Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth,Lymantria dispar

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with M_rs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 M_r heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 M_r heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.     

Rapid responses to high temperature and desiccation but not to low temperature in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera,Tineidae)

A broad definition of rapid cold hardening (RCH) is that it is the process whereby insects increase their survival of a sub-zero temperature after a brief (h) pre-exposure to a less severe low temperature. The effects of various pre-treatments on survival of two h at -7.9 degrees C were investigated in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera: Tineidae), the first time RCH has been investigated in a freeze tolerant arthropod. All caterpillars froze when exposed to -7.9 degrees C, and none of the low temperature pre-treatments (-5, 0, 5 and 15 degrees C, as well as -5 degrees C and 0 degrees C with a delay before freezing) nor slow cooling (0.1 degrees C/min) elicited any improvement in survival of -7.9 degrees C as compared to controls. However, high temperature treatments (25, 30 and 35 degrees C), desiccation and acclimation for 5 days at 0 degrees C did result in significant increases in survival of the test temperature, possibly as a result of heat shock protein production. Haemolymph osmolality was elevated only by the 35 degrees C pre-treatment. It is suggested that the unpredictable environment of Marion Island means that P. marioni must always be physiologically prepared to survive cold snaps, and that this year-round cold hardiness therefore supersedes a rapid cold hardening response.     

Thermotolerance and rapid cold hardening ameliorate the negative effects of brief exposures to high or low temperatures on fecundity in the flesh fly, Sarcophaga crassipalpis

Although the immediate effects of temperature stress are well documented, the longer‐term effects of such stresses are more poorly known. In these experiments, we investigate the effects of suboptimal and supraoptimal temperatures during pharate adult development on fecundity in the flesh fly, Sarcophaga crassipalpis Macquart. A 1 h cold shock at ?10°C during the red‐eye pharate adult stage decreases the fecundity of both sexes. Induction of rapid cold hardening by pre‐treatment at 0°C for 2 h partially prevents reproductive impairment. Heat shock of pharate adults for 1 h at 45°C also reduces fecundity in both sexes, but inducing thermotolerance by pre‐treatment at 40°C for 2 h affords protection only to females. Males heat shocked at 45°C or first pre‐treated at 40°C consistently fail to transfer sperm to the females. The injury inflicted on males by heat shock is most pronounced when the stress is administered to pharate adults or adults; wandering larvae and true pupae are unaffected. The implications of these data for naturally occurring populations are discussed.     

PROTEOMICS ANALYSIS OF OVEREXPRESSED PLASMA PROTEINS IN RESPONSE TO COLD ACCLIMATION IN Ostrinia furnacalis

Many insects in temperate regions overwinter in diapause. In these insects, one of the metabolic adaptations to cold stress is the synthesis of responsive proteins. Using proteomic analysis, an investigation aimed to a better understanding of the molecular adaptation mechanisms to cold stress was carried out in Ostrinia furnacalis larva. Proteins were extracted from the larval hemolymph collected from both control and overwintering larva. By polyethylene glycol precipitation, approximately 560 protein spots were separated and visualized on two‐dimensional (2D) gels after silver staining. Eighteen protein spots were found to be upregulated in overwinter larval plasma in different patterns. As an initial work, 13 of these proteins were identified using MALDI TOF/TOF MS. The differentially overexpressed proteins include heat shock 70 kDa cognate protein, small heat shock protein (sHSP), putative aliphatic nitrilase, arginine kinase, phosphoglyceromutase, triosephosphateisomerase, and glutathione transferase. Alterations in the levels of these proteins were further confirmed by qPCR. This study is the first analysis of differentially expressed plasma proteins in O. furnacalis diapause larvae under extremely low temperature conditions and gives new insights into the acclimation mechanisms responsive to cold stress. Our results also support the idea that energy metabolism, alanine and proline metabolism, and antioxidative reaction act in the cold acclimation of O. furnacalis diapause larvae.     

Identification of DnaJ-like chaperone in Clostridium botulinum type A

Clostridium botulinum type A cells, when challenged to elevated temperature (45°C), increased the expression of at least nine heat shock proteins (HSPs). Simultaneously with the induction of HSPs, changes in the synthesis rates of other cellular proteins were observed. A 40-kDa stress protein was induced and its synthesis rate was enhanced when the cells were shifted to 45°C. Using heterologous antibodies raised against E. coli DnaJ heat shock proteins, the 40-kDa stress protein of C. botulinum type A has been identified as a DnaJ-like chaperone. The DnaJ chaperone might be involved in translocation of the neurotoxin and other cellular proteins across the cell membrane, repair of damaged proteins, and organism survival inside the host. This is the first report of the existence of a DnaJ-like chaperone in this organism.     

