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A Novel 29-kDa Chicken Heat Shock Protein1
MOLECULAR BIOLOGY Research Notes A Novel 29-kDa Chicken Heat Shock Protein1 MIRIAM FRIEDMAN EINAT, ALON HABERFELD, AVI SHAMAY, GUY HOREV, SHMUEL HURWITZ, and SHLOMO YAHAV Institute of Animal Science, Department of Poultry Science, ARO, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel 29-kDa. This protein was induced in broiler chickens' heart muscle and lungs following an in vivo heat stress. The 29-kDa band appears after 3 h of heat stress, much later than the induction of HSP 90, HSP 70, and HSP 27. The late onset of induction suggests that HSP 29 plays a more specific role of a "second stage defense protein".
(Key words: chicken, heat shock proteins, heat stress, metabolic labeling) 1996 Poultry Science 75:1528-1530
INTRODUCTION Heat shock proteins (HSP), also known as stress proteins, are among the most conserved proteins known in phylogeny with respect to both function and structure (reviewed in Lindquist, 1986; Gething and Sambrook, 1992; Jaattela and Wissing, 1992; Caspers et al, 1995). They act as molecular chaperones by binding to other cellular proteins and assisting their folding into the proper secondary structures, thus preventing misfolding and aggregation during stress. The superfamily of HSP includes a number of different molecular weight class families: HSP 110, HSP 90, HSP 70, HSP 60, HSP 47, and a group of small HSP ranging from 16 to 40 kDa in various species (Lindquist and Craig, 1988; White et al, 1994). This evolutionary variable subfamily of proteins is characterized, however, by the presence of a conserved homologous "acrystalline domain" (Ingolia and Craig, 1982) that is present sometimes in duplicate in the protein metabolite. Predictions of that secondary structure and solvent accessibility of this domain, together with hydropathy profiles and intron positions, support the presence of two similar hydrophobic j3-sheet-rich motifs connected by a hydrophilic a-helical region.
Received for publication January 29, 1996. Accepted for publication August 7, 1996. JContribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. Number 1795-E, 1996 series. 2 I. Brown and Sons Ltd., Hod-Hasharon, 45100 Israel. 3 Biological Industries, kibbutz Bet Ha'emek, 25115 Israel. 4 DuPont NEN, Wilmington, DE 19898. 5 Sigma Chemical Co., St. Louis, MO 63178-9916.
We have detected a new member of this small HSP family in heart muscle and lungs of broiler chickens exposed to prolonged high ambient temperature.
MATERIALS AND METHODS Male broiler chickens (Cobb) were obtained from a commercial hatchery. 2 The chicks were raised in battery brooders situated in a temperature-controlled room at 27 C. Water and feed were provided for ad libitum consumption. Feed in mash form was designed according to specifications of the National Research Council (1994). The chickens were exposed to high ambient temperature at the age of 42 d by exposure to 37.2 C and 20 to 30% relative humidity for 4 h. At intervals of 0, 30, 60, 180, and 240 min of high ambient temperature, chickens were killed by neck dislocation and samples of heart muscle and lung were removed into ice-cold methionine-free Dubelcco's Modified Eagle's Medium (DMEM) medium 3 and immediately prepared for labeling as previously described (Shamay et ah, 1987). Briefly, organ samples were minced into pieces of 2 to 4 mg, washed in methioninefree DMEM medium, and placed on impregnated lens paper (about 20 pieces from each explant) in a 50-mm Petri dish. To each dish 80 /*Ci of LPSJmethionine (1,175 Ci/mmol; DuPont, NEN)4 were added in 4 mL of methionine-free DMEM medium. After incubation for 3 h at 37 C, tissues were collected and frozen in liquid nitrogen for further processing. The tissues were homogenized in 2 mL HEPES (N2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) solution containing 0.1 M p H 7.4 and 1 mM phenylmethylsulfonyl fluoride (PMSF),5 diluted with equal volume of
1528
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 22, 2015
ABSTRACT The family of small heat shock proteins is the more variable among the highly conserved superfamily of heat shock proteins (HSP). Using a metabolic labeling procedure with tissue explants, we have detected in chickens a new member of the small HSP family with an a p p a r e n t molecular w e i g h t of
RESEARCH NOTE
1529
B 5
HSP90HSP70-
HSP90HSP70-
6
M
kDa -200 97
-
69
-
46
i-
30
-
21
HSP29HSP27HSP29HSP27
FIGURE 1. Autoradiogram showing induction of heat shock proteins (HSP). Broiler chickens were subjected to high ambient temperature at the age of 42 d. Samples of lung (A), and heart muscle (B), homogenates containing equal amounts of trichloroacetic-acid-precipitable radioactivity were applied to a 10% SDS-PAGE gel. Lanes 1 to 5 represent 0, 30, 60, 180, and 240 min exposure to 37.2 C. Lane 6 represents 48 h recovery from 240 min stress. M = molecular size markers.
