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AMER. ZOOL., 39:901-909 (1999)

Heat Shock Proteins and Physiological Stress in Fish1GE OR GE K. I W AM A, 2 M AT HI L AKAT H M. VI JAYAN, 3 ROB B. F O R S Y T H , AND

PA I G E A. A CK ERM A NFaculty of Agricultural Sciences and the Canadian Bacterial Diseases Network, 208-2357 Main Mall,Vancouver, British Columbia V6T 1Z4 Canada

SYNOPSIS. This paper reviews the generalized stress response in fishat the cellularand neuroendocrine levels. The focus of this review is to examine the possiblerelationships between the stress responses at these two levels in fish. It focusesprimarily on the heat shock protein 70 (hsp70). Thus, the descriptions of the en-docrine and the cellular stress responses are followed by a discussion of how hspsmay be related to the stress hormones adrenaline and cortisol. Preliminary evi-dence shows that adrenaline causes an increase in hsp70 in primary cultures ofrainbow trout hepatocytes. Cortisol does not directly affect hsp70 levels in fishtissues; however, in primary cultures of trout hepatocytes, cortisol decreased thestressor-induced increase in hsp 70. A wide range of abiotic and biological stressorshave been shown to induce hsp induction in many types offish cells, including celllines, primary cell cultures, and in tissues from whole animals. Heat shock proteinshas been implicated in the protection of sulphate transport in the renal epitheliumof the flounder against the damaging effects of heat stress. Heat shock proteinslikely confer thermotolerance in fish, as well as tolerance to cytotoxic effects ofenvironmental contaminants and other non-thermal stressors.

INTRODUCTIONFish are exposed to biological and abioticstressors in the wild, as well as in captivity.Environmental pollutants, disease, and var-

ious aspects of intensive aquaculture aresome examples of those stressors. Fish alsocan become physiologically stressed frompsychological stressors such as exposure topredators and crowding. Like other verte-brates, stressed fish exhibit a generalizedstress response, that is characterized by anincrease in stress hormones and the conse-quent changes at the physiological, organ-ismal and population levels (see WendelaarBonga, 1997; Barton, 1997). Such a gen-eralized stress response also occurs at thecellular level and has been called the cel-lular stress response (see Hightower, 1991).This paper reviews the generalized stressresponse in fish from the cellular to the or-ganismal levels. It focuses on the question1 From the Symposium Organismal, Ecological andEvolutionary Significance of Heat S hock Proteins andthe Heat Shock Response presented at the AnnualMeeting of the Society for Comparative and Integra-tive Biolog y, 610 January 1999, at Denver, Colorado.2 E-mail: [email protected] Present Address: Department of Biology, Univer-sity of Waterloo, Waterloo, Ontario, Canada.

of whether the cellular stress response is re-lated to the neuroendocrine stress responseat the organismal level.At a very broad level, most investigatorssubject their biological subjects to specificstressors. This paper, however, discusses theresponse of cells, tissues and the whole or-ganism that is common to a wide range ofstressors. There are many definitions ofsuch a generalized stress response in fish(see Barton, 1997). Although many defini-tions are restricted to considering only themaladaptive and negative aspects of stress,we must also consider the adaptive aspectsof the stress response. The stress responseto any exogenous or endogenous perturba-tion, from the behavioural to the molecularlevels, usually works to reestablish homeo-stasis. The net outcome of that interactionbetween the stressor and the stress responseof the animal determines the success or fail-ure in reestablishing cellular and physiolog-ical conditions within the normal range forthat organism. T he definitions of stressor asthe causative factor, and stress as the re-sponse of the animal apply to the whole an-imal, as well as to each cell in the organism.

The cellular stress response has been de-scribed in nearly all cells studied to date.901

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90 2 G . K . IWAMA ETALOne of the most common features of thecellular stress response is the production ofheat shock proteins (hsps) in response tostressors that threaten the life of the cell;which is dependent upon the maintenanceof protein integrity and function. Althoughmany investigators have described the cel-lular stress response in different cell types,there is relatively little work on fish cellsand especially on whole organisms (seeIwama et al., 1998). Thus, while it mayseem intuitive that there should be a simpleand direct connection between the organis-mal and cellular stress responses, there islittle direct experimental evidence for this.The emerging evidence supports a rathercomplex relationship between stress hor-mones and hsps.

