muscle-fibre vacuolization and intramuscle-fibre accumulations of multiprotein aggregates, which may result from the demonstrated impairments of the 26S proteasome and autophagy. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal degradation targeting proteins carrying the KFERQ motif. Lysosome-associated membrane protein type 2A (LAMP2A) and the heat-shock cognate protein 70 (Hsc70) constitute specific CMA components. Neither CMA components nor CMA activity has been studied in normal or disease human muscle, to our knowledge. We studied CMA components by immunocytochemistry, immunoblots, real-time PCR and immunoprecipitation in: (a) 16 s-IBM, nine aged-matched normal and nine disease control muscle biopsies; and (b) cultured human muscle fibres (CHMFs) with experimentally inhibited activities of either the 26S proteasome or autophagy. Compared with age-matched controls, in s-IBM muscle, LAMP2A and Hsc70 were on a given transverse section accumulated as aggregates in approximately 5% of muscle fibres, where they (a) colocalized with each other and α-synuclein (α-syn), a CMA-targeted protein; and (b) were bound to each other and to α-syn by immunoprecipitation. By immunoblots, LAMP2A was increased sevenfold P < 0.001 and Hsc70 2.6-fold P < 0.05. LAMP2A mRNA was increased 4.4-fold P < 0.001 and Hsc70 mRNA 1.9-fold P < 0.05. In CHMFs inhibition of either the 26S proteasome or autophagy induced CMA, evidenced by a significant increase of both LAMP2A and Hsc70. Our study demonstrates, for the first time, up-regulation of CMA components in s-IBM muscle, and it provides further evidence that altered protein degradation is likely an important pathogenic aspect in s-IBM.

MRNA expression of heat-shock cognate protein 70 gene (HSC70) in the oyster (Crassostrea hongkongensis) in response to Vibrio algi-nolyticus challenge

We developed a fluorescent real-time quantitative RT-PCR method to evaluate the pattern of HSC70 mRNA expression in response to the bacterial infection in the oyster(Crassostrea hongkongensis).We injected oysters with live Vibrio alginolyticus,and measured HSC70 gene expression in six tissues(mantle,gills,digestive gland,muscle,heart and hemocytes).We measured the accumulation and distribution of V.alginolyticus using the plate counting method.HSC70 mRNA expression was significantly up-regulated in all the six tissues following injection with V.alginolyticus,and the expression was time-dependent.HSC70 gene expression increased signifi-cantly at 6 and 72 h in the gill.Similarly,levels peaked at 6 h in the digestive gland then remained high until 24 h,after which they decreased rapidly to control levels.HSC70 mRNA expression was ~10 fold higher in the haemo-cytes than the control after 3 h,and the expression evels peaked at 6 h(~15 fold higher than the control).The ex-pression of HSC70 was ~2.5 fold higher in the remaining tissues than in the control.The bacterial concentration was higher in the muscle than that in other tissues(digestive gland and hemolymph) at 3 h after the oysters were injected with V.alginolyticus,though levels had declined by 6 h.The accumulation of bacteria in the muscle in-creased at 12 h and remained high up to 72 h.The change in V.alginolyticus concentration was time-dependent in the digestive gland whereas the bacteria increased with time and peaked at 48 h in the hemolymph.We observed a positive correlation between HSC70 mRNA expression and the concentration of bacteria,most obvious in the di-gestive gland.Our results suggest that bacterial challenge induces the up-regulation of HSC70 mRNA expression and suggests that HSC70 plays an important role in protecting the oyster against the bacterial pathogen.

Different levels of hsp70 and hsc70 mRNA expression in Iberian fish exposed to distinct river conditions

Comprehension of the mechanisms by which ectotherms, such as fish, respond to thermal stress is paramount for understanding the threats that environmental changes may pose to wild populations. Heat shock proteins are molecular chaperones with an important role in several stress conditions such as high temperatures. In the Iberian Peninsula, particularly in Portugal, freshwater fish of the genus Squalius are subject to daily and seasonal temperature variations. To examine the extent to which different thermal regimes influence the expression patterns of hsp70 and hsc70 transcripts we exposed two species of Squalius (S. torgalensis and S. carolitertii) to different temperatures (20, 25, 30 and 35 °C). At 35 °C, there was a significant increase in the expression of hsp70 and hsc70 in the southern species, S. torgalensis, while the northern species, S. carolitertii, showed no increase in the expression of these genes; however, some individuals of the latter species died when exposed to 35 °C. These results suggest that S. torgalensis may cope better with harsher temperatures that are characteristic of this species natural environment; S. carolitertii, on the other hand, may be unable to deal with the extreme temperatures faced by the southern species.

