Cell Stress and Chaperones最新文献

MAP kinase phosphatase 3 (MKP3), a member of the dual-specificity protein phosphatase (DUSP) superfamily, has been widely studied for its role in development, cancer, and environmental stress in many organisms. However, the functions of MKP3 in various insects have not been well studied, including honeybees. In this study, we isolated an MKP3 gene from Apis cerana cerana and explored the role of this gene in the resistance to oxidation. We found that AccMKP3 is highly conserved in different species and shares the closest evolutionary relationship with AmMKP3. We determined the expression patterns of AccMKP3 under various stresses. qRT-PCR results showed that AccMKP3 was highly expressed during the pupal stages and in adult muscles. We further found that AccMKP3 was induced in all the stress treatments. Moreover, we discovered that the enzymatic activities of peroxidase, superoxide dismutase, and catalase increased and that the expression levels of several antioxidant genes were affected after AccMKP3 was knocked down. Collectively, these results suggest that AccMKP3 may be associated with antioxidant processes involved in response to various environmental stresses.

Heme homeostasis is of vital importance to many biological processes associated with cell redox activity. However, the role of heme in the doxorubicin (DOX)-induced cardiotoxicity is still not clear. The aim of the present study was to test the hypothesis that heme is related to the DOX-induced oxidative stress and inhibition of heme expression may protect H9c2 cardiomyocytes against DOX-induced cardiotoxicity. For the evaluation of heme changing under doxorubicin treatment, H9c2 cells were treated with 0.5, 1, 2, and 4 mg/mL doxorubicin respectively. H9c2 cells were divided into 5 groups: Control group (cells were cultured without intervention), DOX group (cells were treated with 2 mg/mL doxorubicin for 6 h), Heme depletion+DOX group (cells were cultured with heme-depleted serum media, 0.5 mM succinylacetone and 2 mg/mL doxorubicin), Heme group (cells were treated with 30 μM heme), and Heme depletion+DOX+Heme group. Apoptotic cells were detected by flow cytometry with Annexin V-FITC/PI. The intracellular oxidant levels were measured by DCFH-DA fluorescence. The levels of heme were detected by ELISA. Doxorubicin significantly increased intracellular heme level from 5013 ± 187 ng/mL to the highest level of 11,720 ± 107 ng/mL, as well as the intracellular oxidants and cell apoptosis rate elevated by the increase of doxorubicin concentration. Heme depletion can significantly suppress the DOX-induced apoptosis from 39.8 ± 0.5% to 20.8 ± 0.5% (p < 0.001). Re-supplemented with exogenous heme partially but significantly restored the DOX-induced apoptosis. Heme plays an important role in doxorubicin toxicity-induced cardiomyocyte injury. By appropriate reduction in the accumulation of free heme in cardiomyocytes, doxorubicin-induced cardiotoxicity may be alleviated.

Inositol phosphate synthase (IPS) is a rate-limiting enzyme in myo-inositol biosynthesis, which can regulate stress responses in plants and animals. However, there are few studies on the function of IPS in insects, especially in Apis cerana cerana. In this study, the inositol-3-phosphate synthase 1-B gene (AccIPS1-B) was isolated from Apis cerana cerana, and its connection to antioxidant defence was investigated. The open reading frame of AccIPS1-B was 1542 bp, encoding a 513 amino acid polypeptide. Quantitative real-time PCR analysis revealed that the expression level of AccIPS1-B was highest in pupae of Apis cerana cerana, and it was expressed at higher levels in the thorax than in other tissues tested. Moreover, the expression of AccIPS1-B was significantly upregulated by abiotic stresses. The recombinant AccIPS1-B also displayed significant tolerance to cumene hydroperoxide and HgCl₂. In addition, knockdown of AccIPS1-B significantly suppressed the expression of most of the antioxidant genes and decreased the antioxidant enzymatic activities of SOD, POD, and GST. Taken together, these findings indicate that AccIPS1-B may be involved in the response to antioxidant defence and development in Apis cerana cerana.

