Thermotolerant Cells Research Articles

Mild heat shock at 40°C increases levels of autophagy: Role of Nrf2.

The exposure to low doses of stress induces an adaptive survival response that involves the upregulation of cellular defense systems such as heat shock proteins (Hsps), anti-apoptosis proteins, and antioxidants. Exposure of cells to elevated, non-lethal temperatures (39-41°C) is an adaptive survival response known as thermotolerance, which protects cells against subsequent lethal stress such as heat shock (>41.5°C). However, the initiating factors in this adaptive survival response are not understood. This study aims to determine whether autophagy can be activated by heat shock at 40°Cand if this response is mediated by the transcription factor Nrf2. Thermotolerant cells, which were developed during 3h at 40°C, were resistant to caspase activation at 42°C. Autophagy was activated when cells were heated from 5 to 60min at 40°C. Levels of acidic vesicular organelles (AVOs) and autophagy proteins Beclin-1, LC3-II/LC3-I, Atg7, Atg5, Atg12-Atg5, and p62 were increased. When Nrf2 was overexpressed or depleted in cells, levels of AVOs and autophagy proteins were higher in unstressed cells, compared to the wild type. Stress induced by mild heat shock at 40°C further increased levels of most autophagy proteins in cells with overexpression or depletion of Nrf2. Colocalization of p62 and Keap1 occurred. When Nrf2 levels are low, activation of autophagy would likely compensate as a defense mechanism to protect cells against stress. An improved understanding of autophagy in the context of cellular responses to physiological heat shock could be useful for cancer treatment by hyperthermia and the protective role of adaptive responses against environmental stresses.

Biovalorisation of mixed food waste through newly isolated thermo tolerant fungal cell factories: A step toward transforming waste to wealth

Food waste is a lucrative biomass consisting of abundant complex organic content that can be reduced to simpler compounds and channelled towards bio-manufacturing. In this study, the hydrolysis potential of mixed food and synthetic food waste from the newly isolated fungal enzymes was examined. The screening and characterization of fungal strains with higher starch and protein degrading ability were executed. Among the isolated strains, Aspergillus terreus exhibited a better glucoamylase enzyme production rate (89 ± 4.50 U/g). The random mutagenesis of A. terreus with 150 mM EMS enhanced its glucoamylase activity by 20% as compared with the wild-type strain. The enzyme displayed its optimum activity at 55 °C, pH 5.0 with 20 min of incubation time. Metal ions such as Mn+2 and Ca+2 at 2 mM concentration upregulated the glucoamylase activity and further at 5 mM of Mn+2 concentrations the enzyme activity was augmented by about 53% enhancement as equalled with control. Strain incubation temperature had tremendous impacts on the glucoamylase activity, showing the highest activity of 193.02 ± 6.89 (U/g) and 100.03 ± 4.67 (U/g) in SFW and MFW, respectively at 37 °C under solid state fermentation. In the later stage, the food waste hydrolysis rate was improved by adapting statistical tools such as Box-Behnken design (BBD). An optimum glucose release of 0.43 (g/g) was achieved by BBD. The artificial neural network linked genetic algorithm (ANN-GA) elevated the glucose concentration further to 0.57 (g/g). The mutated strain finally produced glucose of 0.57 g/g, free amino nitrogen of 185 mg/L and 195 mg/L of inorganic phosphate. To the best of our knowledge, this is the first study, where mixed food waste is hydrolysed efficiently using A. terreus. The attained hydrolysate can be further advanced towards integrated biorefinery.

