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.     

Heat shock response of Dictyostelium

In response to a shift from 22 to 30°C the relative rate of synthesis of a small number of proteins is dramatically increased in Dictyostelium discoideum. The cells neither grow nor develop at this temperature but die slowly with a half-life of 18 hr. The major protein synthesized in response to a heat shock to 30°C in either growing cells or developing cells has an apparent molecular weight of 70,000 (70K). An increase in the relative rate of synthesis of 70K can be seen as early as 20 min following heat shock. Synthesis of 70K remains high for 4 hr at 30°C and then decreases. Similar kinetics of 70K synthesis occur during recovery at 22°C following a 1-hr heat shock. RNA synthesis during the first half-hour of heat shock is essential for the high rate of 70K measured 2 hr later. By isoelectric focusing the 70K protein can be separated into two spots, one of which overlaps one of the major heat shock proteins of Drosophila melanogaster. The relative rate of synthesis of several other proteins (82K, 60K, 43K) increases less dramatically in Dictyostelium during heat shock at 30°C. A heat shock to 34°C results in rapid synthesis of these proteins but not of 70K. The relative rates of synthesis of most other proteins made at 22°C decreases, most notably that of actin. Synthesis of heat shock proteins at 30°C does not significantly affect viability at 30°C but dramatically prolongs the period of time the cells can survive at 34°C. Thus, 30°C appears to be a stasis condition for Dictyostelium which elicits a response essential for protection from lethal temperatures. The similarity of the heat shock response in Dictyostelium to that in Drosophila and vertebrate cells suggests that certain aspects of the response may be universal in eukaryotes.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997     

Impact of heat shock on heat shock proteins expression, biological and commercial traits of Bombyx mori

We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB₄D₂, KSO₁, NP₂, CSR₂ and CSR₄and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35°C, 40°C and 45°C for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS‐PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP₂ exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar > second instar > third instar > fourth instar > fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB₄D₂, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.     

Heat shock-induced relaxation of restriction enzyme specificity inEscherichia coli

Methylated and hydroxymethylated cytosine containing DNA was restricted by proteins encoded by themcrBC (rglB) loci ofE. coli. In vivo, RglB proteins recognize and cleave hmCT2 and hmCT4 DNAs at 30°C and 42°C but hmCT6 DNA was unaffected at both temperatures, However, cells carrying therglB genes cloned on pBR322 (pDSS17) did not restrict hmCT6 at 30°C, but hmCT6 DNA was cleaved efficiently at 42°C. Heat shock treatment for five minutes was enough to induce this promiscuity in recognition specificity. We call this activity RglB star. A single copy ofrglB located on the chromosome or cloned on a low copy vector pMU575 failed to show RglB star activity.De novo protein synthesis was not required for the manifestation of RglB star activity.     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.     

Heat shock and cold shock in Deinococcus radiodurans

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42°C maximized survival at the lethal temperature of 52°C and a cold shock at 20°C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I and two proteins of unknown function.     

Heat shock mRNA accumulation during recovery from cold shock in Drosophila melanogaster

Heat shock protein synthesis is induced in response to a variety of chemical and physical stresses. Among these are heating above normal growing temperatures, treatment with heavy metals, amino acid analogues, steroid hormones and a variety of other chemicals (CRC Crit. Rev. Biochem. 18, 239–280). We have shown previously that heat shock proteins are also synthesized during recovery from prolonged 0°C treatment in Drosophila larval salivary glands. In this paper we describe the cold treatments which induce heat shock protein synthesis in more detail, and show that heat shock mRNA does not accumulate during the cold treatment, but rather during the recovery period when the larvae are returned to 25°C. The implications of these results for the regulation of heat shock mRNA levels, and for the role of heat shock proteins in recovery from cold shock are discussed.     

Differential Responses to Environmental Conditions by Fungal Cells Sensitized by Heat Shock or UV Irradiation

Cells of the ascomycele Ophiostoma multianulatum were sensitized to the supra-optimal temperature of 30°C either by heat shock or by UV irradiation. At this incubation temperature the death rate of the heat-shocked cells was higher than that of the irradiated cells. This difference was increased if hydrolysed casein was added to the incubation medium. The heat-shocked cells were also killed faster at 30°C, if nitrogen instead of air was bubbled through the cell suspension. Heat shock, in contrast to UV irradiation, strongly increased the sensitivity to a high concentration of sodium chloride.     

Exposure to heat shock affects thermosensitivity of the locust flight system

The natural habitat of the migratory locust, Locusta migratoria, is likely to result in locusts being heat stressed during their normal adult life. It is known that locusts exhibit a heat-shock response: exposure to 45°C for 3 h induces thermotolerance and the expression of heat-shock proteins. We investigated the effects of exposure to heat-shock conditions on the thermosensitivity of flight rhythm generation in tethered, intact animals and in deafferented preparations. Heat shock had no effect on wingbeat frequency measured at the start of flight sequences, nor did it affect the postimaginal maturation of this parameter. During sustained flight, heat shock slowed the characteristic asymptotic reduction of wingbeat frequency. Wingbeat frequency of heat-shocked animals was less sensitive to temperature in the range 24° to 47°C than that of control animals, and the upper temperature limit, above which flight rhythms could not be produced, was 6° to 7°C higher in heat-shocked animals. These results were mirrored in the response of deafferented preparations, indicating that modifications in the properties of the flight neuromuscular system were involved in mediating the response of the intact animal. We propose that exposure to heat shock had the adaptive consequences of reducing thermosensitivity of the neural circuits in the flight system and allowing them to operate at higher temperatures. © 1996 John Wiley & Sons, Inc.     

