Gene Silencing of Selenoprotein K Induces Inflammatory Response and Activates Heat Shock Proteins Expression in Chicken Myoblasts

In the present study, specific small interfering RNA (siRNA) for selenoprotein K (Selk) gene was designed and transfected into chicken myoblasts. Then, the expressions of inflammatory factors (including induced nitric oxide synthase [iNOS], nuclear factor-kappa B [NF-κB], heme-oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and prostaglandin E synthase [PTGEs]), inflammation-related cytokines (including interleukin [IL]-1β, IL-6, IL-7, IL-8, IL-17, and interferon [IFN]-γ), and heat shock proteins (HSPs) (including HSP27, HSP40, HSP60, HSP70, and HSP90) were examined at 24 and 72 h after transfection. The results showed that messenger RNA (mRNA) expressions of iNOS, NF-κB, HO-1, COX-2, IL-6, IL-7, IL-8, HSP 27, HSP 40, HSP 60, HSP 70, and HSP 90 were significantly increased (p < 0.05) at 24 and 72 h after siRNA transfection, and the mRNA expressions of PTGEs, IL-1β, IL-17, and IFN-γ were significantly increased and decreased (p < 0.05) at 24 and 72 h after siRNA transfection. The results also showed that the protein expressions of iNOS, NF-κB, HO-1, COX-2, HSP60, HSP70, and HSP90 were significantly increased (p < 0.05) at 24 and 72 h after siRNA transfection. The correlation analysis and principal component analysis (PCA) showed that PTGEs, IL-1β, IL-17, IFN-γ, HSP40, and HSP90 might play special roles in response to Selk silencing in chicken myoblasts. These results indicated that Selk silencing induced inflammation response by affecting the expression levels of inflammatory factors and inflammation-related cytokines, and the heat shock proteins might play protective roles in this response in chicken myoblasts.     

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.     

Serum levels of HSP70 and other DAMP proteins can aid in patient diagnosis after traumatic injury

Danger-associated molecular patterns (DAMPs) are activated by endogenous signals that originate from stressed, injured, or necrotic cells, signifying “danger” to the host. In this study, we evaluated the expression of the DAMP heat shock protein 70 (HSP70) in trauma patients with and without secondary infections. Levels of glucose (GLU), procalcitonin (PCT), total cholesterol (T-Chol), and white blood cell (WBC) counts were also evaluated at three time stages after trauma. Our analysis showed that the levels of serum HSP70 in patients with minor, moderate, and severe injuries were significantly higher than in healthy patients at each time point post-injury (P?<?0.01), and levels of serum HSP70 in the severe injury group were significantly higher than in the minor injury group at 1–6 h after trauma (P?=?0.047). HSP70 was correlated with GLU and was negatively correlated with T-Chol in the period 1–6 h after injury (P?=?0.008/0.032). WBC and GLU were elevated after trauma, with mutual positive correlation (P?<?0.001). PCT levels increased later than WBC counts and GLU levels; these levels were correlated at the two later time periods, 24–48 h and 60–90 h (P?=?0.008/0.041). PCT continued to rise in patients with secondary infection, but PCT dropped at the third time period in patients without secondary infection. In summary, our results suggest that danger and stress theory can be used to predict severity of trauma.     

Systematic identification and characterization of stress-inducible heat shock proteins (HSPs) in the salmon louse (<Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>)

Salmon lice (Lepeophtheirus salmonis) are parasitic copepods, living mainly on Atlantic salmon and leading to large economical losses in aquaculture every year. Due to the emergence of resistances to several drugs, alternative treatments are developed, including treatment with hydrogen peroxide, freshwater or thermal treatment. The present study gives a first overview of the thermotolerance and stress response of salmon lice. Sea lice nauplii acclimated to 10 °C can survive heat shocks up to 30 °C and are capable of hardening by a sublethal heat shock. We searched in the genome for heat shock protein (HSP) encoding genes and tested their inducibility after heat shock, changes in salinity and treatment with hydrogen peroxide, employing microfluidic qPCRs. We assessed 38 candidate genes, belonging to the small HSP, HSP40, HSP70 and HSP90 families. Nine of these genes showed strong induction after a non-lethal heat shock. In contrast, only three and two of these genes were induced after changes in salinity and incubation in hydrogen peroxide, respectively. This work provides the basis for further work on the stress response on the economically important parasite L. salmonis.     