Heat shock increases levels of reactive oxygen species, autophagy and apoptosis

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemotherapy. Temperatures above 41.5 °C are cytotoxic and hyperthermia treatments can target a localized area of the body that has been invaded by a tumor. However, non-lethal temperatures (39–41 °C) can increase cellular defenses, such as heat shock proteins. This adaptive survival response, thermotolerance, can protect cells against subsequent cytotoxic stress such as anticancer treatments and heat shock (>41.5 °C). Autophagy is another survival process that is activated by stress. This study aims to determine whether autophagy can be activated by heat shock at 42 °C, and if this response is mediated by reactive oxygen species (ROS). Autophagy was increased during shorter heating times (<60 min) at 42 °C in cells. Levels of acidic vesicular organelles (AVO) and autophagy proteins Beclin-1, LC3-II/LC-3I, Atg7 and Atg12-Atg5 were increased. Heat shock at 42 °C increased levels of ROS. Increased levels of LC3 and AVOs at 42 °C were inhibited by antioxidants. Therefore, increased autophagy during heat shock at 42 °C (<60 min) was mediated by ROS. Conversely, heat shock at 42 °C for longer times (1−3 h) caused apoptosis and activation of caspases in the mitochondrial, death receptor and endoplasmic reticulum (ER) pathways. Thermotolerant cells, which were developed at 40 °C, were resistant to activation of apoptosis at 42 °C. Autophagy inhibitors 3-methyladenine and bafilomycin sensitized cells to activation of apoptosis by heat shock (42 °C). Improved understanding of autophagy in cellular responses to heat shock could be useful for optimizing the efficacy of hyperthermia in the clinic.

118 - Induction of peroxiredoxins by heat shock is mediated by reactive oxygen species and Nrf2

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and/or chemotherapy. It uses heat (>42°C) to kill cancer cells. However, lower temperatures (39-41°C) induce thermotolerance, an adaptive survival response (hormesis) that could interfere with toxicity of cancer treatments. Exposure to low doses of stress (e.g. heat shock, reactive oxygen species (ROS)) can upregulate cellular defenses such as heat shock proteins, antioxidants and anti-apoptosis proteins, which protect cells against subsequent exposure to toxic stressors. Although hyperthermia has proven effective in clinical trials, there are still many things to learn about its cellular and molecular mechanisms. This study examines the role of ROS and antioxidants in cellular stress responses to mild (40°C) and lethal (42-43°C) hyperthermia. Hyperthermia (42-43°C) caused apoptosis and activation of caspases 9 and 3, which were decreased in thermotolerant (40°C) cancer cells. Mild and lethal hyperthermia both increased ROS levels in cells, relative to 37°C. Exposure to mild and lethal hyperthermia increased protein expression of the antioxidants peroxiredoxin-2 (Prx2) and Prx3. Expression of the hyperoxidized form Prx-SO3 increased only at higher temperatures (>42°C). The increases in Prx3 and Prx-SO3 levels at 42°C were inhibited by the antioxidants PEG-catalase and MnTBAP. In thermotolerant (40°C) cells, transcription factor Nrf2 was upregulated. The heat–induced increase in Prx3 expression was diminished in shRNA Nrf2 cells. Furthermore, heat (40-43°C) increased levels of reduced glutathione (GSH), which were inhibited by PEG-catalase and MnTBAP. These results suggest that heat-induced ROS were involved in the induction of antioxidants and shed some light on the role of antioxidants in cellular responses induced by heat shock at both mild and lethal temperatures. Improved knowledge about induction of adaptive survival responses by low-dose heat shock could have potential to protect against toxicity of multiple xenobiotics.

Dicer protein levels elevated by mild hyperthermia promote a pro-survival phenotype.

Cellular exposure to mild stress (39.5°C - 41.5°C) induces thermotolerance, rendering cells resistant to a subsequent heat shock (>42°C) insult. We found that mild hyperthermia at 39.5°C leads to elevations in dicer, a protein well-known for its role in microRNA processing and for its role in cellular stress responses. However, whether elevated dicer protein levels play a role in sustaining a thermotolerant phenotype has, to our knowledge, not been reported. Here we demonstrate that elevated dicer protein is linked to a thermotolerant phenotype in the cervical carcinoma cell line HeLa and in murine embryonic fibroblasts (MEF), and demonstrate that dicer plays a role in mediating PKR and eIF2α phosphorylation. These findings suggest that dicer's role in thermotolerance may be to relay signals to key ER stress pathway components. Moreover, utilizing a MEF cell line defective in microRNA processing, we suggest that dicer's influence on PKR and eIF2α phosphorylation is likely distinct from its microRNA processing role. ATF4 and CHOP are well characterized stress response factors proximal to eIF2α. Evidence is presented that elevated dicer protein in thermotolerant cells differentially modulates ATF4 and CHOP levels to promote a pro-survival phenotype. This work contributes new information on dicer's role in cellular stress responses by defining a pro-survival phenotype in heat stress resistant cells which is sustained, at least in part, by elevated dicer protein levels. Our results suggest an ancillary role for dicer in the cellular stress pathways activated by mild hyperthermia that is likely distinct from its role in microRNA processing.