lysis buffer (Laemmli, 1970), heat-denatured at 95 C for 5 min and centrifuged at 10,000 x g and 4 C for 10 min. The supernatants were collected and aliquots were stored at -20 C. The radioactivity of the radiolabeled cellular proteins was measured by spotting 5 /xL from each sample onto Nitrocellulose filters6 in duplicate or triplicate. The cellular proteins were precipitated on the filters by 15 min incubation at 4 C in 10% trichloroacetic acid (TCA)5 followed by boiling in 5% TCA for 10 min and washed with ethanol before counting in a beta counter. Differences between duplicates were up to 5%. The cellular proteins were separated by onedimension SDS-PAGE (Laemmli, 1970). The amount of proteins loaded on the gels was based on the radioactivity. An equal amount of radioactivity (3 x 104 cpm) was loaded in each lane. Gels containing radiolabeled proteins were processed for fluorography, dried in a SE1160 gel dryer 7 and exposed for 4 d to Kodak XOMAT AR film8 for autoradiography. Molecular weight markers were [ 14 C]Rainbow markers. 9
6
Schleicher & Schuell GmgH, D-3354 Dassel, Germany. Hoefer Scientific Inst., San Francisco, CA 94197. 8 Kodak, New Haven, CT 06511. 9 Amersham Life Science, Arlington Heights, IL 94197. 7
RESULTS AND DISCUSSION As shown by the electrophoresis pattern (Figure 1), an acute 4-h exposure of broiler chickens to 37.2 C (with 20 to 30% relative humidity) resulted in enhanced synthesis of three major HSP in heart muscle arid lungs. The synthesis rate of HSP 90, HSP 70, and HSP 27 accelerated gradually. Maximal rates of synthesis were detected after 4 h, which was the longest duration of heat exposure tested. At this time point most chickens were dying and body temperatures were 47 C ± 0.5. The synthesis of all HSP was hardly detectable in chickens not exposed to the temperature stress. Among the HSP whose rate of synthesis increased, a previously unknown HSP of about 29 kDa (HSP 29) was detected. Synthesis of the newly identified HSP 29 became apparent only after 3 h of exposure to heat stress. It could be detected in lungs (Figure 1A), in heart muscles (Figure IB) and in peripheral blood leukocytes (not shown). The novel HSP 29 had not been detected previously in peripheral leukocytes of turkeys (Wang and Edens, 1993) and broiler chickens (Edens et al., 1992) in which high ambient temperature had been applied both in vitro and in vivo for up to 120 min. This protein was also not detected in cultured skin fibroblasts of broiler chicken (Einat and Yahav, unpublished data) and in heatstressed cultured chick pineal cells (Wolf and Zats,
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 22, 2015
-
1530
EINAT ET AL.
