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Detection of heat shock protein 27, 70, 90 expressions in primary parenchymatous organs of goats after transport stress by real-time PCR and ELISA.

ABSTRACT: Transport stress causes a series of problems to goat production, such as tissue injury and immunity damage. As a pro-survival pathway, the heat shock response protects healthy cells of goat from stressors. To evaluate the effects of transport stress on heat shock protein (HSPs) expression on goat primary parenchymatous organs, a total of three batches of goats were treated in this study. For each batch, 12 healthy adult male goats were randomly and averagely divided into three groups: Control group (non-transported group), 2 hr transported group and 6 hr transported group. Real-time PCR results indicated that the mRNA expression level of heat shock protein 27 (HSP27) in all examined organs of 2 hr transport-treated goats were upregulated (p < .05) except lung, and heat shock protein 70 (HSP70; except spleen) and heat shock protein 90 (HSP90; except liver and lung) were also increased (p < .05). In 6 hr transported group, the transcription levels of HSP27 (except heart and kidney), HSP70 (except heart, liver and lymph nodes) and HSP90 (except heart and spleen) were all backed to the original levels or even reduced (p < .05). Enzyme-linked immunosorbent assay (ELISA) results showed that the protein levels of HSP27 (except lymph nodes), HSP70 (except spleen) and HSP90 (except liver and lung) were all increased after 2 hr transport (p < .05). After 6 hr transport, HSP27 only in kidney and HSP70 only in heart and liver were upregulated (p < .05), while HSP90 in all the examined organs except liver and lung were also maintained in relatively high levels (p < .05). Taken together, these results suggested that the expression of HSPs in goat primary parenchymatous organs may be regulated by transport stress time. Moreover, this study also provides some new data to advocate reducing transport stress of goats and improving animal welfare.

SUBMITTER: Hu W

PROVIDER: S-EPMC7738732 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Detection of heat shock protein 27, 70, 90 expressions in primary parenchymatous organs of goats after transport stress by real-time PCR and ELISA.

Hu Wei W Fang Manxin M Yang Yanzhen Y Ye Tian T Liu Ben B Zheng Wenya W

Veterinary medicine and science 20200725 4

Transport stress causes a series of problems to goat production, such as tissue injury and immunity damage. As a pro-survival pathway, the heat shock response protects healthy cells of goat from stressors. To evaluate the effects of transport stress on heat shock protein (HSPs) expression on goat primary parenchymatous organs, a total of three batches of goats were treated in this study. For each batch, 12 healthy adult male goats were randomly and averagely divided into three groups: Control gr ...[more]

PMID: 32710597

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Project description:Invertebrates are becoming more popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. The humoral immune response in insects acts by melanization, clotting and the production of reactive oxygen species and antimicrobial peptides, while the cellular immunity system is based on nodulation, encapsulation and phagocytosis. An increasingly popular insect model in biological research is Galleria mellonella, whose larvae are sensitive to infection by the entomopathogenic fungus Conidiobolus coronatus, which can also be dangerous to humans. One group of factors that modulate the response of the immune system during infection in mammals are heat shock proteins (HSPs). The aim of this study was to investigate whether infection by C. coronatus in G. mellonella hemolymph is accompanied by an increase of HSP90, HSP70, HSP60 and HSP27. Larvae (five-day-old last instar) were exposed for 24 hours to fully-grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24) or 24 hours later (F48). The concentration of the HSPs in hemolymph was determined using ELISA. Immunolocalization in hemocytes was performed using fluorescence microscopy and flow cytometry. HSP90, HSP70, HSP60 and HSP27 were found to be present in the G. mellonella hemocytes. HSP60 and HSP90 predominated in healthy insects, with HSP70 and HSP27 being found in trace amounts; HSP60 and HSP27 were elevated in F24 and F48, and HSP90 was elevated in F48. The fungal infection had no effect on HSP70 levels. These findings shed light on the mechanisms underlying the interaction between the innate insect immune response and entomopathogen infection. The results of this innovative study may have a considerable impact on research concerning innate immunology and insect physiology.

