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Objective: This study aimed to explore whether acupuncture and moxibustion can enhance the immune response by increasing the expression of the endogenous adjuvant HSP70 mRNA. Method: Forty Wistar rats were divided into four groups: model immune acupuncture group (A), model immune control group (B), normal immune acupuncture group (C), and normal immune control group (D). Model immune groups A and B were induced by injecting d-galactose for 6 weeks. Rats in groups A and C were then treated with low-frequency electroacupuncture (EA) at Zusanli (ST36), Guanyuan (CV4), and Baihui (GV20) and moxibustion for 3 weeks. Subsequently, all rats were observed for 2 more weeks. At the 12th week, diphtheria antitoxin titers were determined using the Vero cell trace neutralization method, CD4+T/CD8+T cell ratios in peripheral blood were examined by flow cytometry, and the relative expression of spleen cell HSP70 mRNA was measured by RT-PCR. Results: Compared with the normal immune control, the diphtheria antitoxin titer, CD4+T/CD8+T cell ratio, and expression of spleen cell HSP70 mRNA significantly decreased in the model immune control group (P < 0.01). However, the model immune acupuncture group showed a significant increase in antitoxin titer (P < 0.01) and elevated CD4+T/CD8+T cell ratio and HSP70 mRNA expression (P < 0.05) after EA and moxibustion intervention. Conclusion: Acupuncture and moxibustion may enhance the humoral immune response (diphtheria antitoxin titer) and cellular immune response (peripheral blood CD4+T/CD8+T cell ratio) by increasing the expression of the endogenous adjuvant HSP70 mRNA, suggesting that acupuncture may serve as a new vaccine adjuvant.

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Neurodegenerative disorders (NDs) are diverse age-related conditions also described as "conformational diseases." The hallmark of NDs is the accumulation of disease-specific proteins as toxic misfolded aggregates in some areas of the brain. They lead to the loss of protein homeostasis (proteostasis) that causes neuronal dysfunction and death. A potential therapeutic strategy for NDs is to prevent the accumulation of misfolded proteins by activating the heat shock response (HSR). The HSR maintains proteostasis through the upregulation of heat shock proteins (HSPs), molecular chaperones that recognize misfolded proteins, and either refold them to their functional conformations and/or target them for degradation. However, how to manipulate the expression of HSPs to obtain a therapeutic effect in neurons remains unclear. Furthermore, the regulation of the HSR in neurons is more complex than what we have learned from culturing somatic nonneuronal cells. This chapter describes a method to investigate the induction of HSP70 in primary hippocampal neurons using single-molecule fluorescence in situ hybridization (smFISH). Quantification of smFISH provides the means to analyze neuron-to-neuron variability in the activation of the HSR and enables us to study the transcriptional induction and localization of HSP70 mRNA in primary neurons. This information might be critical to find the druggable steps for developing effective therapies to treat age-related NDs.

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Objective Through measuring and analyzing blood Heat shock proteins (HSP) 70 and HSP90 mRNA expression of different levels of arsenic exposure population,to explore the relationship between HSP70 and HSP90 mRNA expression and endemic arsenic poisoning.Methods The study subjects included a total of 226 residents exposed to arsenic via drinking water from two townships in the city of Bayannur (Shengfeng Township and Hangjinhouqishahai Township Wuyuan County) in Inner Mongolia Autonomous Region in 2011.The residents were divided into four groups according to drinking water arsenic concentration:control group (drinking water arsenic concentration ＜ 10 μg/L),low exposure group (drinking water arsenic concentration 10-＜ 100 μg/L),middleexposure group (drinking water arsenic concentration 100-＜ 200 μg/L),and high exposure group (drinking water arsenic concentration ≥200 μg/L).Epidemiological investigation was carried out.Real-time PCR technology was used to detect the expression levels of blood HSP70 and HSP90 mRNA.And the relationships between the expression of HSP70 and HSP90 mRNA and water arsenic and urinary arsenic levels were analyzed.Results ①With the increase of arsenic concentration,the expression level of HSP70 mRNA increased first and then decreased.The HSP70 mRNA in the blood of high exposure group [2.44 × 10-3 (1.72 × 10-3)] and middle exposure group [3.01 × 10-3 (1.95 × 10-3)] were significantly different from that in the control group [2.27 × 10-3 (1.09 × 10-3),P ＜ 0.05].There was significant difference between the middle exposure group [3.01 × 10-3 (1.95 × 10-3)] and the low and high exposure groups [2.38 × 10-3 (1.55 × 10-3),2.44 × 10-3 (1.72 × 10-3),P ＜ 0.05].The expression of HSP70 mRNA in blood of male [2.71 × 10-3 (1.90 × 10-3)] was slightly higher than that of female [2.34 × 10-3 (1.35 × 10-3),t =2.523,P ＜ 0.05].② With the increase of arsenic concentration,the expression level of HSP90 mRNA increased first and then decreased.The expression of HSP90 mRNA in the middle exposure group [1.29 × 10-2(1.04 × 10-2)] was significantly different from that in the control group [1.04 × 10-2 (0.83 × 10-2),P ＜ 0.05].The differences of HSP90 mRNA between the high exposure group [1.19 × 10-2 (0.88 × 10-2)] and the low and middle exposure groups [1.23 × 10-2 (0.68 × 10-2),1.29 × 10-2 (1.04 × 10-2)] were statistically significant (P ＜ 0.05).③The relative expression of HSP70 mRNA showed a U-shaped regression relationship with the level of urinary arsenic exposure (R2 =0.031,P ＜ 0.05),and the expression of HSP90 mRNA was also correlated with urinary arsenic and nail arsenic (R2 =0.049,0.036,P ＜ 0.05).Conclusion Chronic arsenic exposure affects blood HSP70 and HSP90 mRNA expression.

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BACKGROUND: When cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress. METHOD: Using arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly. RESULTS: Arsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression. CONCLUSION: In arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly. GENERAL SIGNIFICANCE: Under stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes.

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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 20 min 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 9 months 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 12 months old oocysts that were not viable in the excystation test. Oocysts inactivated at 75°C for 30 min 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 ß-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.

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