Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

The heat shock response in meso- and thermoacidophilic chemolithotrophic bacteria

Abstract The heat shock response was studied in a chemolithotrophic thermoacidophilic archaebacterium Sulfolobus acidocaldarius (shifted from 70° to 85°C) and a mesoacidop0ilic microorganism Thiobacillus ferrooxidans (from 30° to 41°C). When transferred from their normal growth temperature to the stress temperature, cells showed a decrease in the incorporation of Na₂¹⁴CO₃ into proteins, and at the same time, the synthesis of a specific subset of heat shock proteins was observed. Ethanol (4%) at 30°C, also caused a response similar to the heat shock upon T. ferrooxidans cells, whereas Sulfolobus cells at 70°C did not incorporate radioactive CO₂ in the presence of ethanol, apparently being damaged by the organic solvent.     

PROTEIN EXPRESSION DURING HEAT STRESS IN THERMO-INTOLERANT AND THERMO-TOLERANT DIATOMS

To better understand how diatoms are capable of responding to environmental stress, protein expression during heat treatment of a thermo-intolerant ( Phaeodactylum tricornutum ) and thermo-tolerant ( Chaetoceros muelleri ) diatom (Chrysophyta) was investigated. The stress response is a universal and conserved mechanism of cell survival to unfavorable conditions. Typically, a 10 to 15° C temperature elevation above cell growth optimal causes constitutively expressed proteins to decrease and heat shock proteins (HSPs) to increase. HSPs are categorized by molecular weight among five classes with each apparently specialized for a particular function that enhances cell survival. One-dimensional SDS-PAGE of diatoms subjected to heat treatment revealed that P. tricornutum exhibited a typical stress response, but C. muelleri did not exhibit a characteristic response even at a greatly elevated temperature (50° C). This result was confirmed by total soluble protein assays. Chaetoceros muelleri may contain higher basal levels of HSPs than P. tricornutum allowing C. muelleri to better tolerate elevated temperatures. Western blot analysis using pea HSP70 (70 kDa) antisera of heat-treated P. tricornutum and C. muelleri validated the hypothesis that thermo-tolerant cells contain higher levels of constitutively expressed HSPs. Two-dimensional gel electrophoresis of heat-treated cells indicate that the small HSPs (17–30 kDa) played a role in the stress response similar to that found in vascular plants. Ongoing work is focused on the manipulation of the stress response through over-expression of key hsp genes.     

Water-stress-induced heat tolerance in geranium leaf tissues: A possible linkage through stress proteins?

Evidence is accumulating in favor of a linkage at the cellular level between various abiotic stresses. We conducted a study to evaluate the effect of water stress on the heat tolerance of zonal geraniums, Pelargonium × hortorum cv. Evening Glow. Water stress was imposed by withholding irrigation until pots reached 30% (by weight) of well‐watered controls, and by maintaining the pot weight by additions of water for another 7 days. Leaf xylem water potential (XWP, MPa), relative water content (RWC. %), and heat‐stress tolerance (HST; LT₅₀, defined as the temperature causing half‐maximal % injury based on electrolyte leakage) were measured in control, stressed, and recovered plants. Proteins were extracted from the leaves following the above treatments, and SDS‐PAGE and immunoblotting were performed by using standard procedures. Immunoblots were probed with antibodies to dehydrin and 70‐kDa heat shock cognate (HSC70) proteins. Data indicate that XWP and RWC, respectively, were −0.378 MPa and 92.3% for control plants and −0.804 MPa and 78.6% for stressed plants. Water‐stressed plants exhibited a significant increase in HST compared to control (LT₅₀ of 55°C vs 51°C). Water‐stress‐induced HST was not due to heat acclimation (leaf warming in stressed plants). Data also indicate that water‐stress treatment did not increase freezing tolerance of geranium leaves. Increased HST was associated with the accumulation of several heat‐stable, dehydrin proteins (25–60 kDa), and both cytosolic and ER luminal (BiP) HSC70 proteins. Leaf XWP, RWC, and HST reversed to control levels concomitant with the disappearance/reduction of dehydrins and HSC70 proteins in water‐stress‐relieved plants. The possibility of a cellular linkage between water stress and heat‐stress tolerance is discussed.     