Inhibition of autophagy sensitises cells to hydrogen peroxide-induced apoptosis: Protective effect of mild thermotolerance acquired at 40 °C

Various toxic compounds produce reactive oxygen species, resulting in oxidative stress that threatens cellular homeostasis. Yet, lower doses of stress can stimulate defence systems allowing cell survival, whereas intense stress activates cell death pathways such as apoptosis. Mild thermal stress (40°C, 3h) induces thermotolerance, an adaptive survival response that renders cells less sensitive to subsequent toxic stress, by activating defence systems like heat shock proteins, antioxidants, anti-apoptotic and ER-stress factors. This study aims to understand how autophagy and apoptosis are regulated in response to different doses of H2O2, and whether mild thermotolerance can protect cervical carcinoma cells against apoptosis by stimulating autophagy. Autophagy was monitored through Beclin-1 and LC3 expression and acid compartment activity, whereas apoptosis was tracked by caspase activity and chromatin condensation. Exposure of HeLa and C33 A cells to H2O2 for shorter times (15–30min) transiently induced autophagy; apoptosis was activated after longer times (1−3h). Mild thermotolerance at 40°C enhanced activation of autophagy by H2O2. Disruption of autophagy using bafilomycin A1 and 3-methyladenine sensitised cells to apoptosis induced by H2O2, in non-thermotolerant cells and, to a lesser extent, in thermotolerant cells. Inhibition of autophagy enhanced apoptosis through the mitochondrial, death receptor and endoplasmic reticulum pathways. Autophagy was activated by lower doses of stress and protects cells against apoptosis induced by higher doses of H2O2. This work improves understanding of mechanisms that might be involved in toxicity of various compounds and could eventually lead to protective strategies against deleterious effects of toxic compounds.

Mild thermotolerance induced at 40 °C protects cells against hyperthermia-induced pro-apoptotic changes in Bcl-2 family proteins

Purpose: Despite clinical progress, mechanisms involved in cellular responses to low and high doses of hyperthermia are not entirely clear. This study investigates the role of Bcl-2 family proteins in control of the mitochondrial pathway of apoptosis during hyperthermia at 42–43 °C and the protective effect of a low dose adaptive survival response, mild thermotolerance induced at 40 °C.Materials and methods: Levels of Bcl-2 family proteins were detected in HeLa cells by western blotting, caspase activation by spectrofluorimetry and apoptosis by chromatin condensation.Results: Hyperthermia (42–43 °C) decreased total and mitochondrial expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, while expression of pro-apoptotic proteins Bax, Bak, Puma and Noxa increased. Hyperthermia perturbed the equilibrium between these anti- and pro-apoptotic Bcl-2 family proteins in favour of pro-apoptotic conditions. Hyperthermia also caused activation of caspases-9 and -3, and chromatin condensation. Disruption of the balance between Bcl-2 family proteins was reversed in thermotolerant (40 °C) cells, thus favouring cell survival. Bcl-2/Bcl-xL inhibitor ABT-737 sensitised cells to apoptosis, which indicates that Bcl-2 family proteins play a role in hyperthermia-induced apoptosis. The adaptive response of mild thermotolerance (40 °C) was still able to protect cells against hyperthermia (42–43 °C) when Bcl-2/Bcl-xL were inhibited.Conclusions: These results improve knowledge about the role of Bcl-2 family proteins in cellular apoptotic responses to hyperthermia (42–43 °C), as well as the adaptive survival response induced by exposure to mild stresses, such as a fever temperature (40 °C). This study could provide rationale to explore the manipulation of Bcl-2 family proteins for increasing tumour sensitivity to hyperthermia.

Heat shock induces rapid resorption of primary cilia

Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90.