Project description:Heat shock proteins (HSPs) are evolutionarily conserved chaperones occurring in virtually all living organisms playing a key role in the maintenance of cellular homeostasis. They are constitutively expressed to prevent and repair protein damage following various physiological and environmental stressors. HSPs are overexpressed in various types of cancers to provide cytoprotective function, and they have been described to influence prognosis and response to therapy. Moreover, they have been used as a tumor marker in blood serum biochemistry and they represent a potentially promising therapeutic target. To clarify prognostic significance of two canonical HSPs (27 and 70) and less known HSP110 (previously known as HSP105) in colorectal carcinoma (CRC), we retrospectively performed HSP immunohistochemistry on tissue microarrays from formalin-fixed paraffin-embedded tumor tissue from 297 patients with known follow-up. Survival analysis (univariate Kaplan-Meier analysis with the log-rank test and multivariate Cox regression) revealed significantly shorter overall survival (OS, mean 5.54 vs. 7.07, p = 0.033) and borderline insignificantly shorter cancer specific survival (CSS, mean 6.3 vs. 7.87 years, p = 0.066) in patients with HSP70+ tumors. In the case of HSP27+ tumors, there was an insignificantly shorter OS (mean 6.36 vs. 7.13 years, p = 0.2) and CSS (mean 7.17 vs. 7.95 years, p = 0.2). HSP110 showed no significant impact on survival. Using Pearson's chi-squared test, there was a significant association of HSP27 and HSP70 expression with advanced cancer stage. HSP27+ tumors were more frequently mismatch-repair proficient and vice versa (p = 0.014), and they occurred more often in female patients and vice versa (p = 0.015). There was an enrichment of left sided tumors with HSP110+ compared to the right sided (p = 0.022). In multivariate Cox regression adjusted on the UICC stage, grade and right/left side; both HSPs 27 and 70 were not independent survival predictors (p = 0.616 & p = 0.586). In multivariate analysis, only advanced UICC stage (p = 0) and right sided localization (p = 0.04) were independent predictors of worse CSS. In conclusion, from all three HSPs examined in our study, only HSP70 expression worsened CRC prognosis, although stage-dependent. The contribution of this article may be seen as a large survival analysis of HSPs 27 and 70 and the largest analysis of HSP110 described in CRC.

Project description:Heat shock proteins (HSPs) play an essential role in protecting proteins from denaturation and are implicated in diverse pathophysiological conditions like cardiovascular diseases, cancer, infections, and neurodegenerative diseases. Scientific evidence indicates that if HSP expression falls below a certain level, cells become sensitive to oxidative damage that accelerates protein aggregation diseases. On the other hand, persistently enhanced levels of HSP can lead to inflammatory and oncogenic changes. To date, although techniques for measuring HSPs exist, these assays are limited for use in specific sample types or are time consuming. Therefore, in the present study, we developed a single-molecule assay digital ELISA technology (Single Molecule Array-SIMOA) for the measurement of HSPs, which is time effective and can be adapted to measure multiple analytes simultaneously from a single sample. This technique combines two distinct HSP-specific antibodies that recognize different epitopes on the HSP molecule. A recombinant human HSP protein was used as the standard material. The assay performance characteristics were evaluated by repeated testing of samples spiked with HSP peptide at different levels. The limit of detection was 0.16 and 2 ng/mL for HSP27 and HSP70, respectively. The inter- and intra-assay coefficients of variation were less than 20% in all tested conditions for both HSPs. The HSP levels assayed after serial dilution of samples portrayed dilutional linearity (on average 109%, R2 = 0.998, p < 0.001, for HSP27 and 93%, R2 = 0.994, p < 0.001, for HSP70). A high linear response was also demonstrated with admixtures of plasma exhibiting relatively very low and high levels of HSP70 (R2 = 0.982, p < 0.001). Analyte spike recovery varied between 57% and 95%. Moreover, the relative HSP values obtained using Western blotting correlated significantly with HSP values obtained with the newly developed SIMOA assay (r = 0.815, p < 0.001 and r = 0,895, p < 0.001 for HSP70 and HSP27, respectively), indicating that our method is reliable. In conclusion, the assay demonstrates analytical performance for the accurate assessment of HSPs in various sample types and offers the advantage of a huge range of dilution linearity, indicating that samples with HSP concentration highly above the calibration range can be diluted into range without affecting the precision of the assay.

Project description:BackgroundThe tick Dermacentor silvarum Olenev (Acari: Ixodidae) is a vital vector tick species mainly distributed in the north of China and overwinters in the unfed adult stage. The knowledge of the mechanism that underlies its molecular adaptation against cold is limited. In the present study, genes of hsp70 and hsp90 cDNA, named Dshsp70 and Dshsp90, and tubulin were cloned and characterized from D. silvarum, and their functions in cold stress were further evaluated.MethodsThe genome of the heat shock proteins and tubulin of D. silvarum were sequenced and analyzed using bioinformatics methods. Each group of 20 ticks were injected in triplicate with Dshsp90-, Dshsp70-, and tubulin-derived dsRNA, whereas the control group was injected with GFP dsRNA. Then, the total RNA was extracted and cDNA was synthesized and subjected to RT-qPCR. After the confirmation of knockdown, the ticks were incubated for 24 h and were exposed to - 20 °C lethal temperature (LT50), and then the mortality was calculated.ResultsResults indicated that Dshsp70 and Dshsp90 contained an open reading frame of 345 and 2190 nucleotides that encoded 114 and 729 amino acid residues, respectively. The transcript Dshsp70 showed 90% similarity with that identified from Dermacentor variabilis, whereas Dshsp90 showed 85% similarity with that identified from Ixodes scapularis. Multiple sequence alignment indicates that the deduced amino acid sequences of D. silvarum Hsp90, Hsp70, and tubulin show very high sequence identity to their corresponding sequences in other species. Hsp90 and Hsp70 display highly conserved and signature amino acid sequences with well-conserved MEEVD motif at the C-terminal in Hsp90 and a variable C-terminal region with a V/IEEVD-motif in Hsp70 that bind to numerous co-chaperones. RNA interference revealed that the mortality of D. silvarum was significantly increased after injection of dsRNA of Dshsp70 (P = 0.0298) and tubulin (P = 0.0448), whereas no significant increases were observed after the interference of Dshsp90 (P = 0.0709).ConclusionsThe above results suggested that Dshsp70 and tubulin play an essential role in the low-temperature adaptation of ticks. The results of this study can contribute to the understanding of the survival and acclimatization of overwintering ticks.

