Heat Shock Group Research Articles

Physiological high temperatures alter the amino acid metabolism of bovine early antral follicles.

Heat stress reduces the developmental competence of bovine oocytes during the growth phase; however, the detailed mechanisms remain unclear. Amino acids play various critical roles in follicular development, including protein synthesis and as energy sources. We performed in vitro growth (IVG) culture of oocyte-cumulus-granulosa complexes (OCGCs) to assess the amino acid metabolism of small follicles at high temperatures. We isolated OCGCs from early antral follicles (0.5-1.0 mm) and subjected them to IVG culture for 12 days. OCGCs in the heat shock group were cultured under a temperature cycle of (38.5°C: 5 h, 39.5°C: 5 h, 40.5°C: 5 h, and 39.5°C: 9 h) to reproduce the body temperature of lactating cows under a hot environment. OCGCs in the control group were cultured at a constant temperature of 38.5°C for 24 h. Of the surviving OCGCs, those showing similar morphology and size between the groups were selected for amino acid analysis. We analyzed the free amino acids and their metabolites in the culture medium and calculated the depletion or appearance of molecular species. The depletion of three essential amino acids (isoleucine, leucine, and valine), two non-essential amino acids (aspartic acid and glycine), and ornithine was higher in the heat shock group (P < 0.05). Alanine depletion was lower in the heat shock group (P < 0.05). We concluded that heat exposure alters the amino acid metabolism of OCGCs isolated from early antral follicles, which might be involved with the diminished developmental potential of oocytes during summer.

Effect of Pretreatment of Anaerobic Acid Fermentation Sludge from Mixed Swine Wastewater and Food Waste Leachate on Biohydrogen Generation

Objectives:This study aims to investigate the effects of heat shock and 2-bromoethanesulfonate (2-BES) injection on hydrogen production and methanogens inhibition in anaerobic acid fermenter sludge and methane fermenter sludge using mixed swine wastewater and food waste leachate as substrates. Additionally, the analysis of biogas, fermentation products such as volatile fatty acids (VFAs) and alcohols, and microbial community changes were conducted to derive efficient pretreatment conditions for anaerobic hydrogen generation.Methods:Sludge samples were collected from two-phase anaerobic digesters treating swine wastewater and food waste leachate. Heat shock (90°C, 15 min) and 2-BES injection (1, 5, 10, 50, and 100 mmol/L) pretreatments were applied to the each sludge, along with control reactor without pretreatment before anaerobic digestion. Batch experiments were conducted at 30°C to assess biogas, organic compounds (COD, VS, VFAs, and alcohols), and microbial community composition.Results and Discussion:In methanogenic sludge, hydrogen gas was not produced under all conditions, but heat shock method inhibited methane production more effectively than 2-BES injection method. As the concentration of 2-BES increased, methane production decreased. Conversely, hydrogen was produced from the all acid fermentation sludges, mainly attributed to the butyric acid production by dominant Clostridium spp., but the pattern of biogas production varied over time. In the experimental group injected with 2-BES, even at a low concentration of 1 mmol/L, methane gas did not produce due to the inhibition of methanogens. However, in the control group, produced hydrogen was entirely converted to methane by hydrogenotrophic methanogens such as Methanobrevibacter and Methanosphaera. The heat shock group showed delayed hydrogen production due to prolonged lag phase of hydrogen-producing bacteria, with hydrogenotrophic methanogen metabolism starting after 12 days. Heat shock and 2-BES injection reduced bacterial diversity in the acid fermentation process compared to the initial stage.Conclusion:For efficient anaerobic hydrogen production from swine wastewater and food waste leachate, 2-BES injection into the acid fermentation sludge was found to be a preferable method over heat shock, considering hydrogen production efficiency and sustained methanogen inhibition. Further research is needed to find more economical and efficient pretreatment methods and operational strategies considering subsequent methane production processes for practical application in the field.

Thermal tolerance role of novel polyamine, caldopentamine, identified in fifth instar Bombyx mori.