Comparative assessment of native,crossbred and exotic pigs during different seasons (winter,spring and summer) based on rhythmic changes in the levels of serum cortisol,lactate dehydrogenase levels and PBMC HSP70 mRNA expression pattern

The present study was conducted to ascertain the adaptive capability of pigs to different seasons based on changes in serum cortisol and lactate dehydrogenase (LDH) levels, and peripheral blood mononuclear cell (PBMC) heat shock protein 70 (HSP70) mRNA expression. Based on average THI, the seasons were classified as winter (November–February), spring (March–June), and summer (July–October). Hormone cortisol was found to be influenced by season (p < 0.01), age (p < 0.05), and genetics of the animal (p < 0.05). However, level of LDH was not influenced by either of these factors. HSP70 mRNA expression was higher in almost all age groups in crossbred and exotic pigs during summer in comparison to other seasons. Lower HSP70 gene expression was observed in almost all age groups of native pigs in comparison to crossbred and exotic during summer. In conclusion, native pigs were acclimatized for thermal stress in comparison to crossbred and exotic breeds of pigs. Also, the expression pattern of HSP70 gene is breed-specific, most likely due to variations in thermal tolerance and adaptation to different environmental conditions. Both serum cortisol and HSP70 gene may act as reliable biological markers for assessing the adaptive capabilities of pigs to different seasons.     

The expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in peripheral blood of patients with HCC and lung cancer

There are different views of how the immune system participates in the reaction to cancer. Here, we evaluated expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in patients with hepatocellular carcinoma (HCC) and lung cancer to explore tumor immunity. Our analysis showed that levels of HSP70 and SPAG9 antibody were significantly higher in the serum of lung cancer and HCC patients than in the serum of healthy subjects (P < 0.001), but there were no differences in levels of HSP70 antibody in patients and controls. Levels of serum SPAG9 antibody in newly diagnosed lung cancer patients were significantly higher than in treated lung cancer patients (P < 0.05), but there were no differences in levels of HSP70 or HSP70 antibody. Levels of serum HSP70 and SPAG9 antibody, but not HSP70 antibody, were also higher in hepatitis/cirrhosis patients than in healthy subjects (P = 0.005, P < 0.001). Levels of serum SPAG9 antibody were significantly higher in HCC patients than in hepatitis/cirrhosis patients, but there were no differences in HSP70 or HSP70 antibody levels. Finally, levels of serum HSP70 and SPAG9 antibody were significantly higher in HCC patients than in lung cancer patients (P < 0.05, P < 0.001). These results indicate that cancer-testis antigen SPAG9 induces a strong humoral immune response in cancer patients but HSP70 does not. These results show that SPAG9 has potential as a tumor-specific biomarker.     

Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail <Emphasis Type="Italic">Theba pisana</Emphasis> in relation to genetic structure

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.     

Identification of HSP70 gene in <Emphasis Type="Italic">Corythucha ciliata</Emphasis> and its expression profiles under laboratory and field thermal conditions

Previous laboratory studies have demonstrated that insects can tolerate high temperatures by expressing inducible heat shock proteins (HSPs). This HSP-based tolerance, however, has seldom been studied under field conditions. Here, we cloned the HSP70 gene of Corythucha ciliata (Cchsp70), an invasive insect species with substantial thermal tolerance in subtropical China. We also compared the relative mRNA expression levels of Cchsp70 in response to controlled temperature treatments (2 h at 33–43 °C at 2 °C intervals in the laboratory) and to natural increases in temperature (08:00–14:00 at 2-h intervals, 29.7–37.2 °C) on a hot summer day in the field. The complete cDNA of Cchsp70 is 2256 bp long and has a 1917 bp open reading frame that encodes a protein (CcHSP70) with 639 amino acids. The expression levels of Cchsp70 significantly increased in response to high temperatures in both laboratory and field. At similar temperatures, however, the expression levels were much higher in the field than in the laboratory. These results suggest that CcHSP70 contributes to the thermal tolerance of C. ciliata and that factors in addition to thermal stress may induce Cchsp70 expression in the field.     

Conversion of glycerol to 1,3-dihydroxyacetone by glycerol dehydrogenase co-expressed with an NADH oxidase for cofactor regeneration

Objectives

To investigate the efficiency of a cofactor regeneration enzyme co-expressed with a glycerol dehydrogenase for the production of 1,3-dihydroxyacetone (DHA).