BiP, HSP70, NDK and PDI in wheat endosperm. II. Effects of high temperature on protein and mRNA accumulation

The effects of high temperature on accumulation of the 70‐kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17‐kDa NDK were co‐purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants ( Triticum aestivum , cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid‐grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1‐day gradual increase to 40°C. The effects of a 5‐day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post‐anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue.     

Heat shock and thermotolerance of Escherichia coli O157:H7 in a model beef gravy system and ground beef

Duplicate beef gravy or ground beef samples inoculated with a suspension of a four-strain cocktail of Escherichia coli O157:H7 were subjected to sublethal heating at 46 °C for 15–30 min, and then heated to a final internal temperature of 60 °C. Survivor curves were fitted using a linear model that incorporated a lag period (T_L), and D-values and 'time to a 4D inactivation' (T_4D) were calculated. Heat-shocking allowed the organism to survive longer than non-heat-shocked cells; the T_4D values at 60 °C increased 1·56- and 1·50-fold in beef gravy and ground beef, respectively. In ground beef stored at 4 °C, thermotolerance was lost after storage for 14 h. However, heat-shocked cells appeared to maintain their thermotolerance for at least 24 h in ground beef held at 15 or 28 °C. A 25 min heat shock at 46 °C in beef gravy resulted in an increase in the levels of two proteins with apparent molecular masses of 60 and 69 kDa. These two proteins were shown to be immunologically related to GroEL and DnaK, respectively. Increased heat resistance due to heat shock must be considered while designing thermal processes to assure the microbiological safety of thermally processed foods.     

Heat shock response in Actinobacillus actinomycetemcomitans

Abstract The heat shock response in Actinobacillus actinomycetemcomitans , a capnophilic Gram-negative bacterial species that is implicated in the development of certain forms of periodontitis, was characterized. Different strains of A. actinomycetemcomitans were grown at 37, 42 and 48°C in the presence of ³⁵S-methionine. The bacterial cells were lysed, run on SDS-PAGE and subsequently blotted on nitrocellulose paper. After autoradiography of the blots, several protein bands from the cultures at 42°C showed an increased intensity; major bands were observed at 90, 70, and 60 kDa, but increased protein synthesis was also detected at 54, 28 and 17 kDa. Nitrocellulose blots were also incubated with a panel of monoclonal and polyclonal antibodies directed to epitopes on different heat shock proteins. Strong reactivity was found with several antibodies at the position corresponding to a molecular mass of 60 kDa. The protein is probably the GroEL homologue in A. actinomycetemcomitans , a member of the ‘common bacterial antigen’ family.     

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.     

The stress and metabolic responses of juvenile Atlantic cod Gadus morhua L. to an acute thermal challenge

Survival, oxygen consumption (     ), total plasma cortisol and glucose levels and gill heat-shock protein 70 (hsp70) expression were measured in 10 and 50 g juvenile Atlantic cod Gadus morhua during an acute temperature increase (2° C h⁻¹) to their critical thermal maximum. Ninety three per cent of the fish in both size classes survived to 24° C; however, mortality was 100% within 15 min of reaching this temperature. The     for both size classes increased significantly with temperature, reaching peak values at 22° C that were c. 2·8-fold those of control (10° C) fish. Resting plasma cortisol and glucose levels were lower in 10 g as compared to 50 g fish. Plasma glucose levels were highly variable in both size classes, and significant increases were only seen at >22° C for the 10 g fish. In contrast, plasma cortisol showed an exponential increase with temperature starting at 16° C in both size classes, and reached maximum levels at 22° C that were 19-fold (10 g fish) and 35-fold (50 g fish) higher than their respective control groups. Both the constitutive (73 kDa) and inducible (72 kDa) isoforms of hsp70 were detected in both size classes using the widely utilized mouse monoclonal antibody. Expression of these isoforms, however, did not change when Atlantic cod were exposed to elevated temperature, and the 72 kDa isoform was not detected using salmonid-specific antibodies. These results indicate that juvenile Atlantic cod are very sensitive to acute increases in water temperature. In addition, they (1) show that     and plasma cortisol, but not plasma glucose or gill hsp 70 levels, are sensitive indicators of thermal stress in Atlantic cod and (2) support previous reports that the upper critical temperature for this species is 16° C.     