1994). In these cases, the failure to detect HSP 29 could be explained by genetic variation, tissue specificity, or the relatively longer time period required for its induction. For initial clarification of this question, peripheral blood lymphocytes were prepared from broiler chickens subjected to high ambient temperature for 6 h using the same protocol as Edens et al. (1992) and HSP 29 was detected (not shown). Edens et al. (1992) did not detect HSP 29 in peripheral lymphocytes of heatstressed broiler chickens using a heat-stress challenge at a much higher temperature (43 C) but for a shorter duration (60 min). Therefore, lacking information on cloacal temperature, we cannot determine whether their failure to detect HSP 29 is due to a milder hyperthermia of the broiler chickens or to the small difference in genetic background (Arbor Acres vs Cobb).
The procedure used in the present study has been found effective in detecting heat stress response at the level of protein synthesis. HSP 29 and other proteins that could be found using our protocol may provide markers for genetic breeding towards a more heat-stable strain of chicken.
REFERENCES Arrigo, A. P., and J. Landry, 1994. Expression and function of the low-molecular-weight heat shock proteins. Pages 335-373 in: The Biology of Heat Shock Proteins and Molecular Chaperones. R. I. Morimoto, A. Tissieres, and
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 22, 2015
The difference in induction time of HSP 29 suggests that this protein is regulated by a mechanism different than that involved in the induction of other HSP such as HSP 90 and HSP 70. A relatively late onset of induction was observed previously in chickens also for HSP 23 (Lindquist and Craig, 1988; Edens et al, 1992) and in mammals for other small HSP (reviewed by Arrigo and Landry, 1994). These low molecular weight HSP, therefore, may represent an advanced stage defense mechanism activated only when stress becomes severe. Small HSP of around 30 kDa were reported in some cases such as chicken mononuclear cells (HSP 32, Miller and Qureshi, 1992) and chicken chondrocytes (HSP 31, Neri et al., 1992). It is possible that these proteins and HSP 29 are related and that variability in structure is due to differential posttranslational modifications. Posttranslational modifications of HSP isoforms were shown previously for the human HSP 30 family (Cretien and Landry, 1988). Another possibility is that these proteins differ in their primary structure as was shown previously for the family of the fish HSP 30 family (White et al, 1994).
C. Georgopoulos, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Caspers, G. J., J.A.M. Leunissen, and W. W. De-Jong, 1995. The expanding small heat shock proteins family and structure predictions of the conserved "alpha-crystalline domain". J. Mol. Evol. 40:238-248. Cretien, P., and J. Landry, 1988. Enhanced constitutive expression of the 27-kDa heat shock proteins in heatresistant variants from Chinese hamster cells. J. Cell Physiol. 137:157-166. Edens, F. W., C. H. Hill, and S. Wang, 1992. Heat shock protein response in phosphorus-deficient heat-stressed broiler chickens. Comp. Biochem. Physiol. 106B:827-831. Gething, M. J., and J. Sambrook, 1992. Protein folding in cells. Nature 355:33-45. Ingolia, T. D., and E. A. Craig, 1982. Four small Drosophila heat shock proteins are related to each other and to the mammalian a crystalline. Proc. Natl. Acad. Sci. USA 79: 2360-2364. Jaattela, M., and D. Wissing, 1992. Emerging role of heat shock proteins in biology and medicine. Ann. Med. 24:249-258. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Lindquist, S., 1986. The heat shock response. Ann. Rev. Biochem. 55:1151-1191. Lindquist, S., and E. A. Craig, 1988. The heat shock proteins. Ann. Rev. Genet. 22:631-677. Miller, L., and M. A. Qureshi, 1992. Molecular changes associated with heat shock treatment in avian mononuclear and lymphoid lineage cells. Poultry Sci. 71: 473-481. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Neri, M., F. Descalzi-Cancedda, and R. Cancedda, 1992. Heat shock response in cultured chick embryo chondrocytes. Eur. J. Biochem. 205:569-574. Shamay, A., E. Zeelon, and Z. Ghez, N. Cohen, N. MacKinley, and A. Gertler, 1987. Inhibition of casein and fat synthesis and a lactalbumin secretion by progesterone in explants from bovine lactating mammary gland. J. Endocrinol. 113: 81-88. Wang, S., and F. W. Edens, 1993. Stress-induced heat-shock protein synthesis in peripheral leukocytes of turkeys (Meleagris gallopavo). Comp. Biochem. Physiol. 106B: 621-628. White, C. N., L. E. Hightower, and R. J. Schultz, 1994. Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis). Mol. Biol. Evol. 11: 1106-1119. Wolf, M. S., and M. Zats, 1994. Synthesis of heat shock proteins in cultured chick pineal cells. Brain Res. 662:273-277.