Results

In vitro biotransformation of glycerol was achieved with the cell-free extracts containing recombinant GlyDH (glycerol dehydrogenase from Escherichia coli), LDH (lactate dehydrogenase form Bacillus subtilis) or LpNox1 (NADH oxidase from Lactobacillus pentosus), giving DHA at 1.3 g l^?1 (GlyDH/LDH) and 2.2 g l^?1 (GlyDH/LpNox1) with total turnover number (TTN) of NAD⁺ recycling of 6039 and 11100, respectively. Whole cells of E. coli (GlyDH–LpNox1) co-expressing both GlyDH and LpNox1 were constructed and converted 10 g glycerol l^?1 to DHA at 0.2–0.5 g l^?1 in the presence of zero to 2 mM exogenous NAD⁺. The cell free extract of E. coli (GlyDH–LpNox) converted glycerol (2–50 g l^?1) to DHA from 0.5 to 4.0 g l^?1 (8–25 % conversion) without exogenous NAD⁺.

Conclusions

The disadvantage of the expensive consumption of NAD⁺ for the production of DHA has been overcome.

     

Physiological indicators of stress and meat and carcass characteristics in tail bitten slaughter pigs

Background

Tail biting is a common welfare problem in pig production and in addition to being a sign of underlying welfare problems, tail biting reduces welfare in itself. The aim of this study was to evaluate the effects of tail biting on different pre and post mortem indicators of stress in slaughter pigs and on carcass and meat characteristics. A total of 12 tail bitten (TB) and 13 control (C) pigs from a farm with a long-term tail biting problem were selected for salivary cortisol analyses before and after transport to the slaughterhouse. After stunning, samples were taken for the analysis of serum cortisol, blood lactate, intestinal heat shock protein 70 (HSP70), and meat quality characteristics. In addition, body temperature immediately after and muscle temperature 35 min after stunning were measured, as well as lean meat percentage and carcass weight.

Results

TB pigs showed a lower cortisol response to the transport-induced stress than C pigs and also had a lower serum cortisol concentration after stunning. HSP70 content in the small intestine was higher in the TB pigs than in C pigs. TB pigs had a considerably lower carcass weight therefore produced a lower total amount of lean meat per carcass than C pigs.

Conclusions

This study suggests that prolonged or repeated stress in the form of tail biting causes a blunted stress response, possibly a sign of hypocortisolism. In addition, it underlines the importance of reducing tail biting, both from an animal welfare and an economic point-of-view.

     

Stress influenced the aerotolerance of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> hsryfm 1301

Objective

To investigate the aerotolerance of Lactobacillus rhamnosus hsryfm 1301 and its influencing factors.

Results

The growth rate of L. rhamnosus hsryfm 1301 weakened noticeably when the concentration of supplemented H₂O₂ reached 1 mM, and only 2% of all L. rhamnosus hsryfm 1301 cells survived in MRS broth supplemented with 2 mM H₂O₂ for 1 h. After pretreatment with 0.5 mM H₂O₂, the surviving cells of L. rhamnosus hsryfm 1301 in the presence of 5 mM H₂O₂ for 1 h increased from 3.7 to 7.8 log CFU. Acid stress, osmotic stress, and heat stress at 46 °C also enhanced its aerotolerance, while heat stress at 50 °C reduced the tolerance of L. rhamnosus hsryfm 1301 to oxidative stress. Moreover, treatment with 0.5 mM H₂O₂ increased the heat stress tolerance of L. rhamnosus hsryfm 1301 by approximately 150-fold.

Conclusions

Lactobacillus rhamnosus hsryfm 1301 possesses a stress-inducible defense system against oxidative stress, and the cross-adaptation to different stresses is a promising target to increase the stress tolerance of L. rhamnosus hsryfm 1301 during probiotic food and starter culture production.

     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.     

Lutein protects against β-amyloid peptide-induced oxidative stress in cerebrovascular endothelial cells through modulation of Nrf-2 and NF-κb