The heat shock response in hydra: immunological relationship of hsp60, the major heat shock protein of Hydra vulgaris,to the ubiquitous hsp70 family

The major heat shock protein (hsp) of Hydra vulgaris has recently been found to be a 60 kDa protein. Since in all organisms studied so far, the major heat shock protein is a 70 kDa protein, we have analyzed the relationship of hydra hsp60 to the highly conserved 70 kDa heat shock protein family. Genes and proteins related to the 70 kDa class of stress proteins are present in hydra. However, antibodies known to cross-react with hsp70 proteins in several different organisms do not cross-react with hydra hsp60 suggesting that hsp60 is not related to the conserved hsp70 proteins.     

Thermal tolerance and heat shock protein synthesis during development in the anuran Lepidobatrachus laevis

Development of the Paraguayan anuran Lepidobatrachus laevis is unusual in that the larvae are obligate carnivores, facultative cannibals and apparently exist at high environmental temperatures in their natural habitat. In the present study, the effect of environmental temperature on the rate of anuran development was investigated. The larvae have a thermotolerance range of 18°C for normal development between 19 and 37°C. The effect of temperature on the rate of development was dramatic; larvae that were incubated at 36.8°C develop to stage 24 (Gosner) in approximately 9 h compared with 24 h for larvae incubated at 19°C. The ability of larvae to survive heat shock was also examined; larvae did not survive a shock of 45°C for 15 min when it was administered at stages 3, 5, 9, 10 or 20. However, using the same heat shock conditions, 50% survival was observed when larvae were shocked at stage 16. To study protein synthesis during heat shock, larvae were pulsed with [³⁵S]-methionine during heat shock and labeled proteins were analyzed by electrophoresis under reducing and denaturing conditions. Larvae synthesized two sets of heat-shock proteins at doublet molecular weights of 83/78 and 62/59 kDa. These proteins were synthesized independently of the stage of development at which the shock was administered or the magnitude of the heat shock.     

The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.     

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.     

Effect of pre- and post-heat shock temperature on the persistence of thermotolerance and heat shock-induced proteins in Listeria monocytogenes

The effect of incubation temperature, before and after a heat shock, on thermotolerance of Listeria monocytogenes at 58°C was investigated. Exposing cells grown at 10°C and 30°C to a heat shock resulted in similar rises in thermotolerance while the increase was significantly higher when cells were grown at 4°C prior to the heat shock. Cells held at 4°C and 10°C after heat shock maintained heat shock-induced thermotolerance for longer than cells held at 30°C. The growth temperature prior to inactivation had negligible effect on the persistence of heat shock-induced thermotolerance. Concurrent with measurements of thermotolerance were measurements of the levels of heat shock-induced proteins. Major proteins showing increased synthesis upon the heat shock had approximate molecular weights of 84, 74, 63, 25 and 19 kDa. There was little correlation between the loss of thermotolerance after the heat shock and the levels of these proteins. Thermotolerance of heat shocked and non-heat shocked cells was described by traditional log-linear kinetics and a model describing a sigmoidal death curve (logistic model). Employing log-linear kinetics resulted in a poor fit to a major part of the data whereas a good fit was achieved by the use of a logistic model.     

