Heat shock protein 70 response to physical and chemical stress in Chamelea gallina

Intertidal area is characterized by several fluctuations in natural agents and anthropogenic factors (oxygen levels, temperature, salinity, B[a]P presence) that cause a noticeable increase in the expression rate of heat shock protein 70 (HSP70). HSPs acting as molecular chaperones and their induction represent a specific cellular defence mechanism in response to several stress.Chamelea gallina specimens from the North Adriatic coast were exposed to different experimental conditions: varying oxygen levels (48 h of anoxia followed by 24 h of normoxic recovery), temperatures (20, 25, 30 °C for 7 days), salinity (28, 34, 40‰ for 7 days) and B[a]P concentrations (0.5 mg/L for 24 h, 7 and 12 days). Following the extraction of the digestive gland and gills, HSP70 levels were identified in the cytosolic fraction by immunoblotting using primary monoclonal antibodies. An increase in the rate of HSP70 expression under anoxic conditions in the digestive gland was observed at high temperatures, at low salinity and in the presence of B[a]P. The protein was overexpressed in the absence of oxygen and after 12 days of B[a]P exposure, while it was underexpressed in hyposaline conditions in the gills.HSP70 induction can be considered an adaptation mechanism associated with changes in environmental parameters, but also with xenobiotic presence. The overexpression of HSP70 is therefore induced by protein damage due to stressogenic factors. HSP recruitment renders them available for the processes of folding and refolding of denatured proteins or for their transport to a degradation system. The evident sensitivity of HSP70 to natural and anthropogenic stressogenic agents was examined in the present research.The results of this research revealed an interesting response of heat shock protein 70 in C. gallina, underlining the sensitivity of this important commercial species to natural and anthropogenic stress agents.     

Identification of proteins regulated by ABA in response to combined drought and heat stress in maize roots

We adopted a proteomics approach to identify and analyze the differential expression of maize root proteins associated with abscisic acid (ABA) regulation under combined drought and heat stress. Using mass spectrometry, we identified 22 major proteins that were significantly up-regulated under combined drought and heat stress. These 22 proteins were classified into 6 functional categories: disease/defense (8), metabolism (3), cell growth/division (3), signal transduction (2), transporters (2) and unclassified (4). Our previous reports showed that ABA regulates the expression of several small heat-shock proteins (sHSPs) in maize leaves subjected to the combination of drought and heat stress; however, no sHSPs were identified among the root proteins up-regulated in this study. RT-PCR and western blot analyses were used to identify six known sHSPs. The maize roots were pretreated with 100 μM of ABA, and subsequently, the expression of the 22 up-regulated proteins and 6 sHSPs was examined. 11 proteins were up-regulated in an ABA-dependent manner, 13 proteins were up-regulated in an ABA-independent manner, and 4 proteins were up-regulated but inhibited by ABA. The up-regulated proteins are interesting candidates for further physiological and molecular investigations of combination stress tolerance in maize.     

Deficient heat shock protein 70 response to stress in leukocytes at onset of type 1 diabetes

Type 1 diabetes is caused by the immune-mediated destruction of pancreatic beta cells. Animal models of the disease demonstrate an increased susceptibility of beta cells to immunological attacks due to their defective stress-responsiveness. To investigate the stress-responsiveness in human type 1 diabetes we analyzed the heat-inducibility of the dominant stress protein heat shock protein (Hsp)70 in diabetic patients at different disease stages. At diabetes-manifestation heat-induced Hsp70 levels in peripheral blood mononuclear cells (PBMC) reached only about 25% of the levels expressed by heat-treated PBMC from non-diabetic subjects (p < 0.05). Heat-responsiveness improved with disease duration and was re-established at more than eight months after disease-manifestation. Hyperthermia-induced Hsp70 expression was decreased by the T-helper 1-associated cytokine interferon-γ and increased by the T-helper 2-associated transforming growth factor-β. We conclude that impaired cellular stress-responsiveness, aggravated by the inflammatory milieu at the onset of type 1 diabetes, contributes to disease manifestation.     