Silkworms have limited ability to regulate their body temperature; therefore, environmental changes, such as global warming, can adversely affect their viability. Polyamines have shown protection to various organisms against heat stress. This study evaluated the qualitative and quantitative changes in heat-stressed Bombyx mori larvae polyamines. Fifth instar Bombyx mori larvae were divided into two groups; control group,reared at room temperature, i.e., 28 ± 2°C, and the heat shock group, exposed to 40°C. Dansylation of the whole worm polyaminesand subsequent thin-layer chromatography revealed the presence of components with the same Rf value as dansyl-putrescine, spermidine, and spermine. The dansyl-putrescine, spermidine, and spermine polyamines were identified by mass spectrometric analyses. After heat shock, the thin-layer chromatography of the whole-larvae tissue extracts showed qualitative and quantitative changes in dansylated polyamines. A new polyamine, caldopentamine, was identified, which showed elevated levels in heat-stressed larvae. This polyamine could play a role in helping the larvae tolerate various stress, including thermal stress. No significant changes in silk fiber's economic and mechanical properties were observed in our study. This study indicated that PA, caldopentamine, supplementation could improve heat-stress tolerance in Bombyx mori.

Effect of heat exposure on the growth and developmental competence of bovine oocytes derived from early antral follicles

In dairy cows, low fertility caused by summer heat stress continues into the cooler autumn season. This can be caused by impaired oocyte quality in small growing follicles during summer. Here, we subjected oocyte–cumulus–granulosa complexes (OCGCs) derived from early antral follicles (0.5–1 mm) to in vitro growth (IVG) culture under two different temperature settings (the control and heat shock groups), and evaluated effects of heat exposure on growth and developmental competence of oocytes, factors affecting the developmental competence of oocytes (steroidogenesis of granulosa cells, oxidative stress in oocytes, and cell-to-cell communication between oocytes and somatic cells). Oocyte diameters after culture were smaller in the heat shock group. Although nuclear maturation and cleavage rates were similar between the groups, blastocyst rates were lower in the heat shock group (0.0%) than in the control group (27.7%), and reduced glutathione (GSH) levels in oocytes were lower in the heat shock group. Supplementation of cysteine, which stimulates GSH synthesis, increased GSH level and improved blastocyst rate of heat shocked oocytes (27.9%). These results suggest that heat exposure impairs the growth and developmental competence of oocytes in early antral follicles through GSH depletion, which can induce low fertility during summer and the following autumn.

Effects of acute temperature increases on House sparrow (Passer domesticus) pectoralis muscle myonuclear domain.

With rapid climate change, heat wave episodes have become more intense and more frequent. This poses a significant threat to animals, and forces them to manage these physiologically challenging conditions by adapting and/or moving. As an invasive species with a large niche breadth, House sparrows (Passer domesticus) exhibit high phenotypic flexibility that caters to seasonal changes in function and metabolism. For example, their pectoral muscle complex exhibits size and mass plasticity with winter and summer acclimation. Here, we investigated the effects of acute whole-organism heat stress to 43°C on cellular-level changes in House sparrow pectoralis muscle myonuclear domain (MND), the volumetric portion each nucleus is responsible for, that have gone overlooked in the current literature. House sparrows were separated into a control group, a heat-shocked group subjected to thermal stress at 43°C for 24 h, and a recovery group that was returned to room temperature for 24 h after experiencing the same temperature treatment. Here, we found that heat-shocked and recovery groups demonstrated a decrease in number of nuclei per millimeter of fiber and increase in MND, when compared with the control. We also found a significant positive correlation between fiber diameter and MND in the recovery group, suggesting the possibility that nuclei number constrains the extent of muscle fiber size. Together, these results show that acute heat shock alters House sparrow pectoralis muscle cellular physiology in a rigid way that could prove detrimental to long-term muscle integrity and performance.