(Key words: chicken, heat shock proteins, heat stress, metabolic labeling) 1996 Poultry Science 75:1528-1530
INTRODUCTION Heat shock proteins (HSP), also known as stress proteins, are among the most conserved proteins known in phylogeny with respect to both function and structure (reviewed in Lindquist, 1986; Gething and Sambrook, 1992; Jaattela and Wissing, 1992; Caspers et al, 1995). They act as molecular chaperones by binding to other cellular proteins and assisting their folding into the proper secondary structures, thus preventing misfolding and aggregation during stress. The superfamily of HSP includes a number of different molecular weight class families: HSP 110, HSP 90, HSP 70, HSP 60, HSP 47, and a group of small HSP ranging from 16 to 40 kDa in various species (Lindquist and Craig, 1988; White et al, 1994). This evolutionary variable subfamily of proteins is characterized, however, by the presence of a conserved homologous "acrystalline domain" (Ingolia and Craig, 1982) that is present sometimes in duplicate in the protein metabolite. Predictions of that secondary structure and solvent accessibility of this domain, together with hydropathy profiles and intron positions, support the presence of two similar hydrophobic j3-sheet-rich motifs connected by a hydrophilic a-helical region.
Received for publication January 29, 1996. Accepted for publication August 7, 1996. JContribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. Number 1795-E, 1996 series. 2 I. Brown and Sons Ltd., Hod-Hasharon, 45100 Israel. 3 Biological Industries, kibbutz Bet Ha'emek, 25115 Israel. 4 DuPont NEN, Wilmington, DE 19898. 5 Sigma Chemical Co., St. Louis, MO 63178-9916.
We have detected a new member of this small HSP family in heart muscle and lungs of broiler chickens exposed to prolonged high ambient temperature.
MATERIALS AND METHODS Male broiler chickens (Cobb) were obtained from a commercial hatchery. 2 The chicks were raised in battery brooders situated in a temperature-controlled room at 27 C. Water and feed were provided for ad libitum consumption. Feed in mash form was designed according to specifications of the National Research Council (1994). The chickens were exposed to high ambient temperature at the age of 42 d by exposure to 37.2 C and 20 to 30% relative humidity for 4 h. At intervals of 0, 30, 60, 180, and 240 min of high ambient temperature, chickens were killed by neck dislocation and samples of heart muscle and lung were removed into ice-cold methionine-free Dubelcco's Modified Eagle's Medium (DMEM) medium 3 and immediately prepared for labeling as previously described (Shamay et ah, 1987). Briefly, organ samples were minced into pieces of 2 to 4 mg, washed in methioninefree DMEM medium, and placed on impregnated lens paper (about 20 pieces from each explant) in a 50-mm Petri dish. To each dish 80 /*Ci of LPSJmethionine (1,175 Ci/mmol; DuPont, NEN)4 were added in 4 mL of methionine-free DMEM medium. After incubation for 3 h at 37 C, tissues were collected and frozen in liquid nitrogen for further processing. The tissues were homogenized in 2 mL HEPES (N2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) solution containing 0.1 M p H 7.4 and 1 mM phenylmethylsulfonyl fluoride (PMSF),5 diluted with equal volume of
1528
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 22, 2015
ABSTRACT The family of small heat shock proteins is the more variable among the highly conserved superfamily of heat shock proteins (HSP). Using a metabolic labeling procedure with tissue explants, we have detected in chickens a new member of the small HSP family with an a p p a r e n t molecular w e i g h t of
RESEARCH NOTE
1529
B 5
HSP90HSP70-
HSP90HSP70-
6
M
kDa -200 97
-
69
-
46
i-
30
-
21
HSP29HSP27HSP29HSP27
FIGURE 1. Autoradiogram showing induction of heat shock proteins (HSP). Broiler chickens were subjected to high ambient temperature at the age of 42 d. Samples of lung (A), and heart muscle (B), homogenates containing equal amounts of trichloroacetic-acid-precipitable radioactivity were applied to a 10% SDS-PAGE gel. Lanes 1 to 5 represent 0, 30, 60, 180, and 240 min exposure to 37.2 C. Lane 6 represents 48 h recovery from 240 min stress. M = molecular size markers.