In the present study, we determined the protective role of lutein against Aβ _25–35 peptide-induced oxidative stress and apoptosis in bEND.3 cells. Cell viability was determined through MTT assay. Reactive oxygen species, lipid peroxides, and antioxidant enzyme activities were evaluated to analyze the oxidative stress status. NF-κB and Nrf-2 downstream target protein expressions were determined through western blot. Apoptosis was analyzed through caspase activities and subG1 accumulation. The results showed that Aβ _25–35 significantly increased (p < 0.001) oxidative stress biomarkers. Aβ _25–35 significantly up-regulated NF-κB nuclear expression and down-regulated Nrf-2 levels and HO-1 and, NQO-1 expressions. Aβ _25–35 induced apoptosis through decreasing mitochondrial membrane potential and increasing caspase 9 and 3 activities. Lutein pre-treatment significantly (p < 0.001) improved cell viability and decreased ROS levels (p < 0.001) and lipid peroxidation (p < 0.01). Lutein prevented Aβ _25–35-induced NF-κB nuclear expressions and up-regulated Nrf-2 expressions. Further, lutein also improved mitochondrial membrane potential and down-regulated caspase activities and subG1 accumulation. The present study shows the protective role of lutein against Aβ _25–35-induced toxicity by modulating Nrf-2 and NF-κB expressions in cerebrovascular endothelial cells.     

Growth medium standardization and thermotolerance study of the freshwater microalga <Emphasis Type="Italic">Acutodesmus dimorphus—</Emphasis>a potential strain for biofuel production

Microalgal biomass seems to be one of the potential alternative feedstocks for the production of various types of biofuel. In the present study, first of all, suitable growth media and harvesting time were determined for the freshwater chlorophyte microalga Acutodesmus dimorphus. Cultivation of A. dimorphus in BG-11 medium for 15 days resulted in the highest biomass productivity with 24.60 % lipid and 22.78 % carbohydrate contents. Further, thermotolerance property of A. dimorphus was evaluated by heat stressing the cells at 45 °C and 50 °C up to 24 h and determining the cell mortality and pigment composition along with lipid and carbohydrate contents. Chlorophyll and carotenoid contents of cells significantly increased after heat stress at 45 °C. Increasing the heat stress from 8 to 24 h increased the dead cells by 3–4 % at both temperatures, which shows the thermotolerance of A. dimorphus. Lipid content of 27 % and carbohydrate content of 26–28 % even after 24 h of heat stress at 45 and 50 °C suggest A. dimorphus as a potential feedstock for biofuel production.     

Heat stress preconditioning does not protect renal epithelial Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters from their modulation by severe heat stress

This study compares the effects of heat and osmotic stress on heat stress protein (HSP) production while examining the putative protective action of HSPs on modulation of Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters in Madin-Darby canine kidney (MDCK) epithelial cells by severe heat stress (46 degrees C, 15 min). Preconditioning heat stress (43 degrees C, 20 min) followed by 4 h recovery at 37 degrees C led to a 35-fold increase of HSP70 mRNA expression measured by Northern blot analysis. The protein content of HSP70 and HSP27, assessed by Western blots, was augmented by 5- and 2-fold, respectively, after 6 h of recovery. In contrast to preconditioning heat stress, hyperosmotic stress (520 vs. 320 mosm) elevated HSP70 mRNA content only by 7-fold and did not significantly affect the protein content of HSP70 or HSP27. Neither cell survival, assessed as lactate dehydrogenase (LDH) release, nor the basal activities of the ion transporters and their modulation by protein kinase C, P(2)-purinoceptor and cell volume were altered by preconditioning heat stress. Severe heat stress increased extracellular LDH content from 3+/-2 to 23+/-5% and enhanced Na(+),K(+),Cl(-) and Na(+),P(i) cotransport activity by 2-3-fold. The volume- and protein kinase C-dependent regulation of these carriers was abolished by severe heat stress while regulation by P(2)-purinoceptors was preserved. Preconditioning heat stress diminished severe heat stress-induced LDH release to 11+/-4% but did not protect Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters from activation by severe heat stress and did not prevent severe heat stress-induced inactivation of protein kinase C- and volume-dependent signaling pathways. These results show that in MDCK cells, preconditioning heat stress-induced HSPs are not involved in the regulation of Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters and do not protect them from modulation by severe heat stress.     

Symmetric molecules with 1,4-triazole moieties as potent inhibitors of tumour-associated lactate dehydrogenase-A

A series of symmetric molecules incorporating aryl or pyridyl moieties as central core and 1,4-substituted triazoles as a side bridge was synthesised. The new compounds were investigated as lactate dehydro-genase (LDH, EC 1.1.1.27) inhibitors. The cancer associated LDHA isoform was inhibited with IC₅₀?=?117–174 µM. Seven compounds exhibited better LDHA inhibition (IC₅₀ 117–136 µM) compared to known LDH inhibitor – galloflavin (IC₅₀ 157 µM).