Histopathology and oxidative stress responses of Nile tilapia Oreochromis niloticus exposed to temperature shocks

Temperature shock is a major natural cause of mass mortality of global fish. Nile tilapia Oreochromis niloticus, one of the most widely cultured species, with high resistance against severe environmental conditions, is also affected by rapid temperature changes. This research was conducted to investigate histopathological changes and oxidative stress in O. niloticus in response to temperature shock. The malondialdehyde levels in the serum, gills and brain, the histopathology of the gills and brain, and the histochemical characterization of the gills’ mucosubstances were measured in the fish exposed to heat shock and cold shock conditions. The results showed that the fish could not survive under 37 °C for 24 hours. Malondialdehyde levels in the gills, brain and serum increased significantly when compared to those of the controls. Moreover, histopathological changes and a decrease in the number of neutral and acidic mucous cells was observed in the gills of fish in both the heat shock and cold shock groups. Histopathological alteration, vacuolated neuropil in the brain, was observed only in the fish in the cold shock groups. The results from this study indicate that rapid 4 °C changes in water temperature (25–21 and 25–29 °C) evoked oxidative stress and histological damage to O. niloticus, whereas extreme 12 °C changes (25–13 and 25–37 °C) severely affected their oxidative stress and histopathological condition.

Molecular cloning and expression dynamics of UNC-45B upon heat shock in the muscle of yellowtail

As a myosin chaperone, uncoordinated-45B (UNC-45B) is expressed specifically in striated muscle tissues. It has been proved that overheat shock will lead to myosin denaturation, and that UNC-45B is involved in myosin assembly. Yellowtail was chosen for this study, as an important economic aquaculture fish worldwide. To establish the heat shock response of UNC-45B in yellowtail, cDNA cloning and expression analysis were carried out. The cDNA of yellowtail unc-45b was successfully cloned, and consisted of 3051 bp encoding 930 amino acid residues (ORF 2793 bp). Based on the sequence of yellowtail unc-45b, we designed original primers and an anti-peptide antibody for our study. Yellowtail were then exposed to short-term or long-term heat shock, and UNC-45B expression levels were determined by qPCR and western blot analysis. For short-term heat shock exposure, the fish were kept in water tanks at 25 °C (control group) and 30 °C (heat shock group) for up to 30 h. The mRNA and protein expression levels of UNC-45B increased markedly after 8 h and 24 h, respectively. To investigate the effects of long-term heat shock exposure, cultured yellowtail were sampled in summer and winter at sea surface temperatures of 30 °C and 14 °C. The expression of UNC-45B in both mRNA and protein levels was significantly higher in summer than in winter. Our results show that the increase in UNC-45B expression levels can reflect heat shock response sensitively, and it is suggested that UNC-45B can repair the denatured myosin during heat shock exposure in yellowtail. Moreover, we successfully detected the protein expression of UNC-45B in aquaculture fish species in native conditions by using western blot analysis for the first time. Therefore, we assume that UNC-45B can be regarded as a biomarker to predict heat shock resistance level. This would help in aquaculture production, in the selection of juveniles able to endure heat shock.

Comparison of Cytokine Expression in Human PBMCs Stimulated with Normal and Heat-Shocked Lactobacillus plantarum Cell Lysate.

Regulation of immune responses is among the beneficial effects of probiotic bacteria on human health. In this study, we aim to investigate the effect of normal and heat-shocked Lactobacillus plantarum PTCC 1058 cell lysate on cytokine expression by human PBMCs. The mid-exponential phase L. plantarum (108 CFU/mL) were used to prepare cell lysate. Isolated PBMCs were stimulated with 100 µg/mL of each normal and heat-shocked L. plantarum cell lysate for 72 h. Non-stimulated PBMCs were also evaluated as negative control. The mRNA expression of IL-6, IL-10, IFN-ɣ, TNF-α, and TGF-β genes was determined by quantitative RT-PCR amplification of total RNA extracted from PBMCs. Both types of cell lysate were able to increase pro-inflammatory cytokines and decrease anti-inflammatory cytokines. However, this effect was significantly stronger in heat-shocked cell lysate-treated PBMCs. Moreover, comparison of IFN-ɣ/IL-10, IFN-ɣ/TGF-β, IL-6/IL-10, IL-6/TGF-β, and TNF-α/IL-10 ratios in both conditions demonstrated that in the heat-shocked group, all of the above ratios were significantly higher than normal lysate treatment (p˂0.001), suggesting that heat-shocked probiotics are a potent inducer of the immune system in comparison to intact probiotics. Regarding these results, it may be possible to develop a new postbiotic product for the stimulation of immune responses of cancer patients or individuals who suffer from an immune defect.