Heat-shock induces rapid resorption of primary cilia

Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. In this study we investigate the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells cilia loss correlated with a reduction in ligand dependent hedgehog signalling. Heat shock dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by HDAC6 which localises to ciliary axonemes. The rate of cilia resorption was reduced in thermotolerant cells. This implies a role for molecular chaperones in primary cilia maintenance. The cytosolic chaperone Hsp90 localised to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells Hsp90 is known to exist in a complex with HDAC6. Immediately after heat shock Hsp90 levels were reduced in remaining ciliary axonemes. We hypothesise that cilia resorption in response to heat shock is regulated by the disassembly of an HDAC6/Hsp90 complex and would serve to attenuate cilia mediated signalling pathways and reduce the translational load on the cell in times of stress.

Heat shock induces rapid resorption of primary cilia

SummaryPrimary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90.

Activation of ER stress and apoptosis by hydrogen peroxide in HeLa cells: Protective role of mild heat preconditioning at 40 °C

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) during stress conditions causes activation of the unfolded protein response (UPR). If this adaptive response cannot restore ER homeostasis, cells undergo ER-mediated apoptosis. This study determines whether thermotolerance developed at a mild temperature (40°C) can alter induction of ER-mediated stress and apoptosis by H2O2 in HeLa cells. Protein expression of PERK, p-PERK, eIF2α and p-eIF2α was increased in thermotolerant compared to non-thermotolerant cells. Thus, mild thermotolerance enhanced pro-survival effects of the PERK/eIF2α branch of the UPR. A short exposure (15min) of cells to H2O2 (15–50μM) activated the UPR: expression of p-PERK, p-eIF2α and p-IRE1α increased, and ATF6 cleavage occurred. Longer exposure (1–3h) to H2O2 induced ER-mediated apoptosis, whereby CHOP expression increased, and enzymatic activity of calpain, caspase-7, -4, -12 and -9 also increased. These pro-apoptotic events and clonogenic cell killing were all diminished in thermotolerant cells. Activation of caspases-4/-12 was decreased by the calcium chelator BAPTA-AM, and by inhibitors of calpain and caspase-7, confirming the roles of calcium, calpain and caspase-7 in activation of ER-mediated apoptosis by H2O2. In thermotolerant cells with decreased levels of PERK by siRNA, there was partial reversal of resistance to H2O2-induced apoptosis. Hence, a causal connection exists between the ER stress response and resistance to H2O2-induced apoptosis. Mild thermotolerance plays a protective, anti-apoptotic role by increasing the threshold for induction of ER-mediated apoptosis by H2O2. Moreover, the adaptive response (UPR) dominates during milder H2O2 stress, whereas ER-mediated apoptosis occurs during more severe stress.

Mild thermotolerance induced at 40 °C protects HeLa cells against activation of death receptor-mediated apoptosis by hydrogen peroxide

Preexposure to mild temperatures such as 40 °C induces thermotolerance, whereby cells resist subsequent exposure to a toxic insult. This study investigates the protective effect of mild thermotolerance (3 h, 40 °C) against activation of death receptor-mediated apoptosis by H 2O 2 in HeLa cells. H 2O 2 (5–50 μM) caused rapid activation (1–3 h) of the Fas death receptor pathway of apoptosis, which was evident by up-regulation of the death ligand FasL and recruitment of the adaptor protein Fas-associated death domain to the plasma membrane. This resulted in activation of caspase-8 and caspase-2, which led to activation of the cross-talk pathway involving Bid cleavage, t-Bid translocation to mitochondria, and caspase-9 activation. These changes were all diminished in thermotolerant cells. Mild thermotolerance also protected cells against cytotoxicity from H 2O 2 as well as execution-phase events of apoptosis such as caspase-3 activation and chromatin condensation. The antioxidant polyethylene glycol–catalase abolished FasL induction and caspase-8 activation due to H 2O 2. FasL up-regulation; activation of caspases-8, -2, -9, and -3; and chromatin condensation were decreased by the p53 inhibitor pifithrin-α, implicating p53 as an upstream factor in the activation of death receptor-mediated apoptosis by H 2O 2. This study advances knowledge about the protective effect of adaptive responses induced by mild stresses, such as fever temperatures, against induction of apoptosis by oxidative stress.

