In vitro thermal stress induces apoptosis and reduces development of porcine parthenotes

The precise physiological causes that result in reduced development of oocytes after heat shock (HS) are not clear. In this study, apoptosis, heat shock protein70 (hsp70), and in vitro development of porcine oocytes were evaluated after HS. Porcine cumulus-oocyte complexes (COCs) were subjected to in vitro maturation for 42 h. The matured oocytes were then heated at 41.5 degrees C for 0 h (control, C0h), 1 h (HS1h), 2 h (HS2h), or 4 h (HS4h). An additional group of oocytes was cultured for 4 h without HS (control, C4h). In Experiment 1, expression of hsp70 was detected by Western-blotting and no difference between controls and HS groups was observed. In Experiment 2, apoptosis of matured oocytes after HS was examined by Annexin V-FITC and TUNEL. No significant TUNEL-positive signals were detected in the heated oocytes compared to the controls, but the intensity of Annexin V-FITC labeling among different groups increased with length of HS and in vitro culture (P<0.05). Oocytes were parthenogenetically activated by an electric pulse plus 6-DMAP (Experiment 3). Mean (+/-S.E.M.) embryonic development in HS2h (cleavage: 42+/-29%; blastocyst: 11+/-10%) and HS4h (cleavage: 36+/-28%; blastocyst: 11+/-8%) were decreased when compared to those in C0h (cleavage: 63+/-12%; blastocyst: 24+/-14%) and C4h (cleavage: 66+/-8%; blastocyst: 21+/-11%). Numbers of blastocysts with TUNEL-positive signals were similar among groups, but the signals increased before the eight-cell stage in HS groups (P<0.05). In conclusion, developmental competence of matured pig oocytes was compromised after heat shock, but it was not closely associated with the expression of oocyte hsp70. However, there may be a link between apoptosis and developmental competence of porcine oocytes.     

Calmodulin is involved in heat shock signal transduction in wheat

The involvement of calcium and calcium-activated calmodulin (Ca(2+)-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37 degrees C HS. The levels of CaM mRNA and protein increased during HS at 37 degrees C in the presence of Ca(2+). The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl(2) and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl(3) and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca(2+) also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37 degrees C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22 degrees C after HS at 37 degrees C. Taken together, these results indicate that Ca(2+)-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.     

Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70-kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several-fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70-related cDNA probes. Transient ischemia resulted in 5- to 10-fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ascorbic acid and acute heat exposure on heat shock protein 70 expression by young white Leghorn chickens

Dietary ascorbic acid (AA) and heat stress (HS) affect heat shock protein 70 (hsp70) and body temperature (BT) of strain cross white Leghorn chickens. At five weeks of age, chicks fed either no supplemental AA (N-AA) or 200 mg/kg AA (AA) were subjected to either HS (42 degrees C) or maintained in control (CN, 23 degrees C) ambient temperature (T(a)) for 1 h. Body temperature (BT) was recorded for each bird before collection of heart and liver for hsp70 assay. In the CN AA-fed groups, neither the lower constitutive hsc70 nor the decreased hsp70 response to HS in the heart and liver were sex-dependent. The BT was increased by HS, but neither AA nor sex of the bird affected BT response. A diet X T(a) interaction revealed that BT of CN AA-fed chickens was lower than in N-AA-fed chickens, but BT of HS AA-fed chicks was greater than BT in HS N-AA-fed chickens. The BT and hsp70 responses were positively correlated. A lower expression of hsp70 indicated less of a stress response in the AA-fed chickens.     

Mytilus trossulus hsp70 as a biomarker for arsenic exposure in the marine environment: laboratory and real-world results.