85 The effects of heat exposure on the growth and developmental competence of oocytes derived from early antral follicles in dairy cows

Summer heat stress in dairy cows impairs the developmental competence of oocytes from antral follicles (2–8mm) which are used in conventional IVM and IVF systems. Moreover, summer heat stress is considered to impair the oocyte competence derived from smaller follicles; therefore, the impairment of oocyte competence possibly continues into the cooler autumn season. To investigate the thermosensitivity of early antral follicles (&amp;lt;1mm), we evaluated the effects of heat exposure on the growth and developmental competence of oocytes using invitro culture of oocyte–cumulus-granulosa complexes (OCGCs) derived from early antral follicles. OCGCs (n=315) were collected from early antral follicles (0.5–1mm) and cultured for 12 days. OCGCs in the heat shock group were cultured using a temperature cycle of 38.5°C for 5h, 39.5°C for 5h, 40.5°C for 5h, and 39.5°C for 9h, whereas those in the control group were cultured at a constant temperature of 38.5°C for 24h. The diameters of oocytes were measured before culture. Half of the culture medium was replaced every 4 days. Oestradiol-17β (E2) and progesterone (P4) production during the first, second, and third 4-day periods were measured by enzyme immunoassay; the viability of OCGCs was evaluated based on their morphology. Oocytes that survived after 12 days of culture (n=191) were subjected to IVM (38.5°C, 22h); their diameter and nuclear status were evaluated. Some oocytes (n=71) were subjected to IVF (38.5°C, 18h) and embryo culture (39.0°C, 150h). Cleavage and blastocyst rates were evaluated at 48h and 168h after IVF. Effects of treatment groups and culture periods on E2 and P4 production and diameters of oocytes were evaluated by two-way ANOVA followed by Tukey-Kramer or Student’s t-test. The viability of OCGCs, nuclear maturation, cleavage and blastocyst rates between two groups were compared by the chi-squared or Fisher’s exact test. E2 and P4 production and the viability of OCGCs were not different between the 2 groups. Although mean oocyte diameters before culture did not differ between the 2 groups, the mean diameters after IVM were significantly smaller in the heat shock group (108.0µm, n=56) than in the control group (111.7µm, n=61; P&amp;lt;0.05). The nuclear maturation rate in the heat shock group (36.4%, n=55) was significantly lower than in the control group (60.3%, n=58; P&amp;lt;0.05). Cleavage rates were similar between the control (54.5%, n=33) and heat shock groups (45.7%, n=35). However, no oocytes developed to blastocysts in the heat shock group (0%, n=35), whereas 30.3% (n=33) oocytes developed to blastocysts (cell number±s.d.; 92.4±28.4) in the control group (P&amp;lt;0.05). These findings suggest that summer heat stress in dairy cows impairs the growth, nuclear maturation, and developmental competence of oocytes derived from early antral follicles. This experimental model could be used to explore the mechanisms by which heat stress subsequently impairs oocyte competence during the cooler autumn season.