Protein response of insect cells to bioreactor environmental stresses

Protein expression of Spodoptera frugiperda (Sf9) insect cells was characterized upon exposure to environmental stresses typically present in bioreactors including heat shock, oxygen deprivation, shear stress, change of pH, and salinity or ethanol shock. This study fills the void in knowledge as to how bioreactor hydrodynamics, anoxia, small changes in pH as well as salinity alterations due to pH control or exposure to ethanol used in asepsis treatments affect protein expression in Sf9 cells. Heat shock at 43 degrees C induced proteins at 83 kDa, 68-78 kDa and six small heat shock proteins (hsps) at 23-15.5 kDa. Anaerobic conditions in CO2 atmosphere reduced significantly the normal protein synthesis and induced a small subset of heat shock proteins at 70 kDa. Oxygen deprivation in nitrogen atmosphere transiently induces the 70 kDa proteins and had minor effects on the normal protein synthesis. Exposure to increased salinity or ethanol concentration failed to trigger the stress response, but may extensively inhibit the induction of normal proteins even though there was a negligible change in cell viability. Shear stress that had a major reducing effect on cell viability did not change the protein synthesis profile of Sf9 cells. Both long and short term exposures to small pH changes had negligible effects on protein synthesis.     

Heat shock protein synthesis and thermotolerance in Salmonella typhimurium

The resistance of stationary phase Salmonella typhimurium to heating at 55°C was greater in cells grown in nutritionally rich than in minimal media, but in all media tested resistance was enhanced by exposing cells to a primary heat shock at 48°C. Chloramphenicol reduced the acquisition of thermotolerance in all media but did not completely prevent it in any.
The onset of thermotolerance was accompanied by increased synthesis of major heat shock proteins of molecular weight about 83, 72, 64 and 25 kDa. When cells were shifted from 48°C to 37°C, however, thermotolerance was rapidly lost with no corresponding decrease in the levels of these proteins. There is thus no direct relationship between thermotolerance and the cellular content of the major heat shock proteins. One minor protein of molecular weight about 34 kDa disappeared rapidly following a temperature down-shift. Its presence in the cell was thus correlated with the thermotolerant state.     

Water relations and osmotic adjustment in Apium graveolens during long‐term NaCl stress and subsequent relief

Pea plants ( Pisum sativum L. cv. Feltham First) exposed to a heat stress of 37°C for 6 h accumulated two low molecular weight (LMW) heat shock proteins (HSPs) of molecular mass 22 kDa. The two LMW HSPs were associated with purified mitochondria. N‐terminal amino acid sequencing analysis indicates that the more basic of these proteins is a novel protein. The response of other cultivars of P. sativum to heat shock revealed that up to three 22‐kDa HSPs were expressed in a cultivar‐specific manner. Evidence presented suggests that the different 22‐kDa HSPs arise as a result of there being multiple 22‐kDa HSP genes. The expression of the most basic novel HSP was studied in the Feltham First cultivar using two dimensional SDS‐PAGE. Treatment of intact plants with chloramphenicol and cycloheximide prior to heat stress treatment indicated that the LMW HSPs were nuclear encoded and de novo synthesised. The response to heat shock was rapid with protein expression detected within 45 min and the protein remained in excess of 6 days following removal of the stress. The protein accumulated to very high levels with maximal expression being 2% of the total mitochondrial protein. The results are discussed in relation to the likely role of LMW HSPs in thermotolerance.     

Influence of nutrient-limited growth on pathogenesis-associated outer membrane proteins of Yersinia enterocolitica

From studies based on batch culture, it has been postulated that the expression of the virulence-associated proteins of Yersinia spp. is controlled by temperature and Ca²⁺, such that these proteins are synthesized only at the higher temperature (37°C) and calcium-scarce conditions of the intracellular environment. It was found, however, that in Yersinia enterocolitica one of these proteins (140 kDa) is not synthesized at submaximal growth rates under any of the relevant conditions, and that another of the implicated proteins (34 kDa), is synthesized even at 28°C during nutrient-limited growth. Thus, temperature and Ca²⁺ influence the synthesis of these proteins differently under growth conditions that better approximate the natural environments than do batch cultures.