Showing papers on "Heat shock protein published in 1983"

Heat shock proteins, first major products of zygotic gene activity in mouse embryo

Abstract: In many species, the early post-fertilization development of the egg appears to occur mainly under maternal control and does not require transcription of the embryonic genome. In the mouse this situation is restricted to the one-cell stage; activation of the embryonic genome occurs at the late two-cell stage and results in a drastic change in the spectrum of proteins synthesized. This activation is preceded by a decrease in the overall synthesis of proteins at the end of the one-cell stage and the appearance, at the early two-cell stage, of a set of new polypeptides of molecular weight approximately 70,000 (70K) (refs 2, 8, 9). This can be compared with the series of events that occur after hyperthermia in differentiated cells. Heat shock results in an arrest of most transcription and translation; subsequently, expression of a limited set of genes, the heat shock genes, precedes the overall reactivation of cellular genome. Here we show that the 70K early two-cell-specific proteins are identical to two of the mouse heat shock proteins, HSP 68 and HSP 70.

AppppA, heat-shock stress, and cell oxidation

Abstract: Salmonella typhimurium LT2 induces a set of heat-shock proteins analogous to those found previously in Escherichia coli. These are virtually the only proteins synthesized after a temperature shift from 28 degrees C to 50 degrees C. Using a two-dimensional thin-layer chromatographic system developed to resolve adenylylated nucleotides, we have found that S. typhimurium and E. coli accumulate P1,P4-diadenosine-5'-tetraphosphate (AppppA), P1-(adenosine-5')-P3-(guanosine-3'-diphosphate-5')-triphosphate (ApppGpp), P1-(adenosine-5')-P4-(guanosine-5')-tetraphosphate (AppppG), P1-(adenosine-5')-P3-(guanosine-5')-triphosphate (ApppG), and P1,P3-diadenosine-5'-triphosphate (ApppA) after heat shock. These same adenylylated nucleotides accumulate after exposure to ethanol, an agent also known to induce the heat-shock response in a variety of cells. AppppA, ApppGpp, AppppG, ApppG, and ApppA were previously shown to accumulate under conditions of oxidation stress. We proposed that these adenylylated nucleotides may be alarmones--i.e., regulatory molecules, alerting cells to the onset of oxidation stress. The finding that these dinucleotides accumulate in response to heat shock suggests that oxidation and heat shock have a common physiological effect on cells. We hypothesize that these dinucleotides signal the onset of these stresses and trigger the "heat-shock response."

Induction of thermotolerance and enhanced heat shock protein synthesis in Chinese hamster fibroblasts by sodium arsenite and by ethanol.

Abstract: Synthesis of a family of proteins called “heat shock” proteins is enhanced in cells in response to a wide variety of environmental stresses. This suggests that these proteins may have functions essential to cell survival under stressful conditions. A causative relationship between heat shock protein synthesis and development of thermotolerance would imply that agents known to induce heat shock protein synthesis, such as sodium arsenite, also induce thermotolerance. Conversely, agents known to induce thermotolerance, such as ethanol, would also enhance heat shock protein synthesis. To test this hypothesis, I have examined the effect of sodium arsenite or ethanol treatment on protein synthesis and cell survival in Chinese hamster ovary HA-1 cells. After either sodium arsenite or ethanol treatment, the synthesis of heat shock proteins was greatly enhanced over that of untreated cells. In parallel, cell survival was increased as much as 104-fold when cells exposed to either agent were challenged by a subsequent heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis of 70,000 and 87,000 molecular weight proteins most closely mirrored the development of thermotolerance. The results, therefore, strongly reinforce the hypothesis that a causal relationship exists between the enhanced synthesis of heat shock protein and cell survival under specific stresses.

Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection.

Abstract: A cDNA copy of the major human heat shock mRNA was cloned. The clone is complementary to the mRNA encoding the major 70-kilodalton heat shock protein as shown by hybrid arrest translation. The authors utilized the cloned DNA to measure induction of the gene during adenovirus infection. The mRNA increases in abundance approximately 100-fold during a wild-type adenovirus infection but does not increase more than 2-fold during an infection in which there is no E1A gene function (high multiplicity of infection of an E1A (-) mutant). Futhermore, by measuring transcription in isolated nuclei, the authors found that the induction was transcriptional and was mediated by the E1A gene product. The induction was not maintained, however. After a peak level was obtained, transcription returned to preinfection levels. This decline was also reflected in the cytoplasmic mRNA abundance indicating a rapid turnover of the heat shock mRNA. This rapid turnover of the heat shock mRNA appears to be induced by the viral infection since the heat shock mRNA was found to be stable when synthesized in an adenovirus-transformed cell line.

Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.

Abstract: Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.

Spontaneous high expression of heat-shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo.

Abstract: When submitted to a heat-shock, mouse embryonal carcinoma (EC) and fibroblast cells show very different behavior. All the EC cells so far analyzed express very high levels of several heat-shock proteins (HSP) in the absence of stress and independent of their origin and culture conditions. In such cells, the 89-kd, 70-kd and 59-kd HSP are the most prominent proteins after actin. In addition, the 89-kd and 59-kd HSP are not stimulated by an arsenite shock in contrast to what is observed with fibroblasts or cells of the parietal yolk sac type. Arsenite induces the synthesis of a 105-kd polypeptide in fibroblasts but not in EC cells. In vitro differentiation of F9 cells induced by retinoic acid and dibutyryl cAMP is accompanied by a decrease in the spontaneous relative abundance of HSP and restores the arsenite-induced synthesis of the 105-kd polypeptide. EC cells are usually believed to be similar to inner cell mass cells of mouse blastocyst. Furthermore, data in the literature together with our own results suggest that the same three HSP are also spontaneously expressed in high amounts in the early mouse embryo.

Is the major Drosophila heat shock protein present in cells that have not been heat shocked

Abstract: When eukaryotic cells are exposed to elevated temperatures they respond by vigorously synthesizing a small group of proteins called the heat shock proteins. An essential element in defining the role of these proteins is determining whether they are unique to a stressed state or are also found in healthy, rapidly growing cells at normal temperatures. To date, there have been conflicting reports concerning the major heat-induced protein of Drosophila cells, HSP 70. We report the development of monoclonal antibodies specific for this protein. These antibodies were used to assay HSP 70 in cells incubated under different culture conditions. The protein was detectable in cells maintained at normal temperatures, but only when immunological techniques were pushed to the limits of their sensitivity. To test for the possibility that these cells contain a reservoir of protein in a cryptic antigenic state (i.e., waiting posttranslational modification for use at high temperature), we treated cells with cycloheximide or actinomycin D immediately before heat shock. HSP 70 was not detected in these cells. Finally, we tested for the presence of a reservoir of inactive messages by using a high stringency hybridization of 32P-labeled cloned gene sequences to electrophoretically separated RNAs. Although HSP 70 mRNA was detectable in rapidly growing cells, it was present at less than 1/1,000th the level achieved after induction.

Heat shock proteins in maize.

Abstract: The pattern of protein synthesis in roots of 3-day-old maize seedlings (Zea mays L.) is rapidly and dramatically altered when the incubation temperature is raised from 25 to 40 degrees C. One-dimensional sodium dodecyl sulfate gels reveal that although synthesis of the proteins observed at 25 degrees C continues at 40 degrees C, a new set of ;heat shock proteins' (hsp) is induced within 20 minutes of the temperature transition. The hsp have molecular weights of 87, 85, 79, 78, 77, 72, 70, 27, 22, and 18 kilodaltons. The 10 hsp are visible on autoradiograms but not on stained gels, suggesting that the proteins do not accumulate to any great extent.The induction of the hsp is transitory. With prolonged high temperature treatment, the synthesis of hsp continues for 4 hours in excised roots and for 8 hours in the roots of intact seedlings before declining sharply. Coincident to the decline in synthesis of the 10 hsp is the gradual increase in intensity of three new polypeptides having molecular weights of 62, 49.5, and 19 kilodaltons. These proteins begin to appear about the time that synthesis of the other 10 hsp becomes maximal.Shifting the temperature back to 25 degrees C also causes a decline in synthesis of hsp, but this decline occurs more rapidly than that seen during prolonged heat shock. A decrease in hsp synthesis becomes apparent 2 hours after the roots are returned to 25 degrees C.Shifting the temperature from 25 to 45 degrees C results in a pattern of protein synthesis different from that observed after a shift to 40 degrees C. Normal protein synthesis continues, except four proteins, which are produced in small amounts at lower temperatures, show greatly enhanced synthesis at 45 degrees C. These proteins have apparent molecular weights of 83, 81, 68, and 65 kilodaltons. Also, the 10 hsp listed above are not synthesized. It is suggested that at least two distinct high-temperature responses are present in maize, which may reflect the metabolic changes generated at different elevated temperatures.

Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells.

Abstract: During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.

Relationship between hyperthermia-induced heat-shock proteins and thermotolerance in Morris hepatoma cells

Abstract: We recently demonstrated that heat treatments, capable of triggering thermotolerance development in MH-7777 cells, also induce a transiently increased synthesis of a small set of polypeptides, a phenomenon similar to the heat-shock-protein (HSP) induction demonstrated in various organisms. A close temporal relationship was also found between HSP synthesis and thermotolerance development, and the degradation kinetics of the newly synthesized HSPs very closely paralleled the decay of the thermotolerant state. In the present study, we extended our analysis to determine whether various experimental conditions known to affect thermotolerance development (duration and temperature of the conditioning treatment, step-down heating, ethanol exposure) will also influence HSP synthesis and, conversely, whether agents known to induce (sodium arsenite) or to impair (cycloheximide) HSP production will similarly affect thermotolerance. The results suggested that the expression of HSP and thermotolerance in MH-7777 cells are most likely regulated by some interrelated mechanisms, but indicated that HSP synthesis is neither a sufficient or necessary condition for thermotolerance development.

Association of an S1 nuclease-sensitive structure with short direct repeats 5' of Drosophila heat shock genes.

Abstract: The potential for a DNA molecule to assume different tertiary structures has been suggested as a possible determinant of transcriptional regulation in eukaryotes1,2. Such forms include cruciforms3,4 transitions from B to Z DNA in the same molecule2,5 and short unpaired stretches of DNA in an alternating copolymer6. All such structures contain regions which are preferentially sensitive to S1 nuclease4–6. We report here an additional class of S1 nuclease sensitive sites associated with short direct repeats of DNA found in the 5′ flanking regions of certain Drosophila melanogaster heat shock genes.

Genetic regulation during heat shock and function of heat-shock proteins: a review

Abstract: The induction by thermal stress of certain specific genes (heat-shock genes) first described in Drosophila has recently been observed in a wide variety of unicellular and multicellular organisms, e...

Yeast thermotolerance does not require protein synthesis.

Abstract: Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.

Characterization of the synthesis and accumulation of a 71-kilodalton protein induced in rat tissues after hyperthermia.

Abstract: Tissues of rats subjected to brief hyperthermic shock were examined by two-dimensional gel electrophoresis for the synthesis and accumulation of a 71-kdalton stress-induced protein (P71). Tissues of 6-week-old rats, killed immediately or 30 min after hyperthermic shock, contained little or no P71. However, in all tissues tested, synthesis and accumulation of P71 was easily detected as early as 2.5 h after hyperthermic shock. The synthesis of P71 was markedly reduced by 1 and 2 days postshock, while the concentration of P71 in all tissues remained high up to 2 days postshock. After 4 days, P71 accumulation was not detected in brain and was reduced in other tissues; at 8 and 16 days after hyperthermic shock, P71 was still detectable but at ever diminishing amounts in heart, lung, liver, spleen, adrenals, and bladder. The subcellular distribution of P71 in brain and liver was determined 2.5 h and 1 and 2 days after hyperthermic shock. The soluble fractions of brain and liver had the greatest enrichment of P71 at each of these times. These results indicate that P71 is a soluble protein which may be present in at least some tissues of unstressed rats and that relatively high concentrations of P71 are found in most tissues after trauma. The increased synthesis of P71 is transient (persisting for less than 24 h after induction), suggesting that P71 is only slowly degraded in these tissues.

Association between the mammalian 110,000-dalton heat-shock protein and nucleoli.

Abstract: A rabbit antiserum has been prepared using as antigen the 110,000-dalton mammalian heat-shock protein. This protein was purified for injection by two-dimensional PAGE of heat-shocked Chinese hamster ovary cells. Characterization by immunoautoradiography and immunoprecipitation reveals that the antiserum is specific for the 110,000-dalton protein. Both techniques also reveal that the protein against which the antiserum is directed is induced by heat shock. Indirect immunofluorescence shows that the antigen is primarily localized at or near the nucleolus in cultured cells and numerous murine tissues. Treatment of cultured cells with deoxyribonuclease destroys the organization of staining within the nucleus while ribonuclease appears to completely release the antigen from the nucleus. A binding of the antiserum to cytoplasmic structures is also observed by immunofluorescence. This association with nucleoli may have implications in the regulatory aspects of the heat-shock response.

