Influence of selenium sources on age-related and mild heat stress-related changes of blood and liver glutathione redox cycle in broiler chickens (Gallus domesticus)

Selenium is an essential trace element that up-regulates a major component of the antioxidant defense mechanism by controlling the body's glutathione (GSH) pool and its major Se-containing antioxidant enzyme, glutathione peroxidase (GPX). Evidence has emerged suggesting that organic selenium, natural seleno-amino acids found in plants, grains and selenized yeast, maintains the antioxidant defense system more efficiently than inorganic selenium. Inorganic selenium is a pro-oxidant, whereas organic selenium possesses antioxidant properties itself. As a pro-oxidant, inorganic selenium is not suitable for animals or humans. Therefore, we examined the GSH–GPX system in broiler chickens and determined that organic selenium was indeed more beneficial than inorganic selenium. Chickens fed the organic selenium as Sel-Plex^®, a selenized yeast, had elevated GPX activity in both blood and liver in a thermoneutral environment and after heat distress. More importantly, the ability to reduce the oxidized glutathione (GSSG to 2 GSH) was enhanced and facilitated by maintenance of glutathione reductase activity. Organic selenium-fed chickens were less affected by mild heat distress than inorganic selenium-fed chickens, and this assessment was based upon less induction of heat shock protein 70 (hsp70) in organic selenium-fed chickens. Our results clearly show that heat distress, a potent inducer of oxidative stress and hsp70, can be partially ameliorated by feeding organic selenium. We attribute this observation to an enhanced GSH–GPX antioxidant system in organic selenium-fed chickens.     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.     

Influence of organic selenium on hsp70 response of heat-stressed and enteropathogenic Escherichia coli-challenged broiler chickens (Gallus gallus)

The effect of dietary selenium yeast, a source of organic selenium, on heat shock protein 70 (hsp70) responses, redox status, growth and feed utilization were evaluated either in enteropathogenic Escherichia coli-challenged (EPEC) or in heat-stressed (HS) male broiler chickens grown to 42 days of age. One day-old chicks in experiment 1 were challenged orally with EPEC (10(6) cfu/chicken on day 1 and boosted by water application on days 2, 3, and 4) and fed diets with or without selenium yeast. Body weight (BW), feed conversion ratio (FCR), and total mortality were determined at 42 days of age, and this was followed by collection of ileal tissue for the quantification of total glutathione (TGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and hsp70 in randomly selected chickens from each treatment. In experiment 2, male broiler chickens were fed diets with or without selenium yeast under a thermoneutral rearing condition. At four weeks of age, blood and hepatic tissue were collected from chickens maintained in the thermoneutral environment and from chickens subjected to HS (40 degrees C for 1 h) and analyzed for TGSH, GSH, GSSG, and hsp70. Selenium yeast improved BW, FCR, and decreased mortality in both control and EPEC-challenged chicks. Selenium yeast significantly attenuated hsp70 expression in EPEC-challenged chickens and in those subjected to HS. The EPEC challenge increased TGSH and GSSG levels and decreased GSH/GSSG ratio. However, GSSG level accumulated in chickens fed diets without selenium supplementation resulting in a lower GSH/GSSG ratio in the selenium yeast-fed group. Heat stress increased GSSG level and decreased GSH/GSSG ratio. Selenium yeast-fed groups maintained higher levels of GSSG before and after HS with a resultant lower GSH/GSSG ratio. The hsp70 response was significantly less in those chickens fed selenium yeast and challenged with either EPEC or HS than in those chickens given no supplemental selenium. The results of this study suggest that selenium yeast supplementation had imparted resistance to oxidative stress associated with enteric bacteria infection and to high temperature exposure. It is believed that the resistance to the stressors was due to an improved redox status of the selenium yeast-fed chickens.     