Mild thermotolerance induced at 40°C increases antioxidants and protects HeLa cells against mitochondrial apoptosis induced by hydrogen peroxide: Role of p53

Exposure of cells to mild temperatures (40 degrees C) induces thermotolerance, which renders cells resistant to subsequent toxic insults. Thermotolerance is usually associated with accumulation of heat shock proteins. This study determines whether mild thermotolerance (40 degrees C, 3h) can induce other defense proteins (e.g. antioxidants, anti-apoptosis proteins), and protect HeLa cells against apoptosis triggered by H(2)O(2). Protein expression and enzymatic activity of MnSOD and catalase were increased in thermotolerant cells, as well as intracellular glutathione levels and gamma-glutamylcysteine synthetase expression. Furthermore, levels of reactive oxygen species (ROS) were increased in thermotolerant cells, which caused mitochondrial membrane hyperpolarisation. Mild thermotolerance inhibited activation of the mitochondrial cascade of apoptosis by H(2)O(2). This entailed inhibition of mitochondrial Bax translocation, mitochondrial membrane depolarisation, cytochrome c release, activation of caspases-9/-3 and chromatin condensation. Thermotolerance inhibited H(2)O(2)-induced caspase-independent apoptosis involving apoptosis-inducing factor, and activation of p53 and increased expression of its target protein PUMA. Thermotolerance induced at mild physiological temperatures protects cells against both caspase-dependent and caspase-independent apoptosis triggered by oxidative stress.

Anaerosphaera aminiphila gen. nov., sp. nov., a glutamate-degrading, Gram-positive anaerobic coccus isolated from a methanogenic reactor treating cattle waste

A strictly anaerobic, mesophilic and aminolytic strain (WN036(T)) was isolated from a methanogenic reactor treating waste from cattle farms. Cells were Gram-positive cocci, often occurred in pairs and were non-motile. Although spore formation was not confirmed by microscopic observation of cells, the strain produced thermotolerant cells. The optimum temperature for growth was 35-37 degrees C and the optimum pH was 6.7. Oxidase, catalase and nitrate-reducing activities were negative. The novel strain did not ferment carbohydrates and grew in PY medium without additional substrates. The strain utilized l-glutamate, l-glutamine, l-histidine and l-arginine as growth substrates. Major fermentation products were acetate and butyrate with a small amount of propionate. The genomic DNA G+C content was 32.5 mol%. The major cellular fatty acids were C(17 : 1)omega8, C(18 : 1)omega7 DMA and C(16 : 0). The diagnostic diamino acid of the cell-wall peptidoglycan was lysine. Glutamic acid, glycine, alanine and aspartic acid were also detected in the cell-wall peptidoglycan. On the basis of 16S rRNA gene sequences, the most closely related species to strain WN036(T) were Peptoniphilus asaccharolyticus ATCC 14965(T) (89.8 %) and Peptoniphilus indolicus ATCC 29427(T) (89.6 %). Based on the differences in the phenotypic and phylogenetic characteristics of strain WN036(T) compared with those of closely related species, a novel genus and species, Anaerosphaera aminiphila gen. nov., sp. nov., is proposed. The type strain is WN036(T) (=JCM 15094(T)=DSM 21120(T)).

Specific Interaction between Tomato HsfA1 and HsfA2 Creates Hetero-oligomeric Superactivator Complexes for Synergistic Activation of Heat Stress Gene Expression

In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.

Clostridium sufflavum sp. nov., isolated from a methanogenic reactor treating cattle waste

A strictly anaerobic, mesophilic, cellulolytic bacterial strain, designated CDT-1(T), was isolated from rice-straw residue from a methanogenic reactor treating waste from cattle farms. The isolation was performed using enrichment culture with filter paper as a substrate. Cells stained Gram-negative, but reacted Gram-positively in the KOH test. Cells were slightly curved rods and were motile by means of peritrichous flagella. The strain produced yellow pigment when grown on filter-paper fragments. Although spore formation was not confirmed microscopically, thermotolerant cells were produced when the strain was grown on filter paper. The optimum temperature for growth was 33 degrees C and the optimum pH was 7.4. Oxidase, catalase and nitrate-reducing activities were absent. The strain utilized xylose, fructose, glucose, cellobiose, xylooligosaccharide, cellulose (filter-paper fragments and ball-milled filter paper) and xylan. The major fermentation products were acetate, ethanol, H(2) and CO(2). The major cellular fatty acids were iso-C(15 : 0), iso-C(14 : 0) and C(16 : 0) DMA. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The genomic DNA G+C content was 40.7 mol%. On the basis of 16S rRNA gene sequence similarities, strain CDT-1(T) could be placed in cluster III of the genus Clostridium, being closely related to type strains of Clostridium hungatei (96.6 % sequence similarity), Clostridium termitidis (96.2 %) and Clostridium papyrosolvens (96.1 %). On the basis of the cellular, physiological and phylogenetic differences between CDT-1(T) and its close relatives, this strain represents a novel species of the genus Clostridium, for which the name Clostridium sufflavum sp. nov. is proposed. The type strain is CDT-1(T) (=JCM 14807(T)=DSM 19573(T)).