Functional orthopedic treatment is effective for the correction of malformation. Studies demonstrated myoblasts undergo proliferation and apoptosis on certain stretch conditions. MicroRNAs (miRNAs) function in RNA silencing and post-transcriptional regulation of gene expression, and participate in various biological processes, including proliferation and apoptosis. One hypothesis suggested that miRNA was involved into the procedure via suppressing its target genes then triggered endoplasmic reticulum stress-induced apoptosis. Therefore, miRNAs play important roles in the regulation of the proliferation and apoptosis of myoblasts. In our study, the miR-147 has been explored. A cyclic mechanical stretch model was established to observe the features of rat L6 myoblasts. The detection of mRNA and protein levels was performed by qRT-PCR and western blot. L6 cell proliferation/apoptosis was checked by CCK-8 assay, DNA fragmentation assay, and caspase-3 activity assay. MiRNA transfections were performed as per the manufacturer's suggestions: (1) cyclic mechanical stretch induced apoptosis of L6 myoblasts and inhibition of miR-147; (2) miR-147 attenuated cyclic mechanical stretch-induced apoptosis of L6 myoblasts; (3) miR-147 attenuated cyclic mechanical stretch-induced L6 myoblast endoplasmic reticulum stress; (4) BRMS1 was a direct target of miR-147 in L6 myoblasts; (5) miR-147/BRMS1 axis participated in the regulation of cyclic mechanical stress on L6 myoblasts. MiR-147 attenuates endoplasmic reticulum stress by targeting BRMS1 to inhibit cyclic mechanical stretch-induced apoptosis of L6 myoblasts.

The AMP-activated protein kinase (AMPK) has important roles in the regulation of energy metabolism, and AMPK activity and its regulation have been the focus of relevant investigations. However, functional characterization of AMPK is still limited in insects. In this study, the full-length cDNA coding AMPKα (TcAMPKα) was isolated from the red flour beetle, Tribolium castaneum. The TcAMPKα gene contains an ORF of 1581 bp encoding a protein of 526 amino acid residues, which shared conserved domain structure with Drosophila melanogaster and mammalian orthologs. Exposure of female adults to oxidative, heat, and cold stresses caused an increase in TcAMPKα mRNA expression levels and phosphorylation of Thr-173 in the activation loop. The RNAi-mediated knockdown of TcAMPKα resulted in the increased sensitivity of T. castaneum to oxidative, heat, and cold stresses. These results suggest that stress signals regulate TcAMPKα activity, and TcAMPKα plays an important role in enabling protective mechanisms and processes that confer resistance to environmental stress.

In this study, we investigated the function of co-enzyme Q10 (Q10) in autophagy of primary chicken myocardial cells during heat stress. Cells were treated with Q10 (1 μΜ, 10 μΜ, and 20 μM) before exposure to heat stress. Pretreatment of chicken myocardial cells with Q10 suppressed the decline in cell viability during heat stress and suppressed the increase in apoptosis during heat stress. Treatment with 20 μM Q10 upregulated autophagy-associated genes during heat stress. The expression of LC3-II was highest in cells treated with 20 μM Q10. Pretreatment with Q10 decreased reactive oxygen species (ROS) levels during heat stress. The number of autophagosomes was significantly increased by 20 μM Q10 treatment, as demonstrated by electron microscopy or monodansylcadaverine (MDC) fluorescence. SQSTM1 accumulation was diminished by Q10 treatment during heat stress, and the number of LC3II puncta was increased. Treatment with 20 μM Q10 also decreased the activation of the PI3K/Akt/mTOR pathway. Our results showed that co-enzyme Q10 can protect primary chicken myocardial cells by upregulating autophagy and suppressing the PI3K/Akt/mTOR pathway during heat stress.

Phosphorylation is an important event in cell signaling that is modulated by kinases and phosphatases. In Trypanosoma cruzi, the etiological agent of Chagas disease, approximately 2% of the protein-coding genes encode for protein kinases. This parasite has a heteroxenic life cycle with four different development stages. In the midgut of invertebrate vector, epimastigotes differentiate into metacyclic trypomastigotes in a process known as metacyclogenesis. This process can be reproduced in vitro by submitting parasites to nutritional stress (NS). Aiming to contribute to the elucidation of mechanisms that trigger metacyclogenesis, we applied super-SILAC (super-stable isotope labeling by amino acids in cell culture) and LC-MS/MS to analyze different points during NS. This analysis resulted in the identification of 4205 protein groups and 3643 phosphopeptides with the location of 4846 phosphorylation sites. Several phosphosites were considered modulated along NS and are present in proteins associated with various functions, such as fatty acid synthesis and the regulation of protein expression, reinforcing the importance of phosphorylation and signaling events to the parasite. These modulated sites may be triggers of metacyclogenesis.