The highly conserved heat shock protein 70 (hsp70) is induced by heat and chemical toxins, particularly heavy metals such as arsenic (As). The use of Mytilus trossulus (bay mussel) hsp70 as a 'screening' biomarker for marine heavy metals contamination was assessed. Some studies have found high hsp70 sensitivity to heavy metals, while others have found the opposite. Few studies have realistically used low heavy metals exposures, and fewer have used real-world contamination exposures. Clean sub-tidal mussels from the Puget Sound, Washington State (WA), USA, were acclimatized for 2 weeks and exposed for 24 h to As-spiked seawater (n=9) or to contaminated seawater from an arsenical pesticide plant in Tacoma, WA (n=10) followed by a Western blot for hsp70. Hsp70 inductions were insignificant at 10 microg l(-1) As(III), but were strong at 100 microg l(-1) (p<0.05) and 1000 microg l(-1) (p<0.01), with the induction threshold estimated at 30-50 microg l(-1) As(III). Hsp70 induction roughly correlated with arsenical toxicity, with As(III) > As(V) > (CH(3))(2)As(V). Altogether, the inter-individual variability of hsp70 levels tends to mask inductions at low As concentrations, making it a crude toxicity biomarker. In addressing this problem, the following options could prove promising: (1) pre- or post-stressing specimens for greater hsp70 sensitivity, (2) use of internal protein controls such as actin, (3) use of hsp70-reporter gene constructs, and (4) detection with hsp60, heme oxygenase-1, metallothionein, CYP450, MXR or GPx.     

Simultaneous activation of heat shock protein (hsp 70) and nucleolin genes during in vivo and in vitro prereplicative stages of rat hepatocytes.

Rapidly growing cells usually have high levels of ribosome biogenesis. The sequential expression of protooncogenes during the transition of quiescent hepatocytes to the replicative stage was assumed to be followed by activation of cellular genes related to cell growth such as ribosome biosynthesis. First, the expression of major nucleolar protein (nucleolin or C23) and major heat-shock protein (hsp 70) genes was examined during rat liver regeneration. hsp 70 may function in cell growth and has a characteristic nucleolar location after heat shock. Both nucleolin and hsp 70 mRNA began to increase simultaneously after peaks of c-fos and c-myc, showed a peak 6 h after partial hepatectomy, and declined to the control levels around 20 h. That is, the peaks of nucleolin and hsp 70 mRNA precede the peak of ribosome formation (12-20 h) and DNA replication (24 h). Second, the behavior of nucleolin and hsp 70 mRNA was examined in primary cultured hepatocytes during their G0-G1 transition. Although the amounts of c-myc mRNA reached a plateau around 20 h after the initiation of culture and remained at these levels, DNA synthesis has never been found to start without the addition of EGF and insulin to this system. Both nucleolin and hsp 70 mRNA began to increase at around 20 h (prereplicative stage) and simultaneously decreased in inverse proportion to DNA synthesis induced by these growth factors. Thus, it is possible that the simultaneous enhancement of nucleolin and hsp 70 genes as described above is not merely coincidental, but is important biologically during the transition of quiescent hepatocytes to proliferative cells.     

The 70-kDa heat shock protein response in two intertidal sculpins,Oligocottus maculosus and O. snyderi: relationship of hsp70 and thermal tolerance

The role that hsp70 plays in influencing thermal tolerance of a whole animal is not clearly understood. We explored this question by examining liver hsp70 response in the tidepool sculpin (Oligocottus maculosus) and fluffy sculpin (O. snyderi), which have distinct distribution patterns in the intertidal zone. The tidepool sculpin is in upper and lower tidepools, while the fluffy sculpin is exclusively in lower tidepools during a low tide. We conducted experiments in order to investigate: (1) habitat water temperatures; (2) upper thermal tolerance limits; (3) the cellular hsp70 response to changes in water temperature in nature; (4) induction temperatures for hepatic hsp70 and hsp70 mRNA; and (5) effects of long-term heat stress on liver hsp70 levels, in these sculpins. Accordingly, we found: (1) the tidepool sculpin was exposed to a wider temperature range in nature; (2) the tidepool sculpin had higher lethal and induction temperatures for hsp70; (3) the liver hsp70 level of the tidepool sculpin was less sensitive to changes in water temperatures; and (4) the tidepool sculpin had higher constitutive hsp70 levels in nature, compared with the fluffy sculpin. From these results, we proposed that the less thermally sensitive tidepool sculpin may enhance its thermal tolerance by having a large pool of cellular hsp70, thus allowing it to inhabit the upper intertidal zone with relatively large and unpredictable fluctuations in environmental variables.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.     