Melatonin slightly alleviates the effect of heat shock on bovine oocytes and resulting blastocysts

Heat stress is associated with increased production of reactive oxygen species (ROS) and disruption of bovine oocyte function. Here, we examined whether the antioxidant melatonin can alleviate the deleterious effects of heat stress on oocyte developmental competence. Cumulus-oocyte complexes were matured for 22 h at 38.5 °C (control) or for 22 h at 41.5 °C (heat shock) with or without 1.0 × 10−7 M melatonin. At the end of maturation, a subgroup of oocytes was examined for nuclear and cytoplasmic maturation, ROS level and mitochondrial membrane potential. A second subgroup of oocytes underwent fertilization (18 h), and putative zygotes were cultured in an incubator equipped with a time-lapse system for ∼190 h. Cleavage rate and the proportion of blastocysts, as well as embryo kinetics were recorded. Expanded blastocysts were collected and their transcript abundance was evaluated. Heat shock increased ROS and reduced the proportion of oocytes that resumed meiosis and reached the metaphase-II stage. Exposing oocytes to heat shock with melatonin alleviated these effects to some extent, expressed by a marginal reduction in ROS level and increased proportion of metaphase-II stage oocytes. Neither the distribution of oocyte cortical granules nor polarization of the mitochondrial membrane differed between control and heat-shocked oocytes cultured with or without melatonin. Heat shock reduced the proportion of embryos that cleaved and developed to blastocysts, characterized by alterations in kinetics of the developed embryos expressed by a delay in the first cleavage, second cleavage and blastocyst formation for heat-shock vs. control groups. Melatonin did not restore the competence or kinetics of embryos developed from heat-shocked oocytes. However, expanded blastocysts developed from heat-shocked oocytes treated with melatonin expressed a higher transcript abundance of genes associated with mitochondrial function, relative to the control and heat-shock group. In summary, melatonin improved the oxidative status of heat-shocked oocytes to some extent and had a beneficial effect on maternal mitochondrial transcripts in the developed blastocysts.

Transcriptome Profile Changes Associated With Heat Shock Reaction in the Entomopathogenic Nematode, Steinernema carpocapsae.

The entomopathogenic nematode Steinernema carpocapsae has been used for control of soil insects. However, S. carpocapse is sensitive to environmental factors, particularly temperature. We studied an S. carpocapse group that was shocked with high temperature. We also studied the transcriptome-level responses associated with temperature stress using a BGIseq sequencing platform. We de novo assembled the reads from the treatment and control groups into one transcriptome consisting of 43.9 and 42.9 million clean reads, respectively. Based on the genome database, we aligned the clean reads to the Nr, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases and analyzed the differentially expressed genes (DEGs). Compared with the control, the heat-shocked group had significant differential expression of the heat shock protein (HSP) family, antioxidase [glutathione S-transferases (GSTs) and superoxide dismutase (SOD)], monooxygenase (P450), and transcription factor genes (DAF-16 and DAF-2). These DEGs were demonstrated to be part of the Longevity pathway and insulin/insulin-like signaling pathway. The results revealed the potential mechanisms, at the transcriptional level, of S. carpocapsae under thermal stress.

Melatonin protects mouse oocyte from heat stress damage

Objective: To explore the potential effect of melatonin on the in-vitro maturation of mouse oocytes under heat shock condition.Methods: This study used a heat shock model of mouse oocyte maturation. The oocytes were randomly divided into three groups: control group, heat shock group and heat shock + melatonin group, in order to evaluate the effect of 1×10−9 mol/L melatonin on the quality of oocytes after heat shock.Results: In comparison with the control group, the maturation rate of mouse oocytes in heat shock group was significantly decreased [(33.00 ± 0.07)% vs. (85.00 ± 0.03)%, p &lt; .01], with abnormal spindle assembly, and the early apoptosis rate was significantly increased [(59.7 ± 4.5)% vs. (22.0 ± 3.5)%, p &lt; .01]. Compared with heat shock group, the maturation rate ofoocytes was significantly increased in heat shock + melatonin group [(70.00 ± 0.05)% vs. (33.00 ± 0.07)%, p &lt; .01], meanwhile, the spindle abnormality rate and the early apoptosis rate were significantly decreased accordingly [(37.3 ± 6.1)% vs. (59.7 ± 4.5)%, p &lt; .01]. The expression level of heat shock protein 70 was significantly up-regulated in heat shock + melatonin group in comparison with other two groups (p &lt; .01).Conclusions: By regulating the over-expression of heat shock protein 70, melatonin can improve the declined maturation rate of oocytes and the increased rates of spindle assembly abnormality and early apoptosis caused by heat shock.

