Testosterone amplifies HSP70-2a,HSP90 and PCNA expression in experimental varicocele condition: Implication for DNA fragmentation

The DNA fragmentation and failure in post-meiotic maturation of the spermatozoa because of testosterone withdrawal can affect the fertilization potential in varicocele (VCL) patients. To find out the exact mechanism of VCL-induced failure in histone-protamine replacement process and DNA fragmentation, the correlations between the levels of expression of HSP70-2a, HSP90, PCNA, TP1/2 and PCNA genes and the patterns of DNA methylation were investigated before and after testosterone administration in rats. In total, 40 mature male Wistar rats (10 in each group) were assigned between control (with no intervention), control-sham (undergone a simple laparotomy), VCL-induced (VCL-sole), and testosterone-treated VCL-induced (VCLT) groups. The HSP70-2a, HSP90, PCNA, TP1, and TP2 genes expressions and the patterns of global DNA methylation were determined in all groups. A statistically significant (p < 0.05) reduction were found in the HSP70-2a, HSP90, PCNA, TP1 and TP2 genes expressions in VCL-sole group. In VCLT group, testosterone was shown to significantly (p < 0.05) up-regulate the HSP70-2a, HSP90, PCNA, and TP2expression levels, but TP1 expression has not been changed. Furthermore, the VCLT group exhibited higher DNA methylation rates compared to VCL-sole animals. In conclusion, testosterone, by up-regulating the HSP70-2a and HSP90 expressions and maintaining the pre-existing HSP70-2a and HSP90 proteins levels, may be the reason for the significant increment in TP2 expression during post-meiotic stage and can boost the global methylation rates of DNA via up-regulating the PCNA expression, suggesting that administration of testosterone can mitigate the VCL-impaired histone-protamine replacement and DNA methylation rates and protect the cellular DNA content from VCL-induced oxidative stress.     

Analysis of midgut gene expression profiles from different silkworm varieties after exposure to high temperature

The silkworm is a poikilothermic animal, whose growth and development is significantly influenced by environmental temperature. To identify genes and metabolic pathways involved in the heat-stress response, digital gene expression analysis was performed on the midgut of the thermotolerant silkworm variety ‘932’ and thermosensitive variety ‘HY’ after exposure to high temperature (932T and HYT). Deep sequencing yielded 6,211,484, 5,898,028, 5,870,395 and 6,088,303 reads for the 932, 932T, HY and HYT samples, respectively. The annotated genes associated with these tags numbered 4357, 4378, 4296 and 4658 for the 932, 932T, HY and HYT samples, respecti'vely. In the HY-vs-932, 932-vs-932T, and HY-vs-HYT comparisons, 561, 316 and 281 differentially expressed genes were identified, which could be assigned to 179, 140 and 123 biological pathways, respectively. It was found that some of the biological pathways, which included oxidative phosphorylation, related to glucose and lipid metabolism, are greatly affected by high temperature and may lead to a decrease in the ingestion of fresh mulberry. When subjected to an early period of continuous heat stress, HSP genes, such as HSP19.9, HSP23.7, HSP40-3, HSP70, HSP90 and HSP70 binding protein, are up-regulated but then reduced after 24 h and the thermotolerant ‘932’ strain has higher levels of mRNA of some HSPs, except HSP70, than the thermosensitive variety during continuous high temperature treatment. It is suggested that HSPs and the levels of their expression may play important roles in the resistance to high temperature stress among silkworm varieties. This study has generated important reference tools that can be used to further analyze the mechanisms that underlie thermotolerance differences among silkworm varieties.     