Dietary intake of antioxidant supplements modulate antioxidant status and heat shock protein 70 synthesis

The physiological effects and efficacy of dietary intake of antioxidant supplements in humans remains controversial. Experiments involving dietary, often high, intake of a single antioxidant or vitamin may be seriously flawed given the interactive nature of antioxidants in vivo. The present studies were conducted on individuals (35-60 years of age) taking a commercial antioxidant mixture in a double-blind, placebo-controlled, cross-over study. Intake was two capsules per day, for 4 weeks, with a 4-week washout period in between active dose or placebo. Intake of antioxidants was associated with little change in superoxide dismutase activity, but an increase in glutathione peroxidase was noted. Haemolysis of red blood cells (erythrocytes) induced by the free radical generator AAPH was significantly reduced in individuals on antioxidant supplements. In lymphocytes isolated from individuals taking supplements, there was a marked increase, as compared with individuals on placebo, in the synthesis of heat shock protein 70 (hsp70) following heat shock from 37 degrees C to 42.5 degrees C. We conclude that dietary intake of a mixed antioxidant supplement leads to modulation of cellular redox status resulting in decreased oxidative stress and increased ability of lymphocytes to mount a stress response.     

Induction of heat shock protein messenger RNA in maize mesocotyls by water stress, abscisic Acid, and wounding

Exposure of the excised growing region of the mesocotyl of young corn seedlings to heat shock stimulated the production of specific heat shock proteins and the intensification of synthesis of two proteins with a molecular weight of approximately 70,000. Water stress and abscisic acid also stimulated synthesis of these 70,000-dalton proteins, and other unique proteins distinct from those induced by heat shock. Growing tissues of intact corn mesocotyls exposed to heat shock, water stress, or abscisic acid accumulated mRNA species homologous to a cloned genomic probe of the 5′ end of the 70,000-dalton Drosophila heat shock protein gene. Since cut segments of the mesocotyl under unstressed conditions produced a similar mRNA, we suggest that the hsp 70 gene is activated in corn by a variety of diverse stresses. Production of the mRNA is rapid, but transient, being induced within 3 hours of the imposition of the stress, but declining after reaching a maximum at 9 hours.     

Heat shock protein 70 interacts with aquaporin-2 and regulates its trafficking

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.     

Proteomic study of Carissa spinarum in response to combined heat and drought stress

Carissa spinarum is one of the secondary advantage plants grown in dry‐hot valleys in China, which can survive under stress conditions of high temperature and extreme low humidity. Here, we studied the physiological and proteomic changes of C. spinarum in response to 42°C heat stress treatment in combination with drought stress. Dynamic changes in the leaf proteome were analyzed at four time points during the stress treatment and recovery stages. Approximately, 650 protein spots were reproducibly detected in each gel. Forty‐nine spots changed their expression levels upon heat and drought treatment, and 30 proteins were identified by MS and 2‐D Western blot. These proteins were classified into several categories including HSP, photosynthesis‐related protein, RNA‐processing protein and proteins involved in metabolism and energy production. The potential roles of these stress‐responsive proteins are discussed.     

Expression of selected heat shock proteins after individually applied and combined drought and heat stress

Drought and heat stress are among the abiotic factors causing the most severe damage on plant crops. Their combination is quite common in dry and semi-dry regions worldwide and little is known about its effect on heat shock protein (HSP) profile in wheat plants. The expression of four HSP genes (Hsp 17.8, Hsp 26.3, Hsp 70 and Hsp 101b) in Triticum aestivum L. plants subjected to individually applied water deprivation or high temperature and their combination was monitored via one-step RT-PCR analysis. Changes in the expression levels of small HSPs (smHSPs), HSP70 and HSP100 were established also by SDS-PAGE. The combination of drought and heat induced HSP expression more effectively than the individually applied stresses. The induction of HSPs displayed greater rate in the drought-tolerant wheat variety Katya than in the drought-sensitive cv. Sadovo. The results obtained in wheat plants suggested that the effect of separately applied drought and heat shock cannot be extrapolated to their combination.     

Role of nitric oxide in abscisic acid-induced subcellular antioxidant defense of maize leaves