The paper begins with a description ofthe physiological stress response in fish. Itis then followed by a discussion of the cel-lular stress response (primarily the hsp70)in fish and how the neuroendocrine and cel-lular stress responses may be related. Weconclude with a brief discussion of the pos-sible physiological roles for hsps in fish.STRESSORS

The potential stressors to fish discussedhere are grouped as being environmental,physical, or biological. They are presentedhere as a selection of examples in each cat-egory and by no means represent a com-plete or exten sive list of published stressors,as reported by Barton and Iwama (1991).Many stressors are unique to certain speciesor geographic areas. Furthermore, the stressresponse in fish has a genetic component(see Pottinger and Pickering, 1997). Thus,there are differences in the generalizedstress response among different fish species,and different stocks or races of the samespecies differ in their tolerance to appliedstressors. The observed stress response istherefore an expression of both genetic andenvironmental factors such as season, rear-ing history, and nutritional state (see Iwamaet al., 1992). Environmental stressors main-ly include adverse physical and chemicalconditions of the water. Extreme conditionsor changes in water quality such as dis-solved oxygen, ammonia, hardness, pH, gascontent, partial pressures, and temperature

can stress fish. Metals (e.g., copper, cad-mium, zinc, and iron) and other contami-nants (e.g., arsenic, chlorine, cyanide, var-ious phenols, and polychlorinated biphe-nyls) in the water can cause severe stressand death in fish. Other potential environ-mental stressors include insecticides, her-bicides, fungicides, and defoliants. Indus-trial, domestic, and agricultural activitiesadd much of these contaminants to the en-vironment that affect fish at all life stages.Natural changes in water quality, as occursduring low tide in tidepools, may stress theorganisms that live in such environments.Pathophysiological stressors encompass awide range of potential stressors includingthose that disturb the fish physically andpsychologically. Handling, crowding, con-finement, transport, or other forms of phys-ical disturbance to fish also have psycho-logical components. Many of these arepracticed in the intensive culture of fish forboth wild stock enhancement, and for thecommercial production of food. Chasingfish to exhaustion, or holding them in a netout of water for 30-60 sec have been com-mon protocols utilized to study acute stressresponses in fish. Angling also stresses fishin this manner. Other psychological stress-ors can be manifest in dominance hierar-chies, which develop between individualswithin confines such as experimental tanksor possibly in natural environments. Path-ogens and parasites can also be consideredas biological stressors. Fish disease, andoutbreaks leading to massive mortalities,occur in nature as well as in cultured stocks.Plankton blooms can stress and kill fish inthe wild as well as in aquaculture facilities.The plankton may kill the fish directly bytheir toxins; irritate or severely damage thegill epithelium with their spines; or kill thefish indirectly by hypoxic conditions by ei-ther lowering water oxygen levels directlyor by increasing the diffusion distance be-tween blood and water through the stimu-lation of mucus production on the gill sur-face.

THE GENERALIZED STRESS RESPONSEIn response to a stressor such as one ofthose mentioned above, fish undergo a se-ries of biochemical and physiological



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HEAT SHOCK PROTEINS AND STRESS IN FISH 9 0 3changes in an attempt to cope with the chal-lenges imposed upon them. The stress re-sponse in fish has been broadly categorizedinto the primary, secondary and tertiary re-sponses (Barton, 1997; Wendelaar Bonga,1997).The primary response represents the per-ception of an altered state and initiates aneuroendocrine/endocrine response thatforms part of the generalized stress re-sponse in fish. This response includes therapid release of stress hormones, catechola-mines and cortisol, into the circulation.Adrenaline is released from the chromaffintissue in the head kidney of teleosts, andalso from the endings of adrenergic nerves(see Randall and Perry, 1992). Cortisol isreleased from the interrenal tissue, locatedin the head kidney, in response to severalpituitary horm ones, but most potently to ad-renocorticotropic hormone (ACTH) (seeBalm et al, 1994). The resting and stressedlevels of adrenaline and cortisol concentra-tions in the plasma of salmonids are: adren-aline,