ERK1/2 regulates heat stress-induced lactate production via enhancing the expression of HSP70 in immature boar Sertoli cells

Lactate produced by Sertoli cells plays an important role in spermatogenesis, and heat stress induces lactate production in immature boar Sertoli cells. Extracellular signaling regulated kinase 1 and 2 (ERK1/2) participates in heat stress response. However, the effect of ERK1/2 on heat stress-induced lactate production is unclear. In the present study, Sertoli cells were isolated from immature boar testis and cultured at 32 °C. Heat stress was induced in a 43 °C incubator for 30 min. Proteins and RNAs were detected by western blotting and RT-PCR, respectively. Lactate production and lactate dehydrogenase (LDH) activity were detected using commercial kits. Heat stress promoted ERK1/2 phosphorylation, showing a reducing trend with increasing recovery time. In addition, heat stress increased heat shock protein 70 (HSP70), glucose transporter 3 (GLUT3), and lactate dehydrogenase A (LDHA) expressions, enhanced LDH activity and lactate production at 2-h post-heat stress. Pretreatment with U0126 (1?×?10^?6 mol/L), a highly selective inhibitor of ERK1/2 phosphorylation, reduced HSP70, GLUT3, and LDHA expressions and decreased LDH activity and lactate production. Meanwhile, ERK2 siRNA1 reduced the mRNA level of ERK2 and weakened ERK1/2 phosphorylation. Additionally, ERK2 siRNA1 reduced HSP70, GLUT3, and LHDA expressions decreased LDH activity and lactate production. Furthermore, HSP70 siRNA3 downregulated GLUT3 and LDHA expressions and decreased LDH activity and lactate production. These results show that activated ERK1/2 increases heat stress-induced lactate production by enhancing HSP70 expression to promote the expressions of molecules related to lactate production (GLUT3 and LDHA). Our study reveals a new insight in reducing the negative effect of heat stress in boars.     

Differential expression of HSP70 and SAPK/JNK genes in the cabbage aphid upon salicylic and dilute sulfuric acid treatments in canola

Investigating the interaction between insect pests and host plants at molecular level, provides an insight for effective implementation of plant resistance-based strategies in insect pest control programs. Heat shock proteins (HSPs) are found in all living organisms which protect them from biotic and abiotic stresses. Stress-activated protein kinases (SAPKs) are members of MPK kinases which also are activated upon cellular stresses. We investigated the effect of seed-primed canola plants with salicylic acid (SA) and the plants treated with dilute sulfuric acid on the regulation of two stress-related genes, HSP70 and SAPK/JNK genes in the cabbage aphid. The results of qRT-PCR revealed a statistically significant up-regulation of HSP70 gene expression in aphids feeding on sulfuric acid-treated plants in comparison with controls (P < 0.01). However, this gene was not regulated in aphids feeding on salicylic acid-treated plants. In addition, quantification of SAPK/JNK gene showed a significant effect in aphids feeding on SA-primed plants (P < 0.05) and a marginally significant effect in sulfuric acid treatment. Further, the expression of both genes were significantly changed in interaction effect between SA and sulfuric acid treatments. Finally, the induction of HSP70 and SAPK/JNK genes in this research might be related to activation of systemic acquired resistance in canola.     

Oxidative stress-induced expression of HSP70 contributes to the inhibitory effect of 15d-PGJ2 on inducible prostaglandin pathway in chondrocytes

The inhibitory effect of 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) on proinflammatory gene expression has been extensively documented and frequently ascribed to its ability to prevent NF-κB pathway activation. We and others have previously demonstrated that it was frequently independent of the peroxisome proliferator activated receptor (PPAR)γ activation. Here, we provide evidence that induction of intracellular heat shock protein (HSP)70 by oxidative stress is an additional regulatory loop supporting the anti-inflammatory effect of 15d-PGJ₂ in chondrocytes. Using real-time quantitative PCR and Western blotting, we showed that 15d-PGJ₂ stimulated HSP70, but not HSP27 expression while increasing oxidative stress as measured by spectrofluorimetry and confocal spectral imaging. Using N-acetylcysteine (NAC) as an antioxidant, we demonstrated further that oxidative stress was thoroughly responsible for the increased expression of HSP70. Finally, using an HSP70 antisense strategy, we showed that the inhibitory effect of 15d-PGJ₂ on IL-1-induced activation of the NF-κB pathway, COX-2 and mPGES-1 expression, and PGE₂ synthesis was partly supported by HSP70. These data provide a new anti-inflammatory mechanism to support the PPARγ-independent effect of 15d-PGJ₂ in chondrocyte and suggest a possible feedback regulatory loop between oxidative stress and inflammation via intracellular HSP70 up-regulation. This cross talk is consistent with 15d-PGJ₂ as a putative negative regulator of the inflammatory reaction.     