Expression of HSP70 Heat-Shock Proteins under Oxidative Stress

The development of oxidative stress inevitably causes damage to cell proteins in need of timely refolding. The synthesis of the proteins of the heat-shock group is the most ancient and effective mechanism of cell protection. The life-long cellular adaptation to a huge number of cytotoxic factors, both of xenobiotic and natural origin, have provided heat-shock proteins with polyfunctionality: in the processes of programmed death, they act as either anti- or pro-apoptogenic factors or as regulators of the activity of these factors. The important role of heat-shock proteins in adaptation, inflammation, and immune response had also been shown. Various experimental and clinical studies confirmed the important role of heat-shock proteins in the development of pathophysiological phenomena of oxidative stress, aging, tumor formation, and immune reactions. The data presented in the review includes domestic and foreign studies on the physiological significance of heat-shock proteins, their role in the mechanisms of cell, and body stability in general and are relevant to modern medicine and biology.

Time-dependent effects of heat shock on the zona pellucida ultrastructure and in vitro developmental competence of bovine oocytes

The aim of this study was to determine the effects of different exposure lenght to heat shock (HS) during in vitro maturation (IVM) on zona pellucida (ZP) ultrastructure and developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were matured in vitro (IVM) at 38.5 °C for 24 h (control group, CG), or incubated at 41 °C (HS) for 6 h (HS-6h), 12 h (HS-12h), 18 h (HS-18h), and 22h (HS-22h) followed by incubation at 38.5 °C to complete a full 24-h period of maturation. After IVM, oocytes were subjected to scanning electron microscopy (SEM) or in vitro fertilization and culture until the blastocyst stage. For heat-shocked oocytes, with exception of those in the HS-6h group, SEM examinations revealed that ZP surfaces were rough and characterized by a presence of spongy network. Oocytes from the HS-22h group displayed an increase in the number of pores, as well as a higher proportion of oocytes with amorphous ZPs. The proportion of oocytes that reached metaphase II (MII) stage decreased in all HS groups, regardless of the duration of exposure to 41 °C. These results provide evidence that HS during IVM for 12–22 h reduces the developmental competence of bovine oocytes, increasing the percentage of oocytes with abnormal chromosomal organization, and reducing fertilization and blastocysts formation rate. The effects of HS were more pronounced for the 22-h exposure group. The damage induced by HS on oocyte function clearly increased upon exposure to elevated temperature.

Construction of ovine GH-pmKate2N expression vector and its uptake by ovine spermatozoa using different methods.

This study aims to produce transgenic ovine spermatozoa bearing Ossimi sheep growth hormone (Os_GH) cDNA using different methods. The complete coding sequence of Os_GH has been registered in GenBank accession no. KP221575. The sequence of Os_GH cDNA has been subcloned into pmkate2-N expression vectors to construct Os_GH-pmKate2-N vector. Five groups of sperm uptake were submitted. All groups were incubated at 37 °C for 1 h: Control (sperm cells were incubated without vector), Traditional incubation (sperm cells were incubated with vector), Heat shock (sperm cells were incubated with vector at 4 °C for 20 min and heated for 2 min at 42 °C), Heat shock + Dimethyl sulfoxide (DMSO) (sperm cells were incubated with vector and supplemented with 3% of DMSO and then submitted to heat shock regime) and DMSO (sperm cells were incubated with vector and supplemented with 3% DMSO). The sperm genomic DNA in groups was extracted. The Os_GH-pmKate2-N vector was introduced efficiently into the head of sperm cells in all treated groups. Adding DMSO either with or without heat shock increased the sperm uptake. The progressive motility was reduced (P < 0.05) by 29.9% in heat shock group compared to the control. Adding DMSO improved (P < 0.05) the total and progressive motilities by 8.2% and 19.8%, respectively in heat shock group compared to the heat shock group without DMSO. The results documented the ability of ovine spermatozoa to uptake the exogenous vector. Also, sperm incubation with 3% DMSO is the best method to introduce the exogenous vector into spermatozoa without notable adverse effects on sperm motilities.