Synthesis and biological evaluation of C-ring truncated deguelin derivatives as heat shock protein 90 (HSP90) inhibitors

Based on the lead compound L-80 (compound 2), a potent heat shock protein 90 (HSP90) inhibitor, a series of C-ring truncated deguelin analogs were designed, synthesized and evaluated for Hypoxia Inducible Factor-1α (HIF-1α) inhibition as a primary screening method. Their structure–activity relationship was investigated in a systematic manner by varying the A/B ring, linker and D/E ring, respectively. Among the synthesized inhibitors, compound 5 exhibited potent HIF-1α inhibition in a dose-dependent manner and significant antitumor activity in human non-small cell lung carcinoma (H1299), with better activities than L-80. It also inhibited in vitro hypoxia-mediated angiogenic processes in human retinal microvascular endothelial cells (HRMEC). The docking study of 5 showed a similar binding mode as L-80: it occupied the C-terminal ATP-binding pocket of HSP90, indicating that the anticancer and antiangiogenic activities of 5 were derived from HIF-1α destabilization by inhibiting the C-terminal ATP-binding site of hHSP90.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

A protocol comparison for the analysis of heat shock protein A1B +A1538G SNP

Heat shock proteins act as molecular chaperones, assist in peptide maturation, and transport nascent peptides across membranes. One commonly studied single nucleotide polymorphism (SNP) for one of the proteins is HSPA1B (+A1538G). However, several studies of this polymorphism have failed to achieve Hardy–Weinberg equilibrium (HWE) for their sample. We compared various published procedures for analyzing the HSPA1B +A1538G SNP and report reasons for HWE discrepancies. Samples from 141 apparently healthy, physically active, volunteers (99 men and 42 women) were analyzed. The first protocol, initially described by Schröder et al., resulted in a genotypic distribution of 22 GG (15.6%), 119 AG (84.4%), and 0 AA; results were confirmed by reanalysis and sequencing. Two other published protocols, one described by Klausz et al. and another by Fekete et al., were used to confirm these results: both resulted in 22 GG (15.6%), 46 AA (32.6%), and 73 AG (51.7%). Additionally, the results were within HWE and confirmed by sequence analysis. Of the original 119 subjects genotyped as AG by the Schröder protocol, 46 of those were confirmed as AA with the Klausz and Fekete methods. Mixing primers from the Schröder and Klausz protocol resulted in 100% concordance with the data generated by the Klausz and Fekete protocols. Some published data on HSP genotyping deviate from HWE; thus, primers used for analyzing these highly homologous genes must be carefully considered. Our results highlight the importance of reinvestigating data when HWE is not achieved for the HSPA1B, or another, polymorphism.     

Expression of heat shock protein 70 in transport-stressed broiler pectoralis major muscle and its relationship with meat quality

Omics research has indicated that heat shock protein 70 (HSP70) is a potential biomarker of meat quality. However, the specific changes and the potential role of HSP70 in postmortem meat quality development need to be further defined. In this study, Arbor Acres broiler chickens (n=126) were randomly categorized into three treatment groups of unstressed control (C), 0.5-h transport (T) and subsequent water shower spray following transport (T/W). Each treatment consisted of six replicates with seven birds each. The birds were transported according to a designed protocol. The pectoralis major (PM) muscles of the transport-stressed broilers were categorized as normal and pale, soft and exudative (PSE)-like muscle samples according to L* and pH_{24 h} values to test the expression and location of HSP70. Results revealed that the activities of plasma creatine kinase and lactate dehydrogenase increased significantly (P<0.05) in normal and PSE-like muscle samples after transportation. The mRNA expression of HSP70 in normal muscle samples increased significantly (P<0.05) compared with that in the controls after stress. The protein expression of HSP70 increased significantly in normal muscle samples and decreased significantly (P<0.05) in PSE-like muscles. Immuno-fluorescence showed that HSP70 was present in the cytoplasm and on surface membranes of PM muscle cells in the normal samples following stress. Meanwhile, HSP70 was present on the surface membranes and extracellular matrix but was barely visible in the cytoplasm of the PSE-like samples. Principal component analysis showed high correlations between HSP70 and meat quality and stress indicators. In conclusion, this research suggests that the variation in HSP70 expression may provide a novel insight into the pathways underlying meat quality development.     