Supplementation of freezing media with alpha lipoic acid preserves the structural and functional characteristics of sperm against cryodamage in infertile men with asthenoteratozoospermia

The aim of this study was to examine the effects of alpha lipoic acid (ALA) supplementation during semen cryopreservation on the sperm quality, chromatin integrity, oxidative stress, and expression level of BAX, BCL2, HSP70 and iNOS genes in semen samples obtained from infertile men with asthenoteratozoospermia.MethodsTwenty freshly ejaculated semen samples were cryopreserved with sperm freezing medium supplemented with 0.00, 0.02, 0.05, 0.1, 0.5, and 1 mmol/mL of ALA. The samples were analyzed according to the WHO guidelines before and after freezing. Sperm ROS production level, DNA fragmentation and cryo-capacitation were assessed using flow cytometry, TUNEL assay and chlortetracycline (CTC) test, respectively. Expression level of stress protein (HSP70), pro-apoptotic Bax, anti-apoptotic Bcl-2, and iNOS genes was assessed by real-time PCR assay.ResultsThe effective concentrations of ALA (0.02 and 0.5 mM) significantly improved the motility, viability and morphology of the frozen-thawed sperms compared to the control group treated with 0.00 mM of ALA. During cryopreservation, treatment of semen with 0.02 mM of ALA, as the optimal concentration, significantly decreased DNA fragmentation and oxidative stress level (P < 0.05), protected the acrosome integrity, and led to insignificant reduction in BAX gene expression level and significant increase in expression level of BCL2, HSP70, and iNOS genes compared with control group.ConclusionOur findings revealed that the adding ALA to semen samples obtained from infertile men with asthenoteratozoospermia plays a significant protective role against cryodamage by preserving the sperm functional parameters.     

MiR-199a is overexpressed in plasma of type 2 diabetes patients which contributes to type 2 diabetes by targeting GLUT4

Decreased GLUT4 expression and impaired GLUT4 cell membrane translocation are involved in type 2 diabetes mellitus (T2DM) pathogenesis so the factors impacting GLUT4 expression may be associated with T2DM. In this study, we identified four miRNAs: miR-31, miR-93, miR-146a, and miR-199a which suppress GLUT4 expression in HEK293T cells. Subsequently, we determined expression of these four miRNAs in plasma samples of T2DM patients, T2DM susceptible individuals, and healthy controls and found miR-199a was overexpressed in patients’ plasma compared with healthy control. Because the miR-199a binding site in GLUT4 3′UTR is highly conserved among vertebrates, we detected the glucose uptake in rat L6 myoblast cells through gain- and loss-of-function of miR-199a. We found that miR-199a can repress glucose uptake in L6 cells, which was rescued by GLUT4 overexpression. These results indicate that T2DM patients may have a high level miR-199a that reduce GLUT4 expression and contribute to the insulin resistance. Hence, miR-199a may be a novel biomarker for risk estimation and classification in T2DM patients.     

The Stress of Suicide: Temporal and Spatial Expression of Putative Heat Shock Protein 70 Protect the Cells from Heat Injury in Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

Heat stress adversely affects growth, development, and yield of winter wheat (Triticum aestivum). Plants have, however, evolved mechanisms to adapt to such conditions mainly by the expression of stress-associated chaperones, the heat shock proteins (HSPs), for modulating the tolerance level. Here, we report cloning of cytosolic putative HSP70 of 1678 bp from a thermotolerant cultivar (C306) of wheat (T. aestivum). A BLASTn search showed maximum homology with the predicted HSP70 protein reported from Hordeum vulgare (accession no AK354795.1). In silico characterization showed the presence of a nucleotide-binding domain of the sugar kinase/HSP70/actin superfamily in the sequence. Putative HSP70 showed temporal and spatial variations in the expression under heat stress (HS). We observed the abundance of HSP70 protein, H₂O₂, proline, and guaiacol peroxidase activity during the seed-hardening stage under HS; accumulation was, however, higher in the thermotolerant C306 than in thermosusceptible HD2329 cultivar. A gradual decrease in cell membrane stability (CMS) and an increase in total antioxidant capacity (TAC) were observed in both the cultivars at the different stages of growth. The expression of HSP70 showed a negative correlation with CMS and a positive correlation with TAC under HS; changes were less pronounced in C306 than in HD2329 at all the stages of growth studied. HSP70 seems to play diverse roles associated with thermotolerance, and partially protect wheat from terminal HS. Being the important member of family of the HSPs, HSP70 needs to be studied in detail, to be used for developing climate-smart wheat crops, through genetic engineering/breeding approaches.     