热胁迫对双孢蘑菇抗氧化酶及热激蛋白基因的差异表达的影响

抗氧化酶和热激蛋白是双孢蘑菇Agaricus bisporus抵御逆境胁迫的重要蛋白,高温胁迫下菌丝会通过二者基因的差异表达来减少对自身的损伤.通过对双孢蘑菇菌丝进行40℃热胁迫处理0-120min后发现,随着热胁迫时间延长,菌丝生长速度降低、气生菌丝增多和菌丝分叉明显.转录组分析抗氧化酶和热激蛋白基因差异表达发现,在...     

The Role of Antioxidant Systems in the Cold Stress Response of Escherichia coli

The response of aerobically grown Escherichia coli cells to the cold shock induced by the rapid lowering of growth temperature from 37 to 20°C was found to be basically the same as the oxidative stress response. The enhanced sensitivity of cells deficient in two superoxide dismutases, Mn-SOD and Fe-SOD, and the increased expression of the Mn-SOD gene, sodA, in response to cold stress were interpreted as both oxidative and cold stresses are due to a rise in the intracellular level of superoxide anion. The long-term cultivation of E. coli at 20°C was also accompanied by the typical oxidative stress response reactions—an enhanced expression of the Mn-SOD and catalase HPI genes and a decrease in the intracellular level of reduced glutathione (GSH) and in the GSH/GSSG ratio.     

Continuous heat shock enhances translational initiation directed by internal ribosomal entry site

Many cellular mRNAs contain internal ribosomal entry sites (IRES) that become functional under conditions of cellular stress, when the rate of protein synthesis for most cellular mRNA is reduced. Internal ribosomal entry increases in response to hypoxia, cell differentiation, apoptosis, gamma irradiation, and heat shock. Heat shock is the principal cellular stress in which general cap-dependent translation is inhibited. On the other hand, heat shock induces the preferential translation of a small class of mRNA, called heat shock protein (HSP) mRNAs, which probably occurs because little or no eIF4F activity is required for their translation. In this study, we found that continuous heat stress enhances expression of the heat shock protein BiP at the level of translation. Interestingly, heat stress also enhanced the viral IRES-dependent translation of encephalomyocarditis virus and hepatitis C virus but not poliovirus. Although several BiP inducers increased BiP protein expression, BiP IRES-dependent translation was enhanced only during heat shock, suggesting that heat shock is a specific inducer for BiP IRES-dependent translation. Taken together, these results indicate that the mechanism of IRES-dependent translation can be used during heat shock and suggest that this translational mechanism may be critical to the survival and proliferation of cells under stress.     

Modulation of protein phosphorylation and stress protein expression by okadaic acid on heat shock cells

We have demonstrated that pretreatment but not post-treatment with okadaic acid (OA) can aggravate cytotoxicity as well as alter the kinetics of stress protein expression and protein phosphorylation in heat shocked cells. Compared to heat shock, cells recovering from 1 hr pretreatment of OA at 200 nM and cotreated with heat shock at 45°C for the last 15 min of incubation (OA→HS treatment) exhibited enhanced induction of heat shock proteins (HSPs) 70 and 110. In addition to enhanced expression, the attenuation of HSC70 and HSP90 after the induction peaks was also delayed in OA→HS-treated cells. The above treatment also resulted in the rapid induction of the 78 kDa glucose-regulated protein (GRP78), which expression remained constant in cells recovering from treatment with 200 nM OA for 1 hr, heat shocked at 45°C for 15 min, or in combined treatment in reversed order (HS→OA treatment). Enhanced phosphorylation of vimentin and proteins with molecular weights of 65, 40, and 33 kDa and decreased phosphorylation of a protein with a molecular weight of 29 kDa were also observed in cells recovering from OA→HS treatment. Again, protein phosphorylation in cells recovering from HS→OA treatment did not differ from those in cells treated only with heat shock. Since the alteration in the kinetics of stress protein expression and protein phosphorylation was tightly correlated, we concluded that there is a critical link between induction of the stress proteins and phosphorylation of specific proteins. Furthermore, the rapid induction of GRP78 under the experimental condition offered a novel avenue for studying the regulation of its expression. © 1996 Wiley-Liss, Inc.     