Delayed Temporal Increase of Hepatic Hsp70 in ApoE Knockout Mice After Prenatal Arsenic Exposure

Prenatal arsenic exposure accelerates atherosclerosis in ApoE(-/-) mice by unknown mechanism. Arsenic is a hepatotoxicant, and liver disease increases atherosclerosis risk. Prenatal arsenic exposure may predispose to liver disease by priming for susceptibility to other environmental insults. Earlier microarray analyses showed prenatal arsenic exposure increased Hsc70 (HspA8) and Hsp70 (HspA1a) mRNAs in livers of 10-week-old mice. We determined effects of prenatal arsenic exposure on hepatic Hsp70 and Hsc70 expression by Western blot and on DNA methylation by methyl acceptance assay during prenatal and postnatal development. Pregnant ApoE(-/-) mice were given drinking water containing 85 mg/l NaAsO(2) (49 ppm arsenic) from gestation day (GD) 8 to 18. Hsp70 and Hsc70 expression and DNA methylation were determined in GD18 fetuses and 3-, 10-, and 24-week-old mice. Hsc70 expression was unchanged at all ages. Hsp70 induction was observed at 3 and 10 weeks, but was unchanged in GD18 fetuses and 24-week livers of mice. Global DNA methylation increased with age; arsenic had no effects. Bisulfite sequencing of DNA from livers of 10-week-old mice showed Hsp70 promoter region methylation was unchanged, but methylation was increased within the transcribed region. Hsf1 and Nrf2 nuclear translocation were investigated as potential mechanisms of Hsp70 induction and found unaltered. Putative binding sites were identified in HSP70 for in utero arsenic exposure-suppressed microRNAs suggesting a possible mechanism. Thus, prenatal arsenic exposure causes delayed temporal hepatic Hsp70 induction, suggesting a transient state of stress in livers which can predispose the mice to developing liver disease.

In vitro activity of cepharanthine hydrochloride against clinical wild-type and lamivudine-resistant hepatitis B virus isolates

Hepatitis B virus (HBV) infection causes major public health problems worldwide. The clinical limitation of current antiviral drugs for HBV, such as lamivudine, is the emergence of drug-resistant viral strains during prolonged antiviral therapy. Cepharanthine hydrochloride (CH), a natural alkaloid-derived compound, has been reported to possess potent activity against various viruses. The present study was performed to evaluate the in vitro activity of CH against clinical wild-type and lamivudine-resistant HBV isolates in transiently transfected cells. HBV DNA was extracted from serum samples collected both before lamivudine therapy and at the time of viral breakthrough and was amplified by polymerase chain reaction (PCR). The amplicons were cloned into a novel expression vector, pHY106, which can initiate the intracellular HBV replication cycle after cell transfection. Following transfection of the cloned amplicon into HepG2 cells, a drug susceptibility assay was performed. The level of viral antigen, HBeAg, was determined by enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR (Q-PCR) was used for determining the amount of intracellular HBV DNA. Heat stress cognate 70 (Hsc70), a host protein required for HBV replication, was also analyzed by reverse transcription PCR (RT-PCR) to explore the possible antiviral mechanism of CH. The results showed that CH inhibited replication and HBeAg production by either wild-type or lamivudine-resistant HBV clinical isolates in a dose-dependent manner. The Hsc70 mRNA was also downregulated significantly. In conclusion, CH is active against both wild-type and lamivudine-resistant HBV clinical isolates, and its activity may be associated with its inhibition of host Hsc70.