Monitoring Stress-Related Genes during the Process of Biomass Propagation of Saccharomyces cerevisiae Strains Used for Wine Making

Physiological capabilities and fermentation performance of Saccharomyces cerevisiae strains to be employed during industrial wine fermentations are critical for the quality of the final product. During the process of biomass propagation, yeast cells are dynamically exposed to a mixed and interrelated group of known stresses such as osmotic, oxidative, thermic, and/or starvation. These stressing conditions can dramatically affect the parameters of the fermentation process and the technological abilities of the yeast, e.g., the biomass yield and its fermentative capacity. Although a good knowledge exists of the behavior of S. cerevisiae under laboratory conditions, insufficient knowledge is available about yeast stress responses under the specific media and growth conditions during industrial processes. We performed growth experiments using bench-top fermentors and employed a molecular marker approach (changes in expression levels of five stress-related genes) to investigate how the cells respond to environmental changes during the process of yeast biomass production. The data show that in addition to the general stress response pathway, using the HSP12 gene as a marker, other specific stress response pathways were induced, as indicated by the changes detected in the mRNA levels of two stress-related genes, GPD1 and TRX2. These results suggest that the cells were affected by osmotic and oxidative stresses, demonstrating that these are the major causes of the stress response throughout the process of wine yeast biomass production.     

Cadmium-induced synthesis of HSP70 and a role of glutathione in Euglena gracilis

Abstract

The effect of cadmium-induced oxidative stress, with or without glutathione supplementation, was investigated in the single cell eukaryotic phytoflagellate, Euglena gracilis strains Z and its achlorophyllous mutant SMZ as experimental models. Both these strains actively synthesize thiols to prevent or resist cadmium toxicity. The content of glutathione, as a representative antioxidant, was also examined in both strains. Exposure to cadmium induced heat-shock protein 70 (HSP70) synthesis in both strains of E. gracilis. Glutathione supplementation also induced HSP70. Overall, these results indicate that glutathione was closely linked to the induction of stress-related proteins. The sensitivity to cadmium-stress was higher in strain Z than SMZ. The results suggest that chloroplasts may have a role in the regulation of HSP70 expression. The relationship between HSP70 and GSH levels is still far from understood, and further research may shed light upon their up-regulation in the presence of Cd.     

Characterization of HSP70 expression in drones of Apis cerana (Hymenoptera: Apidae)

Heat shock proteins (HSPs) constitute a superfamily of molecular chaperones that are rapidly biosynthesized in response to various biotic and abiotic factors. In this study, we first cloned the full-length HSP70 gene of the Eastern honeybee Apis cerana. Then, using real-time quantitative PCR, we explored HSP70 expression profiles in drones at different developmental stages, ages, and reproductive statuses (with and without semen). The full-length HSP70 cDNA is 2421 bp, including a 1953-bp open reading frame (ORF) that encodes a polypeptide of 650 amino acids. The HSP70 gene consists of one intron and two exons. The phylogenetic analysis revealed that the HSP70 genes of A. cerana and Apis mellifera are the most closely related. We observed HSP70 expression at all selected developmental stages and detected the highest expression in pupae with an unpigmented body cuticle and brown eyes (Pb) and much lower expression in larvae hatched within 72 h. In adult drones of different ages, the highest expression level of HSP70 was observed in 16-day-old drones; significantly lower accumulation of HSP70 mRNA was detected in 4-day-old drones. There was no significant difference in HSP70 expression between drones with and without semen captured at the entrance, while the HSP70 gene expression level strikingly differed between drones captured at the entrance and the drones collected within the hive. Our study suggests that HSP70 might play a critical role in drone development and during reproductive mating events.     

Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.     

Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H₂O₂-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H₂O₂-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.