Global climate change is predicted to intensify thermal stress in marine and coastal organisms, affecting their development, growth, and reproductive functions. In this study, we performed histological observations on ovarian development, immunohistochemical analyses of ovarian heat shock protein-70 (HSP70), nitrotyrosine protein (NTP, an indicator of reactive nitrogen species (RNS)), and dinitrophenyl protein (DNP, an indicator of protein oxidation) expressions, in situ TUNEL assay for cellular apoptosis, biochemical analyses of ovarian caspase-3/7 activity and protein carbonyl (PC, a measure of reactive oxygen species (ROS)) contents, nitrate/nitrite (NOx) levels, and extrapallial fluid (EPF, an important body fluid) pH in the American oyster, Crassostrea virginica. Oysters were exposed to medium (28 °C) and high (32 °C) temperatures under controlled laboratory conditions for 1 week. Oysters exposed to higher temperatures significantly decreased the number and diameter of eggs, and EPF protein concentrations compared with controls (24 °C). In contrast, EPF pH, ovarian HSP70 mRNA levels, and protein expression were increased after heat exposure, consistent with increased ovarian apoptosis. The enhanced apoptosis in ovaries was associated with increased ovarian caspase-3/7 activity, PC contents, NOx levels, and NTP and DNP expressions in heat-exposed oysters. Collectively, these results suggest that higher temperatures drastically increase RNS and ROS levels, increasing incidence of apoptosis and subsequently reducing ovarian functions in oysters.

Heatstroke (HS) is an acute, progressive life-threatening emergency. Animals, including military working dogs (IDFMWD), rapidly activate cytoprotective processes, e.g., heat shock proteins (HSPs) and antioxidative molecules, in response to heat stress. We hypothesized that serum HSPs (eHSP72) and oxidative stress markers would differ in IDFMWD with a history of HS compared with controls and thus could be used to detect susceptibility to recurrent HS. eHSPs concentration, oxidative stress markers, and systemic physiological parameters were studied in dogs with and without histories of HS, undergoing indoor or outdoor training. Treadmill physical performance tests (PPTs) were conducted indoors at 22 °C (groups C-I and HS-I) or outdoors under heat stress conditions of 36 °C; 60% humidity (groups C-O and HS-O). Pre-, immediately post-, and 45 min post-PPT heart rate (HR), respiratory rate, and rectal temperature (T_re) were recorded in all dogs. Likewise, blood samples were collected and eHSP72, venous blood gas analysis, and lactate and creatine kinase activity (CK) were assayed. Serum uric acid (sUA) and total serum redox potential (TRP) were measured only in the indoor group. Immediately post-PPT under both environmental conditions, T_re, HR, eHSP, sUA, and TRP (only measured in indoor PPT) significantly (P < 0.05) increased, whereas venous blood pH and bicarbonate decreased significantly (P < 0.05). Between groups comparisons demonstrated significant differences in basal HR and post-PPT T_re immediately after outdoor PPT. eHSP72 induction, CK, sUA, and serum TRP remained significantly higher in the HS group during post-PPT recovery. Taken together, animals with a history of HS have different results, and this signature of previous HS may predict altered heat sensitivity.

Cardiac microvascular ischemia-reperfusion (IR) injury has been a neglected topic in recent decades. In the current study, we investigated the mechanism underlying microvascular IR injury, with a focus on mitochondrial homeostasis. We also explored the protective role of tanshinone IIA (Tan IIA) in microvascular protection in the context of IR injury. Through animal studies and cell experiments, we demonstrated that IR injury mediated microvascular wall destruction, lumen stenosis, perfusion defects, and cardiac microvascular endothelial cell (CMEC) apoptosis via inducing mitochondrial damage. In contrast, Tan IIA administration had the ability to sustain CMEC viability and microvascular homeostasis, finally attenuating microvascular IR injury. Function studies have confirmed that the SIRT1/PGC1α pathway is responsible for the microvascular protection from the Tan IIA treatment. SIRT1 activation by Tan IIA sustained the mitochondrial potential, alleviated the mitochondrial pro-apoptotic factor leakage, reduced the mPTP opening, and blocked mitochondrial apoptosis, providing a survival advantage for CMECs and preserving microvascular structure and function. By comparison, inhibiting SIRT1 abrogated the beneficial effects of Tan IIA on mitochondrial function, CMEC survival, and microvascular homeostasis. Collectively, this study indicated that Tan IIA should be considered a microvascular-protective drug that alleviates acute cardiac microcirculation IR injury via activating the SIRT1/PGC1α pathway and thereby blocking mitochondrial damage.