lysis buffer (Laemmli, 1970), heat-denatured at 95 C for 5 min and centrifuged at 10,000 x g and 4 C for 10 min. The supernatants were collected and aliquots were stored at -20 C. The radioactivity of the radiolabeled cellular proteins was measured by spotting 5 /xL from each sample onto Nitrocellulose filters6 in duplicate or triplicate. The cellular proteins were precipitated on the filters by 15 min incubation at 4 C in 10% trichloroacetic acid (TCA)5 followed by boiling in 5% TCA for 10 min and washed with ethanol before counting in a beta counter. Differences between duplicates were up to 5%. The cellular proteins were separated by onedimension SDS-PAGE (Laemmli, 1970). The amount of proteins loaded on the gels was based on the radioactivity. An equal amount of radioactivity (3 x 104 cpm) was loaded in each lane. Gels containing radiolabeled proteins were processed for fluorography, dried in a SE1160 gel dryer 7 and exposed for 4 d to Kodak XOMAT AR film8 for autoradiography. Molecular weight markers were [ 14 C]Rainbow markers. 9
6
Schleicher & Schuell GmgH, D-3354 Dassel, Germany. Hoefer Scientific Inst., San Francisco, CA 94197. 8 Kodak, New Haven, CT 06511. 9 Amersham Life Science, Arlington Heights, IL 94197. 7
RESULTS AND DISCUSSION As shown by the electrophoresis pattern (Figure 1), an acute 4-h exposure of broiler chickens to 37.2 C (with 20 to 30% relative humidity) resulted in enhanced synthesis of three major HSP in heart muscle arid lungs. The synthesis rate of HSP 90, HSP 70, and HSP 27 accelerated gradually. Maximal rates of synthesis were detected after 4 h, which was the longest duration of heat exposure tested. At this time point most chickens were dying and body temperatures were 47 C ± 0.5. The synthesis of all HSP was hardly detectable in chickens not exposed to the temperature stress. Among the HSP whose rate of synthesis increased, a previously unknown HSP of about 29 kDa (HSP 29) was detected. Synthesis of the newly identified HSP 29 became apparent only after 3 h of exposure to heat stress. It could be detected in lungs (Figure 1A), in heart muscles (Figure IB) and in peripheral blood leukocytes (not shown). The novel HSP 29 had not been detected previously in peripheral leukocytes of turkeys (Wang and Edens, 1993) and broiler chickens (Edens et al., 1992) in which high ambient temperature had been applied both in vitro and in vivo for up to 120 min. This protein was also not detected in cultured skin fibroblasts of broiler chicken (Einat and Yahav, unpublished data) and in heatstressed cultured chick pineal cells (Wolf and Zats,
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 22, 2015
-
1530
EINAT ET AL.
1994). In these cases, the failure to detect HSP 29 could be explained by genetic variation, tissue specificity, or the relatively longer time period required for its induction. For initial clarification of this question, peripheral blood lymphocytes were prepared from broiler chickens subjected to high ambient temperature for 6 h using the same protocol as Edens et al. (1992) and HSP 29 was detected (not shown). Edens et al. (1992) did not detect HSP 29 in peripheral lymphocytes of heatstressed broiler chickens using a heat-stress challenge at a much higher temperature (43 C) but for a shorter duration (60 min). Therefore, lacking information on cloacal temperature, we cannot determine whether their failure to detect HSP 29 is due to a milder hyperthermia of the broiler chickens or to the small difference in genetic background (Arbor Acres vs Cobb).