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9 0 4 G . K . IWAMA ET ALFish cells are no exception to the cellular

stress response and the expression of vari-ous stress proteins has been reported in celllines, primary cultures of cells, as well asin various tissues from whole animals. Thevast majority of studies have focused onvarious effects of heat shock, but recentstudies have shown that hsps levels increasein fish tissues in response to a variety ofenvironmental and biological stressors. Re-search in this area is very much at a de-scriptive phase. Emerging evidence, such asthat of Deane et al. (1999), supports thepossibility that the cellular and neuroendo-crine stress responses are related in the in-tact organism , but an unequivocal causal re-lationship has not been established.The use of cell lines to study hsp ex-pression h as the convenient features of sim-ple maintenance, uniformity, and abun-dance of the cells. This allows for detailedstudies involving many different experi-mental treatments to be conducted quickly,in contrast to studies with whole animals.Such studies have enabled the separation ofvarious isoforms within families or groupsof induced hsps; the description of the timecourse of novel protein induction; and var-

ious studies of the function of hsps in thecell. Comprehensive studies such as thoseon the chinook salmon embryonic cell line(CHSE-214; Heikkila et al, 1982; Gedamuet al, 1983; Misra et al, 1989), as well asthose on the rainbow trout gonadal cell line(RTG-2; Mosser and Bols, 1988; Kotharyand Candido, 1982; Kothary et al,1984a,fc; Mosser et al, 1986; Burgess,1984), and on the rainbow trout hepatomacell line (RTH-149; Misra et al, 1989;Heikkila et al, 1982) have all shown in-creased expression of various hsps (e.g., 28,41 , 46, 51, 65, 68, 70, and 84 kDa) in re-sponse to heat shock and metal exposure.Cho et al. (1997) reported a novel 90 kDaprotein, that was different from hsp90, toincrease in the cell line CHSE-214 in re-sponse to the infectious haematopoietic ne-crosis virus. Slight differences in the mo-lecular masses of individual hsps may wellbe attributable to differences in techniques,such as in the experimental set-up, isolationof tissues, and the molecular weight mark-ers used in each study. Such factors may

also be particularly important for varioustemporal factors such as time taken to showsignificant increases in hsps or maximumincrease in concentration, or the time takento return to control levels. Focusing onclasses of hsps, rather than on those minordifferences in individual proteins, may bemore meaningful in gaining an understand-ing about the effects of specific stressors onhsps in a particular cell line.Most of the above studies report that theincrease in hsp70 is the most prominent re-sponse to heat shock. In contrast to such ageneralized response, Misra et al (1989)found the metallothionein protein to in-crease only in response to metal stress andonly in CHSE. Studies with transcriptionblockers (e.g., actinomycin D) have sug-gested that the transcription step of proteinsynthesis plays a major role in the regula-tion of which hsps are produced in responseto a given stressor (Heikkila et al, 1982;Currie and Tufts, 1997). Airaksinnen et al.(1998) have recently provided further evi-dence from primary cultures of rainbowtrout hepatocytes and gill epithelial cells, aswell as the RTG-2 cell line that the tran-scriptional step is involved in the regulationof hsp70 levels in those fish cells. There isone report of cold shock inducing a 70 kDaprotein in the RTG-2 cell line (Yamashitaet al, 1996). That protein was differentfrom hsp70, but more closely related insome residues to proteins associated withcell division. Yamashita et al. (1996), there-fore, suggested that the response was a met-abolic compensation to the cold-induced re-duction in cell division. There is a patternfor larger hsps (e.g., 60, 70, 90 kDa) to bemore highly conserved between organismsof different taxa. Smaller hsps (e.g., 16-30kDa) may be more species-specific, and assuch may offer reasonable potential for di-agnostic purposes among species, whereasthe larger hsps may serve as indicators ofnon-specific stressors in a wide range of or-ganisms.

A number of studies with fish cells inprimary culture have been valuable in pro-viding information that is perhaps closer tothe in-vivo condition because such cellsmaintain their differentiated characteristics.The work of Brown et al. (1992), Renfro et