Effect of Different Temperature Upshifts on Protein Synthesis by the Psychrotrophic Bacterium Pseudomonas fragi

Pseudomonas fragi, a psychrotroph bacterium involved in meat product spoilage, was shifted either from 5° to 20°C or 30°C and from 28° to 34°C. The heat-shocked cells in the mid-log phase rapidly reached the characteristic growth rate of the postshock temperature. The patterns of synthesized proteins were compared by autoradiography of two-dimensional gel electrophoregrams. The rates of synthesis, after transfer of cells from 5° to 30°C, 5° to 20°C, and 28° to 34°C, changed for 30, 26, and 21 proteins respectively, of which 19, 17, and 12 were increased respectively. Thirteen proteins changed similarly for the three treatments, and two of the seven overexpressed proteins were immunologically related to the Escherichia coli DnaK and GroEL heat shock proteins. From the four low-molecular-mass proteins, belonging to the family of DNA-binding cold shock proteins (CSPs) such as CS7.4, the major E. coli CSP [15], the amounts of C7.0 and C8.0 decreased rapidly after the upshifts, whereas that of E7.0 and E8.0 increased greatly. Received: 22 November 1995 / Accepted: 22 December 1995     

The heat shock response inLocusta migratoria

Summary Locusta migratoria adults reared at 27–30°C die after 2 h at 50°C, but they survive this temperature stress if first exposed to 45°C for 0.5 to 4.5 h. Fat bodies from adult females produce a set of at least six specific polypeptides with molecular weights of 81, 73, 68, 42, 28, and 24×10³ in reponse to heat shock (39–47°C for 1.5 h). These molecular weights closely match those of the heat shock proteins (hsps) observed inDrosophila, with the possible exception of the 42 kd protein of locusts. The optimal temperature for induction of hsps in locusts is 45°C, which is one of the highest heat shock temperatures reported in metazoans. The correspondence between the optimal temperature for hsp induction and the temperature at which enhanced heat tolerance is acquired (both 45 °C) suggests that the hsps may be associated with thermal protection in these insects.There appears to be no substantial translational control in the locust heat shock response, since other proteins are produced, albeit with some reduction, under heat shock conditions. Vitellogenin synthesis in fat bodies at 45°C is 55% of that observed at 30°C. The high optimal heat shock induction temperature and the continued synthesis of non-heat shock proteins may be adaptive to the locust's natural environment.     

Stage dependent synthesis of heat shock induced proteins in early embryos of Drosophila melanogaster

Summary The synthesis of heat shock proteins (hsp) has been examined during the early embryogenesis of Drosophila melanogaster. Normal protein synthesis stops after heat shock at all developmental stages, while hsp synthesis is induced only after treatment at blastoderm and later stages. The small hsps continue to be synthesised after heat shock for a longer period than the larger ones. Heat shocks at 35°C, 37°C and 40°C were compared for their effect on hsp synthesis and the effect of heat shock on the normal course of development was analysed.     

Starvation reduces the heat shock protein responses in white sturgeon larvae

This study investigates the responses of white sturgeon larvae (Acipenser transmontanus) to starvation and thermal stress, through the measurement of nutritional status (i.e. growth performances) and cellular biomarkers: heat shock proteins (Hsp) 70 and 90. White sturgeon larvae (25 day post hatch; initial weight 179.0 ± 5.1 mg) were fed (20% body weight per day) or starved for 24, 48 or 72 hrs. Every 24 hrs, five larvae from each of the starved or fed treatment replicates were exposed to heat shock resulting from an increase in water temperature from 19°C to 26°C, at a rate of 1°C per 15 min, and maintained at 26°C for 4 hrs. No mortality was observed in this study. Starvation significantly (p < 0.05) decreased the body weight and body contents of energy, protein, and lipid of the experimental larvae, compared to the fed larvae. Heat shock induced the expressions of Hsp70 and Hsp90 in both the fed and starved group; however, starvation reduced the induction at all sampling points. The current study demonstrates that poor larval nutritional status, assessed by the aforementioned parameters, reduced heat shock responses to thermal stress, as measured by heat shock protein levels. Furthermore, Hsp70 and 90 are more sensitive to heat shock and starvation, respectively. This may be, in part, a result of the different functioning of the heat shock proteins in cellular stress response and warrants further study.     

Developmental study of thermotolerance and the heat shock response inLucilia cuprina (Weidemann)

The ability to withstand thermal stress in a laboratory population of the blowflyLucilia cuprina (measured as per cent adult survival following varying periods of exposure to elevated temperature up to a maximum of 48°C) was in the order pupa > larva > adult. Pre-exposure to a mild heat shock (37°C) induced tolerance to temperatures which were otherwise lethal. An analysis of heat shock-induced protein synthesis during development at similar elevated temperatures presented patterns corresponding to the above observations on thermotolerance. The induced level of synthesis of major heat shock proteins (viz., 79, 69, 28, 20 and 19 kDa) were greater in larval tissues than in most of the adult tissues except gonads. The response varied between young (2 days) and old (30 days) adults in a tissue-specific manner. In general, heat shock protein 69 kDa was most abundant in all the tissues studied. Control as well as heat shocked Malpighian tubules of adults uniquely showed two major [³⁵S]methionine labelled bands corresponding to approximately 62 and 64 kDa. Immunoblots showed the 62 kDa protein to cross react with an antibody againstHelioihis HSP60. Although the synthesis of the 62 kDa polypeptide was prominent only in Malpighian tubules of adult blowflies, nearly equal levels of this HSP60 family polypeptide were present in all tissues (control as well heat shocked) except the larval salivary glands.