Rapid thermal adaptation during field temperature variations in Drosophila melanogaster

Under natural conditions, the fruit fly (Drosophila melanogaster) is constantly exposed to variations in temperature and light. Laboratory investigations have demonstrated that D. melanogaster and other insects adapt quickly to temperature variations, but only few studies have investigated this ability under natural temperature variations. Here we placed laboratory raised female D. melanogaster in field cages and exposed them to natural variations in light and temperature over a 2 day period (temperature range: 12–25 °C). During this period we sampled flies every 6 h and measured their ability to survive heat and cold shock. There was a significant positive correlation between field temperature and heat shock survival and a significant negative correlation between field temperature and cold shock survival indicating that D. melanogaster are constantly adapting to their surrounding environment. The results also suggest that heat and cold resistance are obtained at a cost as these two traits were negatively correlated.     

Cell inactivation and membrane damage after long-term treatments at sub-zero temperature in the supercooled and frozen states

The survival of cells subjected to cooling at sub-zero temperature is of paramount concern in cryobiology. The susceptibility of cells to cryopreservation processes, especially freeze-thawing, stimulated considerable interest in better understanding the mechanisms leading to cell injury and inactivation. In this study, we assessed the viability of cells subjected to cold stress, through long-term supercooling experiments, versus freeze-thawing stress. The viability of Escherichia coli, Saccharomyces cerevisiae, and leukemia cells were assessed over time. Supercooled conditions were maintained for 71 days at -10 degrees C, and for 4 h at -15 degrees C, and -20 degrees C, without additives or emulsification. Results showed that cells could be inactivated by the only action of sub-zero temperature, that is, without any water crystallization. The loss of cell viability upon exposure to sub-zero temperatures is suggested to be caused by exposure to cold shock which induced membrane damage. During holding time in the supercooled state, elevated membrane permeability results in uncontrolled mass transfer to and from the cell maintained at cold conditions and thus leads to a loss of viability. With water crystallization, cells shrink suddenly and thus are exposed to cold osmotic shock, which is suggested to induce abrupt loss of cell viability. During holding time in the frozen state, cells remain suspended in the residual unfrozen fraction of the liquid and are exposed to cold stress that would cause membrane damage and loss of viability over time. However, the severity of such a stress seems to be moderated by the cell type and the increased solute concentration in the unfrozen fraction of the cell suspension.     

Characterization of cold-induced heat shock protein expression in neonatal rat cardiomyocytes

Cardiac surgery is usually performed under conditions of cardioplegicischemic arrest. To protect the heart during the ischemic period, themyocardium is exposed to varying degrees of hypothermia. Althoughhyperthermia is known to induce the heat shock response, the moleculareffects of hypothermia on the myocardium have not been investigated. We havestudied the effect of hypothermia on the induction of heat shock proteins inprimary cultures of neonatal cardiomyocytes. Cold stress in cardiomyocytesinduced a 6 fold increase in the heat shock protein HSP70 as compared tocontrol. Increased HSP70 protein levels correlated with induction of HSP70mRNAs. Maximal levels of HSP70 protein appeared 4-6 h following recoveryfrom cold shock, indicating the transient nature of the response. Inductionof HSP25 mRNA was also observed in cold-shocked cardiomyocytes, even thoughincreased HSP25 protein levels were not detected. Our results indicate thathypothermia is capable of inducing the heat shock response in neonatalcardiomyocytes.     

Responses of the bed bug,Cimex lectularius,to temperature extremes and dehydration: levels of tolerance,rapid cold hardening and expression of heat shock proteins

This study of the bed bug, Cimex lectularius, examines tolerance of adult females to extremes in temperature and loss of body water. Although the supercooling point (SCP) of the bed bugs was approximately −20°C, all were killed by a direct 1 h exposure to −16°C. Thus, this species cannot tolerate freezing and is killed at temperatures well above its SCP. Neither cold acclimation at 4°C for 2 weeks nor dehydration (15% loss of water content) enhanced cold tolerance. However, bed bugs have the capacity for rapid cold hardening, i.e. a 1‐h exposure to 0°C improved their subsequent tolerance of −14 and −16°C. In response to heat stress, fewer than 20% of the bugs survived a 1‐h exposure to 46°C, and nearly all were killed at 48°C. Dehydration, heat acclimation at 30°C for 2 weeks and rapid heat hardening at 37°C for 1 h all failed to improve heat tolerance. Expression of the mRNAs encoding two heat shock proteins (Hsps), Hsp70 and Hsp90, was elevated in response to heat stress, cold stress and during dehydration and rehydration. The response of Hsp90 was more pronounced than that of Hsp70 during dehydration and rehydration. Our results define the tolerance limits for bed bugs to these commonly encountered stresses of temperature and low humidity and indicate a role for Hsps in responding to these stresses.     