Intracellular localization of the 90 kDA heat shock protein (HSP90alpha) determined by expression of a EGFP-HSP90alpha-fusion protein in unstressed and heat stressed 3T3 cells

Heat shock protein 90 (Hsp90) is an abundant protein and essential for all eukaryotic cells. The expression of Hsp90 is further enhanced after exposure to stress factors, e.g. a heat shock. Many proteins interacting with Hsp90 as well as the various functions for Hsp90 have been described. In this study, an Hsp90alpha fusion protein along with the enhanced green fluorescence protein (EGFP) was expressed under the control of the human cytomegalovirus immediate early promoter. EGFP-Hsp90alpha was mainly localized in the cytoplasm, with only minor amounts inside the nuclei. No EGFP-Hsp90alpha could be detected inside the nucleoli. Following exposure to elevated temperatures, higher amounts of EGFP-Hsp90alpha are inside the nucleus, but not within the nucleoli. As the most remarkable finding under these conditions, an association of EGFP-Hsp90alpha with the nuclear membrane became visible.     

The effect of heat shock on amino acid transport and cell volume in 3T3 cells

Summary. In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response. Received June 30, 1999 Accepted July 27, 2000     

Investigation of B,C-ring truncated deguelin derivatives as heat shock protein 90 (HSP90) inhibitors for use as anti-breast cancer agents

On the basis of deguelin, a series of the B,C-ring truncated surrogates with N-substituted amide linkers were investigated as HSP90 inhibitors. The structure activity relationship of the template was studied by incorporating various substitutions on the nitrogen of the amide linker and examining their HIF-1α inhibition. Among them, compound 57 showed potent HIF-1α inhibition and cytotoxicity in triple-negative breast cancer lines in a dose-dependent manner. Compound 57 downregulated expression and phosphorylation of major client proteins of HSP90 including AKT, ERK and STAT3, indicating that its antitumor activity was derived from the inhibition of HSP90 function. The molecular modeling of 57 demonstrated that 57 bound well to the C-terminal ATP-binding pocket in the open conformation of the hHSP90 homodimer with hydrogen bonding and pi-cation interactions. Overall, compound 57 is a potential antitumor agent for triple-negative breast cancer as a HSP90 C-terminal inhibitor.     

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.     

朱砂叶螨热激蛋白HSP70基因TCHSP70-4的克隆与表达

为了研究朱砂叶螨Tetranychus cinnabarinus(Boisduval)热激蛋白HSP70的表达与其适应高温和低温胁迫的关系,我们利用物种的同源性及RACE 技术,获得朱砂叶螨热激蛋白HSP70基因1个,命名为TCHSP70-4（GenBank 登录号为EU977182）。该基因全长2 182 bp,包含1 959 bp的开放阅读框,编码653个氨基酸,理论分子量为70.9 kDa,等电点为5.4,含有HSP70家族高度保守的基序。运用real-time PCR分析冷激（4℃）和热激（40℃）1 h后TCHSP70-4在朱砂叶螨体内的表达量。结果显示冷激后TCHSP70-4表达量明显下降,而热激后TCHSP70-4表达量却明显上升。这些结果一方面表明该基因属于诱导型HSP70基因,另一方面揭示了朱砂叶螨分别受到冷和热胁迫后体内TCHSP70-4的表达及所起的保护作用是不同的。     

Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.     

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO₄, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.     

