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Detection and identification of heat-shock proteins
WS2-B1-1-04
DETECTION AND IDENTIFICATION OF HEAT-SHOCK PROTEINS K. Ohtsuka Laboratory of Experimental Radiology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan In mammalian cells, fourteen heat-shock (stress) proteins (hsps) and related proteins are identified so far. Each hsp is named based on its molecular weight. For example, 70-kDa hsp is called hsp70. Usually hsps are detected by one-dimensional (1D) SDS-PAGE and/or two-dimensional (2D) gel electrophoresis. Cultured cells are heat-shocked or treated with chemical stresses such as ethanol, sodium arsenite and amino acid analogs, and labeled with [~S]methionine or [3H]leucine (low molecular weight hsp28 is detected by labeling cells with [3H]leucine but riot with pSS]methionine, because hsp28 does not contain methionine in its moiety). Then the cell lysate and non-treated control cell lysate are analyzed by gel electrophoresis followed by autoradiography. Classical major hsps such as hsp70, hsp90 and hsp110 can be easily detected by 1D SDS-PAGE. However, other hsps are able to be detected only by 2D gel analysis. Since majority of hsps are acidic proteins, they are detected by isoelectric focusing (IEF)/SDS-PAGE 2D gel analysis. Also, the extent of induction level of each hsp is dependent on cell types, heating conditions and stressors used. In order to identify basic hsps, we performed non-equilibrium pH gradient gel electrophoresis (NEPHGE)/ SDS-PAGE 2D gel analysis. Consequently, we found a novel 40-kDa heat-shock protein hsp40 in mammalian and avian cells. We purified human hsp40 by modified 2D gel (Slab-NEPHGE/SDS-PAGE), determined partial amino acid sequence of its N terminal and raised antibody against hsp40. Finally we isolated a cDNA clone from human placenta expression library by immunoscreening with anti-hsp40 antibody and revealed that hsp40 is a mammalian homologue of bacterial heat -shock protein DnaJ. When antibody specific for each hsp are available, detection and estimation of relative amount of the hsp can be obtained by 1 D SDS-PAGE followed by immunoblotting (western blot) and densitometry of the blot. We examined the relationship between thermotolerance and heat-shock proteins. Thermotolerance was assayed by clonogenic survival and the relative amount of hsp70 and hsp40 was measured by western blot with antibodies specific for hsp70 or hsp40. Thus we showed that not only hsp70 but also hsp40 is well correlated with the extent of thermotolerance. Also, we demonstrated the colocalization of hsp70 with hsp40 in heat-shocked cells and direct interaction between hsp70 and hsp40 as shown by immunoprecipitation. These results suggested that both hsp70 and hsp40 are involved in the development of thermotolerance.
WS2-BI-I-05
INTERACTION BETWEEN HEAT SHOCK PROTEINS (HSPs) AND INTERLEUKIN-I DURING FEVER M.KOSAKA, J.M.LEE, T.MATSUMOTO*, K.TSUCHIYA, N.OHWATARI AND M.SHIMAZU Department o f Environmental Physiology and Thermal Adaptation*, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852, Japan
Institute of Tropical Medicine,
INTRODUCTION: The s e c r e t i o n o f IL-1 i s an i n i t i a l r e s p o n s e t h a t i n t e r v e n e s and p r o m o t e s many other immune responses (Duff and Durum, 1982). The reaction as follows is important during bacterial infection: LPS--~macrophage-~IL-]-*PGE2-->fever. One might wonder why even extremely high fevers seldom exceed 4 1 ~ or 42°C. It is postulated that there is a self-limiting mechanism, such as a negative feedback loop. However, the existence of a negative feedback loop has not yet been proven. It would thus be quite important to investigate whether or not fever is responsible for this role. On the other hand, it is well-known that the heat shock protein (hsp 70) can be induced by heat stress (Hatayama et al., 1985, 1986; Otsuka and Kano, 1986). Many reports have discussed about the relationship between heat tolerance and hsp 70 including our previous reports (Landry, 1982; Lee et al., 1990, 1991). Whether hsp 70 relates to a febrile response or not however, has been seldom discussed (Ciavarra and Simeone, 1990). The present study, therefore, investigated influences of heat shock on macrophage proliferation and 11-1~ secretion, studied the induction of hsp 70 in the macrophage, and examined the correlation between them. METHODS: Effects of heat, IL-I B and LPS on proliferation of macrophage were measured with [JH]t-~ne uptake. Hsp70 induction in RAW 246.7 cells (mouse macrophage cell line) by heat, IL-I~ and LPS was examined by Western- and Northern- blot analysis. Hsp70 induction in U-937 cells (human monocytelike histiocytic lymphoma) by heat was examined by Western blot analysis. RESULTS: ]. The proliferation of macrophage was suppressed by a heat load of 39~C for 2 hours. T1s~ppression was also observed upon treatment with high concentration of LPS, but not observed upon IL-I ~ treatment. 2. The induction of the hsp70 family by heat shock in RAW cells and U-937 cells were identified. 3. The induction of the hsp70 family and the increase of hsp70 mRNA transcription by IL-I~ and LPS in RAW ceils were identified. DISUCUSSION: The results of this experiment actually showed suppression of macrophage proliferation subjected to mild heat load such as 39°C for 2 hours. It is assumed that LPSinduced fever may act as a negative feedback control on macrophage's activation. The correlation between hsp and immunity has been reported in many studies. Therefore, in the present study, we confirmed that IL-] B and LPS may induce hsp70 family and positively regulate hsp70 mRNA expression. It suggests that hsp may play an important role correlated to IL-I B function. In addition to the present results, the hsp70 in the pika rabbit was demonstrated and discussed from the view point of thermal acclimatization.