低温胁迫下虎纹蛙的生存力及免疫和抗氧化能力

全球气候变化是造成世界范围内两栖类种群衰退和灭绝的重要因素之一。随着极端天气出现变得日趋频繁,非季节性的、短期且剧烈的气温变化可能会严重干扰两栖类动物的生存与种群稳定。监测了浙江省金华市南山野生虎纹蛙(Hoplobatrachus rugulosus)分布区冬季的环境气温,并参考监测数据在实验室条件下研究了虎纹蛙在短期梯度降温(2℃/24 h)和急性冷暴露(即冷休克)(2℃)下的生存力及冷休克对机体免疫功能和抗氧化能力的影响。结果表明,虎纹蛙在冬季(2009-12-01—2010-03-31)经历的温度范围普遍在0—14℃之间,主要遭遇的低温区间在0—4℃,主要高温区间在10—14℃。通过梯度降温实验,发现温度降至12℃累积死亡率约为28.1%,10℃为87.5%,8℃为100%。在一定温度范围内,虎纹蛙死亡率与环境温度呈显著负相关(Pearson test,r=-0.952,P<0.05)。经曲线拟合,回归方程计算可得半数致死温度为11.5℃。虎纹蛙在冷休克处理下,在第6 h累积死亡率为45%,12 h为80%,24 h达到100%。虎纹蛙死亡率与冷休克时间呈显著正相关(Pearson test,r=0.91,P<0.05),半数致死时间为7.6 h。此外,冷休克(2℃,6 h)显著抑制了虎纹蛙脾脏巨噬细胞呼吸爆发强度(t=3.827,df=6,P<0.05)、全血吞噬活性(t=5.388,df=3.037,P<0.05)及胃溶菌酶活力(t=6.37,df=6,P<0.05);肝脏(t=0.773,df=8,P>0.05)和肾脏(t=0.164,df=4.542,P>0.05)组织脂质过氧化物产物丙二醛(MDA)的含量虽无明显变化,但肝脏(t=-2.817,df=6,P<0.05)和肾脏(t=-11.302,df=6,P<0.05)组织抗氧化物谷胱甘肽(GSH)含量及肝脏(t=-3.3,df=6,P<0.05)超氧化物歧化酶(SOD)活性均显著升高。上述结果表明虎纹蛙对低温较为敏感,冷休克能够诱导机体的免疫抑制,并导致机体对抗氧化物质需求的增加。可以推测,当遭遇极端低温天气时,低温胁迫可能会严重干扰虎纹蛙生理机能,加大种群的生存压力和疾病感染的风险。     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42°C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.     

Cold shock during liquid production increases storage shelf-life of Cryptococcus nodaensis OH 182.9 after air-drying

Fermentation, formulation and drying studies are necessary and important in order to simplify production, transportation, storage and application of biocontrol agents. Air-drying is a convenient and economical drying method for developing microbial biocontrol products. Experiments were designed to determine the effect of temperature shock during liquid cultivation on cell survival of a Fusarium head blight biocontrol agent Cryptococcus nodaensis OH 182.9 after air-drying. OH 182.9 cultures were grown at various temperatures in semi-defined complete liquid media, with cultures grown at 25°C for 48 h serving as the standard control culture condition. Harvested cultures were mixed with 10% diatomaceous earth (DE), vacuum filtered, air dried for 20 h at 60-70% RH, and stored at 4°C. In general, cells grown at 25°C for 20 h followed by cultivation at 15°C for 28 h survived air-drying better than control cells. The survival of cells subjected to heat shock at 31°C generally did not differ from control cells regardless of whether heat shock was applied at the late exponential or early stationary stage of growth. In another experiment designed to optimize the effect of cold temperatures during cultivation on subsequent survival of air-dried cells in DE at 4°C and room temperature (25°C), prolonged (28 h) cold shock at 10 and 15°C after incubation at 25°C for 20 h enhanced the storage stability (shelf-life) of a DE-formulated OH 182.9 product. In greenhouse tests, air-dried cells of OH 182.9 stored for 6 weeks at 4°C maintained a higher biocontrol efficacy than cells stored for 6 weeks at 25°C.     

Preservation of frozen yeast cells by trehalose.

Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.