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HEAT SHOCK PROTEINS AND STRESS IN FISH 9 05al . (1993), and Sussman-Turner and Renfro(1995) using the primary culture of renalproximal tubule cells of the winter flounder,Pleuronectes americanus, have shown bothmild heat stress (+5C) and zinc to abolishthe negative effects of severe heat shock(+10C) or 0.5 mM 2,4-dichlorophenoxy-acetic acid on sulphate transport across thatepithelium. Since several hsps (28, 68-70,and 90 kDa) were induced with the heatshock, and blockage of protein synthesis bycyclohexamide abolished the protective ef-fect, those hsps may be vital to the transportfunctions of that renal epithelium. The useof primary cultures of hepatocytes from cat-fish, Ictalurus punctatus, (Koban et al,1991), rainbow trout, Oncorhynchus mykiss(M . M. Vijayan, C. Pereira and G. K. Iwa-ma, in preparation), and desert topminnow,Poeciliopsis spp., (White et al, 1994; Nor-ris et al., 1995) have shown the inductionof a number of hsps (e.g., 30kDa, 60kDa,70kDa class, 90kDa class, and lOOkDa) inresponse to heat shock and various environ-mental contaminants. The studies on thetopminnows have shown that the constitu-tive hsc70 was identical among various spe-cies of this genus, while the heat-induciblehsp70 had significant polymorphism amongspecies. They concluded that a high hsc70content and the ability to quickly increasethe synthesis of hsp70 were vital to ther-motolerance in that genus. While primarycell cultures may be closer to the in-vivocondition, relative to cell lines, there aresome discrepancies between results fromprimary culture studies and in-vivo experi-ments. Koban et al. (1991), for example,found that the acclimation temperature ofcatfish hepatocytes did not affect the hspinduction temperature. However, Dietz andSomero (1992) showed in whole goby fish(genus Gillichthys), that the acclimationtemperature did affect the induction tem-perature of a 90 kDa hsp; this showed thatthe hsp induction temperature was notstrictly determined by genetic code, butcould be modified by environmental fac-tors.

There are several studies of hsp expres-sion in intact fish. The extensive range ofspecies, tissues, and stressors, as well as thehsp responses precludes a detailed descrip-

tion here. Studies in a number of fish spe-cies (Koban et al., 1991; Dyer et al, 1991;Dietz and Somero, 1992, 1993; Mazur,1996; Forsyth et al, 1996; Koban et al,1991; Kikuchi et al, 1993; and Vijayan etal, 1991b; 1998) have shown hsp levels ofvarious classes (e.g., small hsps, 60 kDa,70kDa, 90kDa) increased in a wide rangeof tissues in response to stressors such asheat shock, environmental contaminants,and bacterial disease (see Iwama et al,1998). Liver, kidney and gill tissues seemto be sensitive tissues to the hsp response.It is also noteworthy, from the perspectiveof the potential use of this response as abiomarker of environmental quality, thathandling stress does not elicit hsp70 ex-pression (Vijayan et al, \991b). The poten-tial artifact that handling and sampling pro-cedures can induce in stress studies is aconstant and significant problem. Vijayan etal . (1998) showed that the induction ofhsp70 in the liver of rainbow trout exposedto two toxicants occurred at toxicant con-centrations several-fold lower than the LC50value (lethal concentration to 50% of thepopulation in 96h; a standard index of tox-icity). They concluded that changes in hspmay be a sensitive indicator of stressedstates in fish. The study of Forsyth et al.(1996) was the first to document an hsp re-sponse to a fish disease (Renibacterium sal-moninarum in coho salmon, Oncorhynchuskisutch).

THE GENERALIZED STRESS RESPONSE ANDHSPS

The relationship between the cellular andneuroendocrine stress responses in tissuesof intact animals is important in several re-gards. Whether it is the study of the phys-iological and ecological significance of hspexpression in fish in natural environments,or the development of hsp-based probes fordetermining stressed states in fish, elucida-tion of the relationship between the stresshormones and hsps is an important area ofresearch. Most of the evidence about howthe generalized stress response, as dis-cussed, and hsp expression may be relatedcomes from studies on mammals, but someevidence is available for fish.The interaction of hsp70 and hsp90, with



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9 0 6 G . K . IWAMA ET AL.the steroid receptor has been characterizedusing cell line models (see Bohen and Ya-mamoto, 1994). A number of studies onheat stressed and restraint-stressed rats haveshown that various components of a stateof physiological stress can act as stressorsand do elicit hsps. Blake et al. (1990) dem-onstrated hsp gene expression in rats ex-posed to heat shock. Blake et al. (1991) andUdelsman et al. (1993) have shown the ex-pression of hsp70 after 3h to 6h of restraintstress in adrenal cortical tissue and thoracicaorta tissue of rats. The observation that hy-pophysectomized rats did not show the hsp-gene expression in response to restraintstress, and that addition of ACTH to thoserats induced hsp70 expression in the adre-nals, (Blake et al, 1991) indeed supportsthe possibility that a functional relationshipbetween hsp expression and the hypothal-amus-pituitary-adrenal axis exists. Udels-man et al. (1994b) have shown an a,-me-diated adrenaline effect on hsp70 expres-sion in aortic tissue of rats. Udelsman et al.(1994a) showed that the glucocorticoiddexamethasone attenuated the induction ofhsp70 mRNA expression in the adrenal, butnot in the aortic tissue of rats undergoingrestraint stress. Thus, such responses can betissue specific.