138
DETECTION AND IDENTIFICATION OF HEAT-SHOCK PROTEINS K. Ohtsuka Laboratory of Experimental Radiology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan In mammalian cells, fourteen heat-shock (stress) proteins (hsps) and related proteins are identified so far. Each hsp is named based on its molecular weight. For example, 70-kDa hsp is called hsp70. Usually hsps are detected by one-dimensional (1D) SDS-PAGE and/or two-dimensional (2D) gel electrophoresis. Cultured cells are heat-shocked or treated with chemical stresses such as ethanol, sodium arsenite and amino acid analogs, and labeled with [~S]methionine or [3H]leucine (low molecular weight hsp28 is detected by labeling cells with [3H]leucine but riot with pSS]methionine, because hsp28 does not contain methionine in its moiety). Then the cell lysate and non-treated control cell lysate are analyzed by gel electrophoresis followed by autoradiography. Classical major hsps such as hsp70, hsp90 and hsp110 can be easily detected by 1D SDS-PAGE. However, other hsps are able to be detected only by 2D gel analysis. Since majority of hsps are acidic proteins, they are detected by isoelectric focusing (IEF)/SDS-PAGE 2D gel analysis. Also, the extent of induction level of each hsp is dependent on cell types, heating conditions and stressors used. In order to identify basic hsps, we performed non-equilibrium pH gradient gel electrophoresis (NEPHGE)/ SDS-PAGE 2D gel analysis. Consequently, we found a novel 40-kDa heat-shock protein hsp40 in mammalian and avian cells. We purified human hsp40 by modified 2D gel (Slab-NEPHGE/SDS-PAGE), determined partial amino acid sequence of its N terminal and raised antibody against hsp40. Finally we isolated a cDNA clone from human placenta expression library by immunoscreening with anti-hsp40 antibody and revealed that hsp40 is a mammalian homologue of bacterial heat -shock protein DnaJ. When antibody specific for each hsp are available, detection and estimation of relative amount of the hsp can be obtained by 1 D SDS-PAGE followed by immunoblotting (western blot) and densitometry of the blot. We examined the relationship between thermotolerance and heat-shock proteins. Thermotolerance was assayed by clonogenic survival and the relative amount of hsp70 and hsp40 was measured by western blot with antibodies specific for hsp70 or hsp40. Thus we showed that not only hsp70 but also hsp40 is well correlated with the extent of thermotolerance. Also, we demonstrated the colocalization of hsp70 with hsp40 in heat-shocked cells and direct interaction between hsp70 and hsp40 as shown by immunoprecipitation. These results suggested that both hsp70 and hsp40 are involved in the development of thermotolerance.
WS2-BI-I-05
INTERACTION BETWEEN HEAT SHOCK PROTEINS (HSPs) AND INTERLEUKIN-I DURING FEVER M.KOSAKA, J.M.LEE, T.MATSUMOTO*, K.TSUCHIYA, N.OHWATARI AND M.SHIMAZU Department o f Environmental Physiology and Thermal Adaptation*, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852, Japan
Institute of Tropical Medicine,
INTRODUCTION: The s e c r e t i o n o f IL-1 i s an i n i t i a l r e s p o n s e t h a t i n t e r v e n e s and p r o m o t e s many other immune responses (Duff and Durum, 1982). The reaction as follows is important during bacterial infection: LPS--~macrophage-~IL-]-*PGE2-->fever. One might wonder why even extremely high fevers seldom exceed 4 1 ~ or 42°C. It is postulated that there is a self-limiting mechanism, such as a negative feedback loop. However, the existence of a negative feedback loop has not yet been proven. It would thus be quite important to investigate whether or not fever is responsible for this role. On the other hand, it is well-known that the heat shock protein (hsp 70) can be induced by heat stress (Hatayama et al., 1985, 1986; Otsuka and Kano, 1986). Many reports have discussed about the relationship between heat tolerance and hsp 70 including our previous reports (Landry, 1982; Lee et al., 1990, 1991). Whether hsp 70 relates to a febrile response or not however, has been seldom discussed (Ciavarra and Simeone, 1990). The present study, therefore, investigated influences of heat shock on macrophage proliferation and 11-1~ secretion, studied the induction of hsp 70 in the macrophage, and examined the correlation between them. METHODS: Effects of heat, IL-I B and LPS on proliferation of macrophage were measured with [JH]t-~ne uptake. Hsp70 induction in RAW 246.7 cells (mouse macrophage cell line) by heat, IL-I~ and LPS was examined by Western- and Northern- blot analysis. Hsp70 induction in U-937 cells (human monocytelike histiocytic lymphoma) by heat was examined by Western blot analysis. RESULTS: ]. The proliferation of macrophage was suppressed by a heat load of 39~C for 2 hours. T1s~ppression was also observed upon treatment with high concentration of LPS, but not observed upon IL-I ~ treatment. 2. The induction of the hsp70 family by heat shock in RAW cells and U-937 cells were identified. 3. The induction of the hsp70 family and the increase of hsp70 mRNA transcription by IL-I~ and LPS in RAW ceils were identified. DISUCUSSION: The results of this experiment actually showed suppression of macrophage proliferation subjected to mild heat load such as 39°C for 2 hours. It is assumed that LPSinduced fever may act as a negative feedback control on macrophage's activation. The correlation between hsp and immunity has been reported in many studies. Therefore, in the present study, we confirmed that IL-] B and LPS may induce hsp70 family and positively regulate hsp70 mRNA expression. It suggests that hsp may play an important role correlated to IL-I B function. In addition to the present results, the hsp70 in the pika rabbit was demonstrated and discussed from the view point of thermal acclimatization.
138