The procedure used in the present study has been found effective in detecting heat stress response at the level of protein synthesis. HSP 29 and other proteins that could be found using our protocol may provide markers for genetic breeding towards a more heat-stable strain of chicken.
REFERENCES Arrigo, A. P., and J. Landry, 1994. Expression and function of the low-molecular-weight heat shock proteins. Pages 335-373 in: The Biology of Heat Shock Proteins and Molecular Chaperones. R. I. Morimoto, A. Tissieres, and
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 22, 2015
The difference in induction time of HSP 29 suggests that this protein is regulated by a mechanism different than that involved in the induction of other HSP such as HSP 90 and HSP 70. A relatively late onset of induction was observed previously in chickens also for HSP 23 (Lindquist and Craig, 1988; Edens et al, 1992) and in mammals for other small HSP (reviewed by Arrigo and Landry, 1994). These low molecular weight HSP, therefore, may represent an advanced stage defense mechanism activated only when stress becomes severe. Small HSP of around 30 kDa were reported in some cases such as chicken mononuclear cells (HSP 32, Miller and Qureshi, 1992) and chicken chondrocytes (HSP 31, Neri et al., 1992). It is possible that these proteins and HSP 29 are related and that variability in structure is due to differential posttranslational modifications. Posttranslational modifications of HSP isoforms were shown previously for the human HSP 30 family (Cretien and Landry, 1988). Another possibility is that these proteins differ in their primary structure as was shown previously for the family of the fish HSP 30 family (White et al, 1994).
C. Georgopoulos, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Caspers, G. J., J.A.M. Leunissen, and W. W. De-Jong, 1995. The expanding small heat shock proteins family and structure predictions of the conserved "alpha-crystalline domain". J. Mol. Evol. 40:238-248. Cretien, P., and J. Landry, 1988. Enhanced constitutive expression of the 27-kDa heat shock proteins in heatresistant variants from Chinese hamster cells. J. Cell Physiol. 137:157-166. Edens, F. W., C. H. Hill, and S. Wang, 1992. Heat shock protein response in phosphorus-deficient heat-stressed broiler chickens. Comp. Biochem. Physiol. 106B:827-831. Gething, M. J., and J. Sambrook, 1992. Protein folding in cells. Nature 355:33-45. Ingolia, T. D., and E. A. Craig, 1982. Four small Drosophila heat shock proteins are related to each other and to the mammalian a crystalline. Proc. Natl. Acad. Sci. USA 79: 2360-2364. Jaattela, M., and D. Wissing, 1992. Emerging role of heat shock proteins in biology and medicine. Ann. Med. 24:249-258. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Lindquist, S., 1986. The heat shock response. Ann. Rev. Biochem. 55:1151-1191. Lindquist, S., and E. A. Craig, 1988. The heat shock proteins. Ann. Rev. Genet. 22:631-677. Miller, L., and M. A. Qureshi, 1992. Molecular changes associated with heat shock treatment in avian mononuclear and lymphoid lineage cells. Poultry Sci. 71: 473-481. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Neri, M., F. Descalzi-Cancedda, and R. Cancedda, 1992. Heat shock response in cultured chick embryo chondrocytes. Eur. J. Biochem. 205:569-574. Shamay, A., E. Zeelon, and Z. Ghez, N. Cohen, N. MacKinley, and A. Gertler, 1987. Inhibition of casein and fat synthesis and a lactalbumin secretion by progesterone in explants from bovine lactating mammary gland. J. Endocrinol. 113: 81-88. Wang, S., and F. W. Edens, 1993. Stress-induced heat-shock protein synthesis in peripheral leukocytes of turkeys (Meleagris gallopavo). Comp. Biochem. Physiol. 106B: 621-628. White, C. N., L. E. Hightower, and R. J. Schultz, 1994. Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis). Mol. Biol. Evol. 11: 1106-1119. Wolf, M. S., and M. Zats, 1994. Synthesis of heat shock proteins in cultured chick pineal cells. Brain Res. 662:273-277.