Gene expression of HSP90 and HSP70 in four silkworm hybrids (Bombyx mori L.) in response to severe thermal shock

Usually silkworm egg producers provide several silkworm hybrids with different qualities of productivity and viability traits. This study was conducted to compare the expression of HSP70 and HSP90 genes among different silkworm hybrids. In the fourth day of fifth larval instar of two Japanese maternal parents (103×104 and 107×110) and two Chinese maternal parents (110×107 and 104×103), silkworm larval fat body was sampled from heat shock (45 °C for 35 min) and non-heat shock groups. Sampling was done at 0, 2, 4 and 24 h after heat shock. Gene expression of HSP70 and HSP90 (target genes) were measured by qRT-PCR using RPL27a as reference gene. The prolonged heat shock (8 h at 39 °C) was utilized to examine the thermal tolerance of larvae in comparison with control group (24°C). The results showed both HSP70 and HSP90 have been up-regulated in the treated larvae. The hybrids with Japanese female parents (107×110 and 103×104) were more sensitive than two others; moreover in these hybrids, HSP90 and HSP70 were expressed significantly more than others after heat exposure. The maximum expression was occurred in the time zero, then a decreasing trend was observed over time. Comparison of larval mortality among four hybrids revealed that (103×104) and (110×107) hybrids had the highest and the lowest mortality rate.

Modifications in the proteome of rainbow trout (Oncorhynchus mykiss) embryo and fry as an effect of triploidy induction.

Two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry, and database searching were used to analyze the effects of triploidization heat shock treatment on protein expression in rainbow trout eyed embryo and fry. After fertilization, the eggs were incubated at 10°C for 10min. Half of the eggs were then subjected to heat shock for 10min submerged in a 28°C water bath to induce triploidy. The remainder was incubated normally and used as diploid controls. Specimens of eyed embryos and fry were taken on 18 and 76days post-fertilization, respectively. In the eyed embryo extracts, seven protein spots were significantly changed in abundance between the control and heat-shocked groups and one of these was decreased while the others were increased in the heat shock-treated group. Of the spots that were shown to change in abundance in the eyed embryos with heat shock treatment, two were identified as vitellogenin, while the others were creatine kinase and angiotensin I. In the 2-DE from the fry muscle extraction, 23 spots were significantly changed in abundance between the diploid and triploid groups. Nineteen of these showed a decreased abundance in diploids, while the remaining four spots had an increased abundance. Triploidization caused differential expression of muscle metabolic proteins including triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and beta-enolase. Myosin heavy chain as a structural protein was also found to change in abundance in triploids. The altered expression of both structural and metabolic proteins in triploids was consistent with their increased cell size and lower growth performance.

Effect of patchouli alcohol on the regulation of heat shock-induced oxidative stress in IEC-6 cells

Purpose Patchouli alcohol (PA) is used to treat gastrointestinal dysfunction. The purpose of this study was to ascertain the function of PA in the regulated process of oxidative stress in rat intestinal epithelial cells (IEC-6). Materials and methods Oxidative stress was stimulated by exposing IEC-6 cells to heat shock (42 °C for 3 h). IEC-6 cells in treatment groups were pretreated with various concentrations of PA (10, 40, and 80 ng/mL) for 3 h before heat shock. Results Heat shock caused damage to the morphology of IEC-6 cells, and increased reactive oxygen species (ROS) level and malondialdehyde (MDA) content. Moreover, mRNA and protein expression by target genes related to oxidative stress in heat shock were also altered. Specifically, the mRNA expression by HSP70, HSP90, GSH-px, NRF2 nd HO-1were all increased, and Nrf2 and Keap1 protein expression were increased after heat shock. However, pretreatment with PA weakened the level of damage to the cellular morphology, and decreased the MDA content caused by heat shock, indicating PA had cytoprotective activities. Pretreatment with PA at high dose significantly increased generation of intracellular ROS. Compared with the heat shock group alone, PA pretreatment significantly decreased the mRNA expression by HSP70, HSP90, SOD, CAT, GSH-px, KEAP1 and HO-1. Furthermore, the high dose of PA significantly increased Nrf2 protein expression, while both the intermediate and high dose of PA significantly increased HO-1 protein expression. Conclusion Heat-shock-induced oxidative stress in IEC-6 cells, and PA could alleviate the Nrf2-Keap1 cellular oxidative stress responses.