The sources of nitric oxide (NO) production in response to abscisic acid (ABA) and the role of NO in ABA-induced hydrogen peroxide (H(2)O(2)) accumulation and subcellular antioxidant defense in leaves of maize (Zea mays L.) plants were investigated. ABA induced increases in generation of NO and activity of nitric oxide synthase (NOS) in maize leaves. Such increases were blocked by pretreatment with each of the two NOS inhibitors. Pretreatments with a NO scavenger or NR inhibitors inhibited ABA-induced increase in production of NO, but did not affect the ABA-induced increases in activity of NOS, indicating that ABA-induced NO production originated from sources of NOS and NR. ABA- and H(2)O(2)-induced increases in expression of the antioxidant genes superoxide dismutase 4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by pretreatments with the NO scavenger, inhibitors of NOS and NR, indicating that NO is involved in the ABA- and H(2)O(2)-induced subcellular antioxidant defense reactions. On the other hand, NO donor sodium nitroprusside (SNP) reduced accumulation of H(2)O(2) induced by ABA, and c-PTIO reversed the effect of SNP in decreasing the accumulation of H(2)O(2). SNP induced increases in activities of subcellular antioxidant enzymes, and the increases were substantially prevented from occurring by the pretreatment with c-PTIO. These results suggest that ABA induces production of H(2)O(2) and NO, which can up-regulate activities of the subcellular antioxidant enzymes, to prevent overproduction of H(2)O(2) in maize plants. There is a negative feedback loop between NO and H(2)O(2) in ABA signal transduction in maize plants.     

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.     

不同抗旱性玉米幼苗根系抗氧化系统对水分胁迫的反应

以抗旱性不同的2个玉米品种为材料,研究不同程度水分胁迫下玉米根系活性氧清除系统的变化及膜脂过氧化水平。明确了轻度水分胁迫下玉米根系POD、CAT、APX等保护酶活性明显提高;中、重度胁迫下其活性急剧下降,但几种酶对水分胁迫的敏感程度不同。SOD对水分胁迫表现最不敏感,在中度水分胁迫下仍保持上升趋势;抗氧化剂GSH含量变化趋势与保护酶相似;而AsA含量在不同程度水分胁迫下持续下降;MDA含量随水分胁迫程度加剧而增加。其中抗旱性强的鲁玉14与抗旱性弱的掖单13相比具有较高的保护酶活性和抗氧化剂含量,膜脂过氧化程度较轻,除POD外,品种间抗氧化酶活性（抗氧化剂含量）呈极显著差异,说明抗氧化能力强是抗旱性品种具有较强抗旱性的重要原因之一。     

Expression of heat shock protein70 in pig oocytes: heat shock response during oocyte growth

The heat shock response of growing and fully-grown pig oocytes was analyzed in vitro by determining heat shock protein70 (HSP70) synthesis under both normal conditions (39 degrees C; 0 and 6h) and after heat shock (43 degrees C; 1, 4 and 6h). The expression of HSP70 in oocytes was detected by immunoblotting analysis. Growing oocytes measuring 80-99 microm synthesized a high number of HSP70 without heat shock effect, and these were capable of increasing the synthesis of HSP70 after heat shock to a maximum after 1h. Growing oocytes measuring 100-115 microm also synthesized HSP70 without heat shock and after it, but the HSP70 synthesis was not statistically changed by increasing duration of heat shock. In fully-grown oocytes, great amounts of HSP70 were found without heat shock treatment, and the contents of HSP70 significantly decreased after heat shock. These results indicate that growing oocytes are able to synthesize HSP70 after heat shock. This ability declines at the end of the growth period, and fully-grown oocytes are unable to induce HSP70 synthesis after heat shock. HSP70 is synthesized and stored during oocyte growth. The high HSP70 synthesis in non-heat-treated growing oocytes and a great amount of HSP70 in fully-grown oocytes support the hypothesis that HSP70 is important for oocyte growth and maturation.     

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite leishmania

Antimony-containing drugs are still the drugs of choice in the treatment of infections caused by the parasite Leishmania. Resistance to antimony is now common in some parts of the world, and several mechanisms of resistance have been described. By transfecting cosmid banks and selecting with potassium antimonyl tartrate (SbIII), we have isolated a cosmid associated with resistance. This cosmid contains 2 copies of the heat shock protein 70 (HSP70) and 1 copy of the heat shock cognate protein 70 (HSC70). Several data linked HSP70 to antimony response and resistance. First, several Leishmania species, both as promastigotes and amastigotes, increased the expression of their HSP70 proteins when grown in the presence of 1 or 2 times the Effect Concentration 50% of SbIII. In several mutants selected for resistance to either SbIII or to the related metal arsenite, the HSP70 proteins were found to be overexpressed. This increase was also observed in revertant cells grown for several passages in the absence of SbIII, suggesting that this increased production of HSP70 is stable. Transfection of HSP70 or HSC70 in Leishmania cells does not confer resistance directly, though these transfectants were better able to tolerate a shock with SbIII. Our results are consistent with HSP70 and HSC70 being a first line of defense against SbIII until more specific and efficient resistance mechanisms take over.