Until recently, there has been no workrelating stress hormone levels and cellularhsp levels in fish. Vijayan et al. (1997Z?)showed that physical handling, whichcaused an increase in circulating cortisollevels, did not affect liver hsp70 levels, oraffect the (J-naphthoflavone (BNF)-inducedhsp70 increase in rainbow trout. Most re-cently, Deane et al. (1999) also found thatdaily intraperitoneal injections of 4ug/g cor-tisol did not affect either hsp70 mRNA orhsp70 levels in the liver tissue of the silversea bream (Spams sarba). Deane et al.(1999) also observed that similar injectionswith 1 ug/g recombinant bream growth hor-mone, and in another group 6 ug/g ovineprolactin, both caused decreases in both he-patic hsp70 mRNA (42% and 54%, respec-tively) and hsp70 (76% and 64%, respec-tively) of the sea bream. However, the po-tential state of physiological stress in theirfish, due to the daily handling, was not as-sessed (e.g., through the measurement of

plasma cortisol levels in control and treat-ment fish). In contrast to the above findings,cortisol significantly decreased the hsp 70induction associated with temperature orBNF exposure in primary cultures of trouthepatocytes (M. M . Vijayan, C. Pereira andG. K. Iwama, in preparation). It is theoret-ically possible that higher levels of circu-lating cortisol could inhibit hsp70 synthesisby binding with cortisol receptors in the cy-tosol. Glucocorticoid receptor complexescontain hsp70 which would be releasedupon binding with cortisol (see Pratt, 1993).Increased levels of free hsp70 as a resultmay inhibit the likelihood of HSF1 trimer-ization and subsequent hsp70 expressionthrough: HSF1 binding to the promoter re-gion of the hsp70 genes; stressor-inducedactivation of HSF1 through phosphoryla-tion; and hsp70 transcription (see M orimotoet al., 1996). The relative quantities of cel-lular glucocorticoid receptor com plexes rel-ative to the total hsp70 pool, would likelyvary from tissue to tissue, and among dif-ferent species. This theoretical speculation,however, has no supporting experimentalevidence, and warrants further research.

We are clearly at the very early stages ofunderstanding what seem to be complex in-teractions among stressors, stress hor-mones, and hsp expression in fish. Eluci-dation of the various signal pathways thatregulate hsp production and degradationwill contribute significantly in this regard.

PHYSIOLOGICAL ROLE OF HSPS IN FISHSome of the vital roles that hsps play inthe cell include the maintenance of proteinintegrity, preventing premature folding andaggregation of proteins, protein transloca-tion, and mediating steroid and receptorbinding (see Chapter 1 of this volume). Al-

though these intracellular roles have beenclearly shown for the cell, the body ofknowledge concerning the physiologicaland ecological importance of hsps to thesurvival of fish and other animals is rela-tively small. There are data showing the in-crease in hsps in various tissues of manydifferent fish species subjected to stressors.However, few studies clearly show thephysiological significance of changes in hspconcentration in various cells. The imposed



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HEAT SHOCK PROTEINS AND STRESS IN FISH 907degree of the stressor, in many cases, is be-yond that which the animal would encoun-ter in nature. Most of the data from studieswhere the animals are exposed to stressorswithin an ecologically normal range, arecorrelative. Thus, one can only imply fromsuch data that the increase in hsps must beplaying som e role in enhancing the survivalof the stressed fish. While most of thesestudies have involved temperature as astressor, many studies show that stressorsother than heat shock can increase hsp con-centration. Several studies show that hsp in-duction can increase tolerance to subse-quent stressors. A good example is thework showing that hsp28, hsp70, and hsp90induction in the renal epithelium (in pri-mary culture) of the white flounder protectsthe cells against the damaging effects of se-vere heat and 2,4-dichlorophenoxyaceticacid on sulphate transport (Brown et al.,1992; Renfro et al, 1993; Sussman-Turnerand Renfro, 1995).