The lost correlation between heat shock protein 70 (HSPA1A) and plasminogen activator inhibitor-1 in patients with type 2 diabetes and albuminuria

We aimed to study the relation between plasma levels of stress-induced heat shock protein 70 (HSPA1A) with plasminogen activator inhibitor-1 (PAI-1) and high-density lipoprotein cholesterol (HDL-C), apolipoprotein A1 (Apo-A1), and HDL-C/Apo-A1 ratio. In a matched case-control study on patients with diabetes (40 patients with albuminuria and 40 without albuminuria matched for age, sex, and body mass index), we observed that plasma levels of HSPA1A and PAI-1 are increased in patients with albuminuria (0.55 ± 0.02 vs. 0.77 ± 0.04 ng/ml, p value <0.001 for HSPA1A; 25.9 ± 2 vs. 31.8 ± 2.4 ng/ml, p value <0.05 for PAI-1). There was a significant correlation between HSPA1A and PAI-1 in diabetic patients without albuminuria (r = 0.28; p value = 0.04), but not in those with albuminuria (r = 0.07; p value = 0.63). No association was found between HSPA1A and HDL-C, between HSPA1A and Apo-A1, or between HSPA1A and HDL-C/Apo-A1 ratio. We concluded that there is a direct correlation between plasma HSPA1A and PAI-1 levels in patients with diabetes, which is lost when they develop albuminuria.     

Physiological impact of acute molybdenum exposure in juvenile kokanee salmon (Oncorhynchus nerka)

A series of experiments were conducted to determine the physiological impact of acute sublethal molybdenum exposure to juvenile kokanee salmon (Oncorhynchus nerka Kennerlyi). Molybdenum was found to be relatively non-toxic to kokanee as the 96 h LC(50) was greater than 2,000 mg Mo l(-1). Exposure to either 25 or 250 mg Mo x l(-1) for 7 days was found to stimulate a significant 1.6- to 1.7-fold increase in ventilation which was later characterized to be dose-dependent between 5 and 250 mg Mo l(-1). Acute sublethal molybdenum exposure was found to have little or no impact on kokanee oxygen consumption at rest or immediately following a bout of forced activity or on physiological indicators of stress such as plasma lactate, sodium and cortisol. Despite these findings, prior exposure to 25 or 250 mg Mo l(-1) resulted in post-exercise loss of equilibrium and exercise-induced delayed mortality that were not observed in controls. Molybdenum accumulation in gill and liver of kokanee was also characterized. The findings of this study suggest that despite the non-toxic nature of molybdenum, acute sublethal exposure to this metal has physiological consequences to those fish exposed even for only a brief period. Further studies are needed to more fully elucidate the metabolism and mode of action of this metal in fish.     

Analysis of serum heat shock protein 70 (HSPA1A) concentrations for diagnosis and disease activity monitoring in patients with rheumatoid arthritis

Heat shock proteins (HSPs) have been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA). The authors aimed to study applicability of heat shock protein 70 (HSPA1A) serum levels as a diagnostic factor and a severity indicator in patients with RA and to quantify cut-off point that predicts status of RA with highest specificity. A total of 76 patients with RA and 36 healthy adults were studied in this case-control analysis. Patients had a higher HSPA1A level than the control group (0.78 ± 0.13 vs. 0.12 ± 0.02 ng/mL, p = 0.006), irrespective of presence of absence of rheumatoid factor or anti-citrullinated cyclic peptide. Next, diagnostic accuracy of the HSPA1A in diagnosis of RA was evaluated (area under curve 0.71; p < 0.05). HSPA1A predicted status of having RA in levels above 0.42 ng/mL with more than 90 % specificity. In addition to diagnostic value, HSPA1A can distinguish between high disease activity (1.66 ± 0.75 ng/mL) and low (0.49 ± 0.1 ng/mL), moderate (0.52 ± 0.12 ng/mL), or remission phase (0.48 ± 0.11 ng/mL). Moreover, patients in remission still had a higher HSPA1A level compared to normal subject (0.48 ± 0.11 vs. 0.12 ± 0.02 ng/mL, p < 0.05). Our results showed that serum HSPA1A could be implemented as a specific tool to facilitate diagnosis and monitoring disease activity in patients with rheumatoid arthritis.

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