Effects of emodin and vitamin C on growth performance, biochemical parameters and two HSP70s mRNA expression of Wuchang bream (Megalobrama amblycephala Yih) under high temperature stress

In order to study the effects of dietary emodin, high-dose vitamin C (Vc) and their combination on growth of Wuchang bream (Megalobrama amblycephala Y.) and its resistance to high temperature stress, 1200 healthy Wuchang bream with initial body weight of 133.44 ± 2.11 g were randomly divided into four groups: a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin, 700 mg/kg Vc, and the combination of 60 mg/kg emodin + 700 mg/kg Vc, respectively. After feeding for 60 days, the growth performance of Wuchang bream was measured. Then 25 fish per tank were exposed to heat stress of 34 °C. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that before stress, compared with the control, the weight gain (WG) and specific growth rate (SGR), serum total protein (TP), lysozyme (LSZ), and alkaline phosphatase (ALP) levels, liver superoxide dismutase (SOD) activity and expression level of HSP70 mRNA significantly increased in emodin and Vc groups while feed conversion rate (FCR), serum cortisol (COR), triglyceride (TG) and liver malondialdehyde (MDA) contents decreased (P < 0.05); liver catalase (CAT) activity also significantly increased in emodin group (P < 0.05). Although serum TP, LSZ, and liver HSP70 mRNA levels significantly increased and liver MDA level decreased in combination group (P < 0.05), no synergism was observed.After heat stress, compared with the control, the serum TP, LSZ, ALP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P < 0.05) while the difference between the combination and control groups was not significant (P > 0.05). Thus, the basal diet supplemented with 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, reduce FCR, increase non-specific immunity of fish, antioxidant capacity, and two HSP70s mRNA expression levels, and enhance resistance to heat stress in fish. However, the combination of emodin and high-dose Vc showed no better effect.

Synthesis, structure−activity relationship and biological evaluation of novel N-substituted matrinic acid derivatives as host heat-stress cognate 70 (Hsc70) down-regulators

Oxymatrine (1) is a natural anti-hepatitis B virus (HBV) drug that down-regulates host heat-stress cognate 70 (Hsc70) expression through a mechanism different from that of nucleosides. Taking Hsc70 as a target against HBV, 26 novel N-substituted matrinic acid analogs were designed, synthesized and evaluated for their regulation of Hsc70 mRNA expression with 1 as the lead. The SAR analysis revealed that (i) the carboxyl group at the 11-position was required for activity; (ii) introducing of a substituent on the nitrogen atom at the 12-position of 3, especially substituted benzyl, might significantly improve the activity. Among these analogs, compound 9p possessing N-p-methoxylbenzyl afforded an increased anti-HBV effect in comparison with 1. We consider 9p a promising anti-HBV candidate.

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Cold stenothermal cave-dwelling beetles do have an HSP70 heat shock response

The response to high temperatures in adults of two cold stenothermal cave-dwelling leptodirins, Neobathyscia mancinii and Neobathyscia pasai (Coleoptera, Cholevidae) was evaluated by determinating levels of gene expression of two members of the family of heat shock proteins 70 kDa by qPCR. In both species, hsc70 mRNA level was constant with increasing temperature, whereas a significant increase in the inducible member ( hsp70) mRNA was observed, higher in N. pasai. This difference could be due to their in-cave distribution: N. pasai colonizes the cave entrance where the temperature is more variable than the internal part where N. mancinii is confined. These results demonstrated for the first time the occurrence of a heat shock response in troglobite insects and suggest the correlation between the intensity of this response and the adaptation to the cave environment.

Characterization of two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): Evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes

Two cDNAs, encoding the stress-inducible 70-kDa heat shock protein (Hsp70) and the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), were isolated from grass carp. The Hsp70 and Hsc70 cDNAs were 2250 bp and 2449 bp in length and contained 1932 bp and 1953 bp open reading frames, respectively. Tissue distribution results showed that Hsp70/Hsc70 was highly expressed in gill, kidney, head kidney and peripheral blood lymphocytes (PBLs). Using grass carp PBLs as a cell model, effects of lipopolysaccharide (LPS) on the mRNA and protein levels of Hsp70/Hsc70 were examined. In this case, LPS increased the mRNA expression of Hsp70 in a time- and dose-dependent manner, but had no effect on Hsc70 mRNA expression. In agreement with this, LPS elevated the intracellular Hsp70 markedly, but not the Hsc70 protein levels in parallel experiments. Furthermore, Hsp70 protein was also detected in culture medium. Moreover, inhibition of LPS on Hsp70 release in a time-dependent manner was observed, indicating that there may be a dynamic balance between Hsp70 stores and Hsp70 release in grass carp PBLs following exposure to LPS. Taken together, these results not only shed new insights into the different regulations of LPS on Hsp70/Hsc70 gene expression, protein synthesis and release, but also provide a basis for further study on the functional role of Hsp70 in fish immune response.