Stress-induced increases in hsps in fishmay occur in a threshold manner, ratherthan in a graded way dependent on the de-gree of the stressor. Evidence for this comesfrom studies using the primary culture ofhepatocytes from salmonids exposed to var-ious environmental contaminants (Vijayanet al., 1998), as well as from the study ofCurrie and Tufts (1997) on rainbow trouterythrocytes exposed to heat shock in to-nometers.Stress-induced increases in hsps in fishmay occur in a threshold manner, ratherthan in a graded way dependent on the de-gree of the stressor. Evidence for this comesfrom studies using the primary culture ofhepatocytes from salmonids exposed to var-ious environmental contaminants (Vijayanet al., 1998), as well as from the study ofCurrie and Tufts (1997) on rainbow trouterythrocytes exposed to heat shock in to-nometers. However, those studies were notdesigned to specifically investigate that re-sponse.

The studies on the desert topminnows byWhite et al. (1994) and Norris et al. (1995)suggest that the combination of high hsc70and the ability to quickly induce hsp70 ex-pression are vital to the tolerance of hightemperatures in the studied species of that

genus. Although there are many studiesshowing a correlation between the induc-tion temperatures for hsps and various as-pects of thermal tolerance or thermal pref-erence in fish, none of them unequivocallyshow that hsps cause the observed ther-motolerance or influence geographic distri-bution.

CONCLUDING REMARKSLike all other organisms, fish face thechallenges of maintaining a constant "mi-lieu interieur" in the face of environmentalchange, at all levels of biological organi-zation. The generalized stress response atthe cellular level as well as at the wholeanimal level share some common proper-ties, and comprise the attempt to maintainand reestablish homeostasis in the face ofany stressor. A wide range of abiotic andbiological stressors elicits these responses.They both have genetic components but aremodified by environmental factors. Currentevidence points to a complex relationshipbetween the cellular and neuroendocrinestress responses in fish, as is the case formammals. While there are similarities be-tween the cellular stress responses of fish

and other vertebrates, there are notable dif-ferences that have to be reconciled. Whydoes restraint stress increase hsp levels inmice, but handling stress does not cause achange in fish tissues? Clearly, further re-search is needed to study the nature of therelationship between these two stress re-sponses as well as between vertebrategroups.There are many possible applications ofmeasuring the stress response in fishes, andother aquatic organisms. They range frombeing able to resolve the generalized stressresponse in our experimental animals, sep-arate from treatment-specific effects, to themonitoring of the quality of the aquatic en-vironment through the stressed states of theorganisms that live there. However, theseapplications can only be developed if thereis unequivocal evidence for a relationshipbetween the stressed state of the animal andthe cellular stress response. The fact thatstressors cause the induction of specificproteins offers the possibility of developingdiagnostic probes for monitoring the con-



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908 G. K. IWAMA ETAL.dition of fish and their environment. Theevidence showing that increased levels ofhsps induce tolerance of cells, tissues, andwhole fish to subsequent stressors suggeststhat it may be possible to develop strategiesto enhance tolerance to stressors by induc-ing the cellular stress response.

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sitive, age-dependent response. Proc. Natn. Acad.Sci. U.S.A. 88:9873-9877.Blake, M. J., T. S. Nowak, and N. J. Holbrook. 1990.In vivo hyperthermia induces expression ofHSP70 mRNA in brain regions controlling theneuroendocrine response to stress. Mol. BrainRes. 8:89-92.Bohen, S. P. and K. R. Yamamoto. 1994. Modulationof steroid receptor signal transduction by heatshock proteins. In R. I. Morimoto, A. Tissieres,and C. Georgopoulos, (eds.), The biology of heatshock proteins and molecular chaperones, ColdSpring Harbor Monograph Series, Vol. 26, pp.313-334. Cold Spring Harbor Laboratory Press,Plainview, N.Y.Brown, M. A., R. P. Upender, L. E. Hightower, and J.L. Renfro. 1992. Thermoprotection of a functionalepithelium: heat stress effects on transepithelialtransport by flounder renal tubule in primarymonolayer culture. Proc. Natl. Acad. Sci. U.S.A.89:3246-3250.Cho, W., S. Cha, J. Do, J. Choi, J. Lee, C. Jeong, K.Cho, W. Choi, H. Kang, H. Kim, and J. Park.1997. A novel 90-kDa stress protein induced infish cells by fish rhabdovirus infection. Biochem.Biophys. Res. Coram. 233:316-319.Currie, S. and B. L. Tufts. 1997. Synthesis of stressprotein 70 (Hsp70) in rainbow trout (Oncorhyn-chus mykiss) red blood cells. J. Exp. Biol. 200:607-614.

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