Evolution of the response to heat shock in genus Drosophila

Thermotolerance was studied in a wide spectrum of Drosophila species and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilis group exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogaster Oregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters of Drosophila and variation in the numbers of gene copies and hsp70 isoforms in group virilis were found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genus Drosophila is discussed.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.     

An instructive function for Notch in promoting gliogenesis in the zebrafish retina

The Gal4-UAS technique has been used to misexpress a constitutively active Notch receptor variant (notch1a-intra) in the developing zebrafish retina. This is the first study to use this technique to misexpress genes and assess their function in neural development of the zebrafish. Expression of activated Notch1a either ubiquitously, driven by a heat-shock70 promoter, or in a spatially regulated manner, controlled by the deltaD promoter, causes a block in neuronal differentiation that affects all cell types. Developing cells take on either a glial fate or remain undifferentiated. A large number of cells eventually undergo apoptosis. These phenotypic effects of activated Notch1a are expressed cell autonomously. Cells within central regions of the retina adopt a glial fate if they express activated Notch1a in a time window that extends from 27 to 48 hours postfertilization. This period corresponds mainly to the time of origin of ganglion cells in the normal retina. Activation of notch1a at later stages results in defects in cell type specification that remain restricted to the ciliary marginal zone, whereas neuronal types are specified normally within the central region. These observations indicate that glial differentiation is initiated by Notch1a-intra expressing cells, which become postmitotic in the same time window. Our results strongly suggest that Notch1a instructs a certain cell population to enter gliogenesis, and keeps the remaining cells in an undifferentiated state. Some or all of these cells will eventually succumb to apoptosis.     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of IL-10 and inhibition of experimental arthritis are specific features of microbial heat shock proteins that are absent for other evolutionarily conserved immunodominant proteins

Bacterial heat shock proteins (hsp) are evolutionary conserved immunodominant proteins that manifest amino acid homologies with hsp present in mammalian cells. Preimmunization with mycobacterial hsp65 has been found to protect against various forms of experimental arthritis. As these protective effects have previously been attributed to induction of self homologue cross-reactive T cell responses, the question was raised as to whether this protective effect could be extended to other highly conserved and immunodominant microbial Ags with mammalian homologues. Therefore, we immunized Lewis rats with conserved bacterial Ags (superoxide dismutase, aldolase, GAPDH, and hsp70). Although all Ags appeared highly immunogenic, we only found a protective effect in experimental arthritis after immunization with bacterial hsp70. The protective effect of hsp70 was accompanied with a switch in the subclasses of hsp70-specific Abs, suggesting the induction of Th2-like response. The most striking difference between immunization with hsp70 and all other immunodominant Ags was the expression of IL-10 found after immunization with hsp70. Even more, while immunization with hsp70 led to Ag-induced production of IL-10 and IL-4, immunization with aldolase led to increased production of IFN-gamma and TNF-alpha. Thus, the protective effect of conserved immunodominant proteins in experimental arthritis seems to be a specific feature of hsp. Therefore, hsp may offer unique possibilities for immunological intervention in inflammatory diseases.     

Negative elongation factor accelerates the rate at which heat shock genes are shut off by facilitating dissociation of heat shock factor

Promoter-proximal pausing of RNA polymerase II (Pol II) occurs on thousands of genes in animal cells. This pausing often correlates with the rapid induction of genes, but direct tests of the relationship between pausing and induction rates are lacking. hsp70 and hsp26 in Drosophila are rapidly induced by heat shock. Contrary to current expectations, depletion of negative elongation factor (NELF), a key factor in setting up paused Pol II, reduced pausing but did not interfere with rapid induction. Instead, depletion of NELF delayed the time taken for these genes to shut off during recovery from heat shock. NELF depletion also delayed the dissociation of HSF from hsp70 and hsp26, and a similar delay was observed when cells were depleted of the histone acetyltransferase CBP. CBP has been reported to associate with Pol II, and acetylation of HSF by CBP has been implicated in inhibiting the DNA-binding activity of HSF. We propose that NELF-mediated pausing allows Pol II to direct CBP-mediated acetylation of HSF, thus causing HSF to dissociate from the gene. Activators are typically viewed as controlling Pol II. Our results reveal a possible reciprocal relationship in which paused Pol II influences the activator.