Compression Loading Induced Cellular Stress Response of Intervertebral Disc Cells in Organ Culture

Introduction Mechanical stress is often associated to interverterbal disc (IVD) degeneration and the effect of mechanical loading on IVD has been studied and reviewed.1,2 Previously, expression of heat shock proteins, HSP70 and HSP27 has been found in pathological discs.3 However, there is no direct evidence on whether IVD cells respond to the mechanical loading by expression of HSPs. The objective of this study is to investigate the stress response of IVD cells during compressive loading in an organ culture. Materials and Methods Fresh adult bovine caudal discs were cultured with compressive loading applied at physiological range. Effect of loading type (static and dynamic) and repeated loading (2 hours per day for 2 days) were studied. Nucleus pulposus (NP) and annulus fibrosus (AF) of the IVD were retrieved at different time points: right after loading and right after resting. Positive control discs were heat shocked (43°C). Cell activity was assessed and expression of stress response genes (HSP70 and HSF1) and matrix remodeling genes (ACAN, COL2, COL1, ADAMTS4, MMP3 and MMP13) were studied. Results Cell activity was maintained in all groups. Both NP and AF expressed high level of HSP70 in heat shock groups, confirming their expression in response to stress. In NP, expression of HSP70 was up-regulated after static loading and dynamic loading with higher fold change was observed after static loading. During repeated loading, HSP70 appeared to be upregulated right after loading and decreased after resting. Such trend was not observed in AF and HSF1 levels. Expressions of matrix remodeling genes did not change significantly with loading except ADAMTS4 decreased in AF during static loading. Conclusion This study demonstrated that NP cells upregulate expression of HSP70 in response to loading induced stress without changing cell activity and matrix remodeling significantly. Acknowledgments This project was funded by AO Spine (AOSPN) (grant number: SRN_2011_14) and a fellowship exchange award by AO Spine Scientific Research Network (SRN). References Chan SC, Ferguson SJ, Gantenbein-Ritter B. The effects of dynamic loading on the intervertebral disc. Eur Spine J 2011;20(11):1796–1812 Neidlinger-Wilke C, Galbusera F, Pratsinis H, et al. Mechanical loading of the intervertebral disc: from the macroscopic to the cellular level. Eur Spine J 2014;23(Suppl 3):S333–S343 Sharp CA, Roberts S, Evans H, Brown SJ. Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining. Eur Spine J 2009;18(11):1587–1594

Changes in α7β1 integrin signaling after eccentric exercise in heat-shocked rat soleus.

α7β1 integrin links the extracellular matrix to the focal adhesion (FA) in skeletal muscle and serves as a stabilizing and signal relayer. Heat shock (HS) induces expression of proteins that interact with the FA. Male Wistar rats were assigned to 1 of 3 groups: control (CON); eccentric exercise (EE); or EE+HS (HS). Soleus muscle was analyzed at 2 h and 48 h post-exercise. The 120-kDa α7 integrin decreased in the EE and HS groups, and the 70-kDa peptide decreased in the EE group at 2 h post-exercise. Total expression of focal adhesion kinase (FAK) and RhoA were decreased in EE and HS at 2 h post-exercise. Expression of phosphorylated FAK(397) decreased in the EE group but not the HS group at 2 h post-exercise. Long-duration EE may cause alterations in the FA in rat soleus muscle through the α7 integrin subunit and FAK.