Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

A comprehensive analysis of heat shock protein synthesis in human peripheral lymphocytes: the effect of penicillin/streptomycin.

A reliable experimental procedure is described for the simultaneous characterisation of a comprehensive range of heat shock proteins (hsps) in human peripheral lymphocytes. In this system, a mild heat shock from 37 to 42 degrees C for 1 h induced the synthesis of hsps 105, 90, 70, 60, 57, 47, 40, 27 and 16. Densitometric analyses of 35[S]-methionine labelled protein gels indicated that levels of these hsps peaked at 3 to 4 h, following post-heat shock recovery at 37 degrees C. The presence of penicillin and streptomycin in the cell culture medium, appeared to have little effect on the kinetics of hsp synthesis. The present method can be used for relatively small blood samples and its relative ease of application and reproducibility make it appropriate for screening the expression of hsps in human lymphocytes from a range of individuals.     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.     

Temperature stress induces notochord abnormalities and heat shock proteins expression in larval green sturgeon (Acipenser medirostris Ayres 1854)

The effects of thermal stress on survival, development and heat shock protein (hsp) expression of green sturgeon (GS) yolk‐sac larvae, from hatching through yolk depletion were investigated to provide insight into effects of highly altered natural river hydrographs. Hatched GS larvae were reared at constant water temperatures 18°C (control) through 28°C at 2°C increments. Larval survival significantly decreased at 26–28°C, with 28°C being lethal. Significant proportions of deformed larvae were found at sub‐lethal (20–26°C) and lethal 28°C rearing temperatures, with kyphosis (i.e. backward flexion of notochord) accounting for >99% of morphological deformities. Histological analysis of larvae preparations indicate that elevated water temperature affects notochord cell function and physiology. At rearing temperatures 20–28°C, thermal stress elicited a quick (24 h) and long lasting (yolk‐sac absorption) significant over‐expression of measured heat shock proteins (hsps), all of which are known components of intracellular protein repair and stabilization mechanism. Thermal sensitivity, as indicated by the incidence of abnormalities and expression of different hsps, varied significantly between crosses. Thermally tolerant progeny exhibited a short but rapid hsp72 (size in kDa) over‐expression, and more pronounced hsp60 and hsp90 over‐expression, than less tolerant progeny which exhibited a prolonged hsp72 and hsp78 over‐expression. At environmentally relevant water temperatures bent larvae exhibited spiral swimming, which in the wild would compromise the ability of emerging larvae to forage, avoid predators, and migrate downstream, ultimately compromising survival and recruitment. Before larvae hsp content can be used as a thermal‐stress biomarker for GS, field validation studies are needed.     

Heat shock proteins and chilling sensitivity of mung bean hypocotyls

Excised mung bean (Vigna radiata L.) hypocotyl sections wereexposed to 40 C for up to 4 h in the presence or absence of50 µM cycloheximide (CHX) before being held at a non-chilling(20 C) or chilling (2.5 C) temperature. Mung bean hypocotyltissue is chilling sensitive, and the rate of solute leakageis highly correlated with the extent of chilling injury. A 3h heat shock at 40 C reduced chilling-induced solute leakageby up to 40%, but leakage was similar to non-heat-shocked hypocotylswhen CHX was present. Specific proteins were labelled when hypocotylswere exposed to [³⁵S] methionine during the last hour of heatshock. The nine most intense bands on the autoradiographs ofSDS-PAGE gels of extracted protein corresponded to molecularweights of 114, 79, 73, 70, 60, 56, 51, 46, and 18 kDa. The18 kDa band reached a maximum after 1 h at 40 C and then rapidlydecreased in intensity as the heat shock continued, becomingundetectable at 4 h. The four most intense bands after 3 h at40 C corresponded to molecular weights of 79, 70, 51, and 46kDa. The synthesis of these four hsps was markedly reduced whenthe hypocotyl sections were exposed to CHX during heat shock.During chilling for 6 d, the levels of hsps 79 and 70 remainedsignificantly higher in tissue that was heat shocked prior tochilling than in tissue that was not heat shocked. In contrast,the levels of hsps 51 and 46 were similar in bothheat-shockedand control tissues. Heat-shock-induced chilling tolerance waslost between 6 and 9 d ofstorage at 2.5 C; this loss coincidedwith the decay of hsps 79 and 70 to control levels. These resultssuggest that heat shock induces an increase in both chillingtolerance and the de novo synthesis of specific heat shock proteins;namely hsps 79 and 70. This is the first report showing a relationshipbetween heat-shock-induced chilling tolerance and specific heat-shock-inducedproteins. Key words: Ion leakage, protein synthesis, Vigna radiata     