Mice that overexpress human heat shock protein 27 have increased renal injury following ischemia reperfusion

We previously showed that activation of the A1 adenosine receptor protected the kidney against ischemia-reperfusion injury by induction and phosphorylation of heat shock protein 27 (HSP27). Here, we used mice that overexpress human HSP27 (huHSP27) to determine if kidneys from these mice were protected against injury. Proximal tubule cells cultured from the transgenic mice had increased resistance to peroxide-induced necrosis compared to cells from wild-type mice. However, after renal ischemic injury, HSP27 transgenic mice had decreased renal function compared to wild-type mice, along with increased renal expression of mRNAs of pro-inflammatory cytokines (TNF-alpha, ICAM-1, MCP-1) and increased plasma and kidney keratinocyte-derived cytokine. Following ischemic injury, neutrophils infiltrated the kidneys earlier in the transgenic mice. Flow cytometric analysis of lymphocyte subsets showed that those isolated from the kidneys of transgenic mice had increased CD3(+), CD4(+), CD8(+), and NK1.1(+) cells 3 h after injury. When splenocytes or NK1.1(+) cells were isolated from transgenic mice and adoptively transferred into wild-type mice there was increased renal injury. Further, depletion of lymphocytes by splenectomy or neutralization of NK1.1(+) cells resulted in improved renal function in the transgenic mice following reperfusion. Our study shows that induction of HSP27 in renal tubular cells protects against necrosis in vitro, but its systemic increase counteracts this protection by exacerbating renal and systemic inflammation in vivo.

The heat-induced γ-H2AX response does not play a role in hyperthermic cell killing

Purpose: The goal of this study was to determine whether the heat-induced formation of γ-H2AX foci is involved in hyperthermic cell killing.Materials and methods: The heat-induced γ-H2AX response was determined in cells exhibiting various degrees of heat sensitivity. The panel of cells tested included cells that are transiently thermotolerant, permanently heat resistant, permanently heat sensitive, and permanently resistant to oxidative stress. Cells exposed to non-thermal environmental conditions that lead to protection from, or sensitization to, heat were also tested. The heat sensitivity of cells in which H2AX was knocked out was also ascertained.Results: The protein synthesis independent state of thermotolerance, but not the protein synthesis dependent state of thermotolerance, was found to be involved in the attenuation of the γ-H2AX response in thermotolerant cells. The initial magnitude of the γ-H2AX response was found to be the same in all cell lines with altered heat sensitivity. Furthermore, no differences in the resolution of γ-H2AX foci were found among the cell lines tested. We also found that H2AX knock-out cells were not more heat sensitive.Conclusions: We conclude that the heat-induced γ-H2AX response does not play a role in heat-induced cell killing, thereby adding further evidence that the heat-induced γ-H2AX foci are not due to DNA double strand breaks.

Re-induction of hsp70 synthesis: An assay for thermotolerance

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41°C, 4 h) and then incubated at 37°C for 0–72 h. At various times after the first heat treatment, cells were either challenged with a 45°C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [35S]methionine before or after an additional test heat dose (e.g. 43.5°C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.

Hyperthermia classic article commentary: ‘Re-induction of hsp70 synthesis: An assay for thermotolerance’ by Gloria C. Li and Johnson Y. Mak,International Journal of Hyperthermia1989;5:389-403

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells’ thermal sensitivity during the development and decay of thermotolerance in model systems. Specifically, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance in tumor cells in vitro and in animal tumor models. We found that the cells’ ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. These data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test heat shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during fractionated hyperthermia.