A physiologically relevant increase in body temperature from 39.7 to 42.50C, which was generated after the intravenous injection of D-lysergic acid diethylamide (LSD), caused the induction of synthesis of a 74,000-dalton heat shock protein in the brain, heart, and kidney of the young adult rabbit. A marked increase in the relative labeling of a 74,000-dalton protein was noted after analysis of both in vivo labeled proteins and cell-free translation products of isolated polysomes. A temporal decrease in the synthesis of this protein was noted as LSD-induced hyperthermia subsided. The 74,000-dalton protein, which is induced in various organs of the intact animal at a body temperature similar to that attained during fever reactions, may play a role in homeostatic control mechanisms.Increase of ambient temperature has been reported to induce the synthesis of a specific set of heat shock proteins in a wide range oftissue culture systems and unicellular organisms (1-9). The exact number and molecular weight ofthe induced proteins is dependent on the particular system under investigation; however, the induction of several proteins in the molecular mass range of 68,000-74,000 daltons and 95,000 daltons is common to most systems (1,3,5).Understanding the functional role that these proteins may play in mammalian cells is particularly important given the suggestion raised by Kelley and Schlesinger (3) that these proteins may be involved in the response of the body to fever reactions. An intriguing question that requires examination is whether heat shock proteins are induced within organs of the intact mammal during physiologically relevant increases in body temperature. In this report we demonstrate that an increase in body temperature similar to that attained during fever reactions induces the synthesis ofa 74-kilodalton (kDal) protein in the brain, heart, and kidney of the young adult rabbit. 0.20C prior to treatment were selected for experimentation. Animals that were injected with LSD had rectal temperatures of 42.5 ± 0.30C 1 hr after drug administration. It has been shown (10) that rectal temperature in rabbits is an accurate representation of the body temperature of organs and that rectal temperature and organ temperature increase in parallel during hyperthermia.
MATERIALS AND METHODSIn Vivo Labeling of Proteins. Proteins in the cerebral hemisphere of the brain were labeled by injection of 0.5 mCi (1 Ci = 3.7 x 10'°becquerels) of [3S]methionine (New England Nuclear) 20 min after the drug injection. The isotope was administered into the lateral ventricles of the brain through stereotaxically implanted stainless steel cannulae. The animals were pulsed for 1 hr and then dispatched by cervical dislocation. The cerebral hemispheres were dissected out and homogenized in 2 vol of 0.32 M sucrose/50 mM Hepes/KOH, pH 7.5/140 mM potassium acetate/5 mM magnesium acetate/2.5 mM dithiothreitol. The homogenate was centrifuged at 10,000 rpm for 10 min in a Sorvall SS34 rotor at 40C to obtain a postmitochondrial supernatant....
A cell-free protein synthesis system, derived from brains of 3 mo-old male Fischer-344 rats, has been characterized. The optimum conditions for amino acid incorporation in the system were 5 mM magnesium ion and 200 mM potassium ion. Incorporation depended on the addition of ATP, GTP, and an energy-generating system, and was sensitive to addition of the drugs aurintricarboxylic acid and sodium fluoride, inhibitors of initiation of protein synthesis. Both 40S and 80S initiation complexes were labeled in vitro, using [35S]methionine. Such labeling was sensitive to the protein synthesis inhibitors, aurintricarboxylic acid and sodium fluoride. The system, which can initiate protein synthesis, should be of use for examining mechanisms which underlie alterations in rat brain protein synthesis induced by various treatments.
Free and membrane-bound polysomes and polyadenylated mRNA isolated from rabbit brain were translated in an mRNA-dependent rabbit reticulocyte lysate system. Electrophoretic analysis of the cell-free translation products demonstrated that although most of the nascent proteins were common to both free and membrane-bound brain polysomes, qualitative and quantitative differences were observed. Compared with the results obtained with purified polyadenylated mRNA, the addition of intact polysomes to the cell-free translation assay was more efficient and produced higher molecular weight products. Analysis of the translation products of free and membrane-bound polysomes revealed the appearance of 74K protein following neither LSD administration or hyperthermia induced by elevated temperature treatment. The presence of this 74K protein was verified by analysis of the translation products by two-dimensional gel electrophoresis.
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