Acidic extracellular environment induces only a subset of heat-shock proteins in primary mouse kidney cell cultures

Exposure of primary mouse kidney cell cultures to acidic medium (pH 5.5) induced the expression of a 70 kilodalton (kDa) protein. This protein was identified as the major inducible heat-shock protein 70 (hsp70) by immunoprecipitation with anti-hsp70 serum and Northern blot analysis with a hsp70 cDNA probe. Maximum induction of the 70-kDa protein at pH 5.5 after 240 min was about 30% of that observed after 60 min of thermal treatment at 43 degrees C. In addition, there was an apparent induction of the glucose-regulated proteins (GRPs) of 76-78 and 98-100 kDa, but not of the other hsps. This subset induction of the heat-shock response by acidic medium suggests that different mechanisms are responsible for the induction of the various families of hsps.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Purification and Partial Amino Acid Sequence of the Major 70,000-Dalton Heat Shock Protein in Neurospora crassa

Fracella, F., Mohsenzadeh, S., and Rensing, L. 1993. Purification and partial amino acid sequence of the major 70,000-Dalton heat shock protein in Neurospora crassa. Experimental Mycology , 17, 362-367. The major heat shock protein of 70 kDa (hsp70) from heat-shocked mycelial extracts of Neurospora crassa was purified to near homogeneity employing DEAE anion-exchange chromatography followed by affinity chromatography on ATP-agarose. The isolated hsp70 migrates as a single band on one-dimensional sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), with a molecular mass of ∼69 kDa. On two-dimensional gels it is resolved into two polypeptides with isoelectric points in the acidic range of ∼pH 5.2. The first 53 amino terminal amino acids of the major protein were sequenced and compared with hsp70 of other species. The amino acids aspartic acid, arginine, and phenylalanine occur at positions 27, 28, and 44 (from the methionine terminus) in contrast to the main consensus sequence. These three differing amino acids are shared by yeast, and, in addition, the first two by Arabidopsis, petunia, and maize.     

Induction of the heat shock response and translational thermotolerance in day 15 ovine trophectoderm

The objectives of this study were to determine the ability of trophectoderm from preimplantation ovine embryos to synthesize hsp70 in response to heat shock and to identify conditions which induce translational thermotolerance in this tissue. Day 15 embryos were collected, and proteins synthesized in 1.5-mm sections of trophectoderm were radioactively labeled with (35)S-methionine. One-dimensional SDS-PAGE gels, two-dimensional gel electrophoresis and Western blots were utilized to characterize the heat shock response and to examine the induction of translational thermotolerance. Increased synthesis of the 70 kDa heat shock proteins and a protein with an approximate molecular weight of 15 to 20 kDa was observed with heat shock (> or = 42 degrees C). Total protein synthesis decreased (P < 0.05) with increased intensity of heat shock. At 45 degrees C, protein synthesis was suppressed with little or no synthesis of all proteins including hsp70. Recovery of protein synthesis following a severe heat shock (45 degrees C for 20 min) occurred faster (P < 0.05) in trophectoderm pretreated with a mild heat shock (42 degrees C for 30 min) than trophectoderm not pretreated with mild heat. In summary, trophoblastic tissue obtained from ovine embryos exhibit the characteristic "heatshock" response similar to that described for other mammalian systems. In addition, a sublethal heat shock induced the ability of the tissue to resume protein synthesis following severe heat stress. Since maintaining protein synthesis is crucial to embryonic survival, manipulation of the heat-shock response may provide a method to enhance embryonic survival.     

Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25°C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25°C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42°C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25°C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25°C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25°C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times.     

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     

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.     

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, α-amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Heat shock in barley ( Hordeum vulgare L. cv. Himalaya) aleurone layers induces the synthesis of heat shock proteins (hsps) and suppresses the synthesis and secretion of α-amylase, the principal secretory protein. This is accompanied by the destabilization of α-amylase mRNA and a concomitant dissociation of ER lamellae. In the absence of heat shock α-amylase mRNA is extremely stable (Belanger et al. 1986. Proc. Natl. Acad. Sci. USA 83: 1354–1358). In most organisms there is a direct correlation between the synthesis of hsps and thermotolerance. The ability of hsps to provide thermoprotection to secretory protein synthesis, α-amylase mRNA and ER lamellae was analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pulse-chased, [³⁵S]-methionine-labeled proteins revealed that the half-life of hsps in barley aleurone cells recovering from heat shock was approximately 12 h. Within approximately 6 h, there was a recovery of α-amylase mRNA and a reformation of ER lamellae. Heat shock protein synthesis was induced by either heat shock (40°C) or arsenite, the cells were allowed to recover for 8 h, then were re-exposed to heat shock. Results from SDS-PAGE showed that, despite the presence of hsps, α-amylase synthesis was suppressed. Northern blot hybridizations showed that α-amylase mRNA levels were reduced in heat-shocked tissues. Transmission electron microscopy demonstrated that ER lamellar structures were dissociated. The synthesis of hsps did not enable barley aleurone cells to sustain the synthesis of any proteins at lethal temperature. In contrast, similar conditions established thermotolerance and provided thermoprotection to protein synthesis in germinating barley embryos. Our findings suggest that the aleurone layer does not become thermotolerant following the induction of hsp synthesis.     

Chelation affects the conformation, lability and aggregation of channel catfish (Ictalurus punctatus) phosphorylcholine-reactive protein (PRP).

1. Phosphorylcholine-reactive protein (PRP) affinity-purified from channel catfish (Ictalurus punctatus) serum on phosphorylcholine-Sepharose, eluted from Bio-Gel A-5M as a 94.6 +/- 2.4 kDa protein when the gel filtration column buffer (Tris-saline) contained 25mM ethylenediaminetetraacetic acid (EDTA). 2. PRP chelated with EDTA immediately after affinity purification and gel-filtered in Tris-saline-EDTA, eluted as a 75.5 +/- 2.67 kDa protein referred to as fast-PRP (F-PRP). 3. PRP and F-PRP were identical on SDS-PAGE. Both resolved as a broad band of protein (ca 86-100 kDa) on non-reducing gels or as a ca 100 kDa protein after reduction with 2-mercaptoethanol (2-ME). 4. After gel-filtration in Tris-saline-EDTA, nearly complete reduction of 100 kDa PRP was achieved on SDS-PAGE. However, the protein regained its resistance to reduction upon storage at -60 degrees C. 5. SDS-PAGE and native PAGE also revealed that during storage, PRP and F-PRP combined to form 3 different aggregates referred to as aggregated-PRP (aggPRP). These aggregates are readily dissociated in the presence of 2-ME, suggesting a covalent interaction between adjacent pentamers comprising decameric aggPRPs. 6. PRP, F-PRP, and aggPRP have similar amino acid compositions.     

Heat-shock proteins during growth and sporulation of Bacillus subtilis

Four major heat-shock proteins (hsps) with apparent molecular masses of 84, 69, 32 and 22 kDa were detected in exponentially growing stationary phase and sporulating cells of Bacillus subtilis heat-shocked from 30 to 43 degrees C. The most abundant, hsp69, is probably analogous to the E. coli groEL protein. These proteins were transiently inducible by heat-shock. Partial purification of RNA polymerase revealed several other minor hsps. One of these, a 48 kDa polypeptide probably corresponds to sigma 43. The synthesis of this polypeptide and at least two other proteins appeared to be under sporulation and heat-shock regulation and was affected by the SpoOA mutation.