Project description:BackgroundHeat shock proteins (HSPs) are found in all prokaryotes and most compartments of eukaryotic cells. Members of the HSP family mediate immune responses to tissue damage or cellular stress. However, little is known about the immune response induced by the oriental liver fluke, Clonorchis sinensis, even though this organism is carcinogenic to humans. We address this issue in the present study in mouse bone marrow dendritic cells (mBMDCs), using recombinant HSP70 and 90 from C. sinensis (rCsHSP70 and rCsHSP90).MethodsrCsHSP70 and rCsHSP90 were produced in an E. coli system. Purified recombinant proteins were treated in BMDCs isolated from C57BL/6 mice. T cells were isolated from Balb/c mice and co-cultured with activated mBMDCs. Expression of surface molecules was measured by flow cytometry and cytokine secretion was quantified using ELISA. C57BL/6 mice were divided into four groups, including peptide alone, peptide/Freund's adjuvant, peptide/CsHSP70, peptide/CsHSP90, and were immunized intraperitoneally three times. Two weeks after final immunization, antibodies against peptide were measured using ELISA.ResultsBoth proteins induced a dose-dependent upregulation in major histocompatibility complex and co-stimulatory molecule expression and increased secretion of pro-inflammatory cytokines including interleukin (IL)-1β, -6, and -12p70 and tumor necrosis factor-α in mBMDCs. Furthermore, when allogenic T cells were incubated with mBMDCs activated by rCsHSP70 and rCsHSP90, the helper T cell (Th)1 cytokine interferon-γ was up-regulated whereas the level of the Th2 cytokine IL-4 was unchanged. These results indicate that rCsHSPs predominantly induce a Th1 response. Over and above these results, we also demonstrated that the production of peptide-specific antibodies can be activated after immunization via in vitro peptide binding with rCsHSP70 or rCsHSP90.ConclusionThis study showed for the first time that the HSP or HSP/peptide complexes of C. sinensis could be considered as a more effective vaccine against C. sinensis infection as results of the activator of host immune response as well as the adjuvant for antigenic peptide conjugate to induce peptide-specific antibody response in mice.

Project description:It has been established that the synthesis of heat shock protein 70 (Hsp70) is temperature-dependent. The Hsp70 response is considered as a cellular thermometer in response to heat stress and other stimuli. The variation in Hsp70 gene expression has been positively correlated with thermotolerance in Drosophila melanogaster, Caenorhabditis elegans, rodents and human. Goats have a wide range of ecological adaptability due to their anatomical and physiological characteristics; however, the productivity of the individual declines during thermal stress. The present study was carried out to analyze the expression of heat shock proteins in different tissues and to contrast heat stress phenotypes in response to chronic heat stress. The investigation has been carried out in Jamunapari, Barbari, Jakhrana and Sirohi goats. These breeds differ in size, coat colour and production performance. The heat stress assessment in goats was carried out at a temperature humidity index (THI) ranging from 85.36-89.80 over the period. Phenotyping for heat stress susceptibility was carried out by combining respiration rate (RR) and heart rate (HR). Based on the distribution of RR and HR over the breeds in the population, individual animals were recognized as heat stress-susceptible (HSS) and heat stress-tolerant (HST). Based on their physiological responses, the selected animals were slaughtered for tissue collection during peak heat stress periods. The tissue samples from different organs such as liver, spleen, heart, testis, brain and lungs were collected and stored at -70 °C for future use. Hsp70 concentrations were analyzed from tissue extract with ELISA. mRNA expression levels were evaluated using the SYBR green method. Kidney, liver and heart had 1.5-2.0-fold higher Hsp70 concentrations as compared to other organs in the tissue extracts. Similarly, the gene expression pattern of Hsp70 in different organs indicated that the liver, spleen, brain and kidney exhibited 5.94, 4.96, 5.29 and 2.63-fold higher expression than control. Liver and brain tissues showed the highest gene expression at mRNA levels as compared to kidney, spleen and heart. HST individuals had higher levels of mRNA level expression than HSS individuals in all breeds. The Sirohi breed showed the highest (6.3-fold) mRNA expression levels as compared to the other three breeds, indicating the better heat stress regulation activity in the breed.

Dataset Information

Detection of heat shock protein 27, 70, 90 expressions in primary parenchymatous organs of goats after transport stress by real-time PCR and ELISA.

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Detection of heat shock protein 27, 70, 90 expressions in primary parenchymatous organs of goats after transport stress by real-time PCR and ELISA.

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