ADP-ribosylation of the Mr 83,000 stress-inducible and glucose-regulated protein in avian and mammalian cells: modulation by heat shock and glucose starvation

Abstract: ADP-ribosylation of proteins was analyzed by in vivo labeling of cells with [3H]adenosine, followed by separation of their protein components by two-dimensional isoelectric focusing/NaDodSO4 polyacrylamide gel electrophoresis. We show here that in several cell types of avian and mammalian origin the major [34H]adenosine acceptor in vivo is a polypeptide with a Mr of 83,000 and isoelectric point of approximately equal to 5.3. This polypeptide is identical to one of the stress-inducible and glucose-regulated proteins (here called SP83) previously described in avian and mammalian cells. Snake venom phosphodiesterase digestion of purified 3H-labeled SP83 releases 5'-AMP and a minor fraction of 2'-(5"-phosphoribosyl)-5-AMP. In vitro labeling with [32P]NAD+ of total cell lysates made in the presence of non-ionic detergents also results in incorporation of radioactivity into SP83. Both of these results strongly suggest that the modification is an ADP-ribosylation. Heat shock and glucose starvation of cells induce a rapid and extensive decrease in the incorporation of ADP-ribose into SP83, suggesting that ADP-ribosylation may be important for the regulation of the function of this protein.

Localization of the hsp83 transcript within a 3292 nucleotide sequence from the 63B heat shock locus of D. melanogaster.

Abstract: We have determined the complete nucleotide sequence of a 3292 bp cloned segment derived from the 63B heat shock cytogenetic locus of D. melanogaster. Within this segment we have positioned the start of transcription and RNA splice sites of the unique gene that encodes the 83,000 d heat shock polypeptide (hsp83 gene) by S1 mapping and synthesis of cDNA from restriction fragment primed mRNA. The sequence begins at a point 879 bp upstream from the transcription start and includes the 149 bp nontranslated first exon, the 1139 bp intron and extends 1125 bp into the protein coding region. These data identify a single open translation reading frame for the first 375 amino acids of the 83,000 d polypeptide, beginning with the first ATG codon located at the 3' intron-exon junction. We discuss and demonstrate the use of E. coli exonuclease III generated single-strand DNA probes as an alternative to strand separation for S1 mapping of mRNA. We also use homology search criteria based upon known protein-DNA binding sites to compare our hsp83 sequence with other sequenced Drosophila heat shock genes. These comparisons indicate that a large region of approximately 80 bp centered around the transcription initiation point of the hsp83 gene shares only a 31% homology with the corresponding region of the hsp70 gene, whereas the hsp22, 23, 26, and 27 genes share a 54% homology with hsp70 in this region. The lower homology of the hsp83 gene is consistent with the deviant nature of this heat shock gene.

Heat shock protein in mammalian brain and other organs after a physiologically relevant increase in body temperature induced by D-lysergic acid diethylamide.

Abstract: A physiologically relevant increase in body temperature from 39.7 to 42.5 degrees C, 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.

In vivo phosphorus-31 nuclear magnetic resonance reveals lowered ATP during heat shock of Tetrahymena

Abstract: Cells synthesize a characteristic set of proteins--heat shock proteins--in response to a rapid temperature jump or certain other stress treatments. The technique of phosphorus-31 nuclear magnetic resonance spectroscopy was used to examine in vivo the effects of temperature jump on two species of Tetrahymena that initiate the heat shock response at different temperatures. An immediate 50 percent decrease in cellular adenosine triphosphate was observed when either species was jumped to a temperature that strongly induces synthesis of heat shock proteins. This new adenosine triphosphate concentration was maintained at the heat shock temperature.

Heat-stress proteins and thermal resistance in rat mammary tumor cells.

Abstract: Exposure of clone MTC rat 13672NF mammary adenocarcinoma cells to continuous heating at 42 degrees C or to heating for 20 min at 45 degrees C followed by incubation at 37 degrees C induced or enhanced synthesis of several heat-stress proteins (hsp) detectable by [3H]leucine pulse labeling, gel electrophoresis, and fluorography. Hsp synthesis occurred during development and expression of thermal resistance. For example, continuous heating of MTC cells at 42 degrees C after a rapid temperature transient (about 4 min) produced thermal resistance within 4 to 5 hr of initiation of heating. Hsp synthesis was observed within 2 hr of the cells reaching 42 degrees C and continued throughout 8 hr of heating; four major hsp appeared at nominal molecular weights of 112, 90, 70, and 22 kDa. A slow temperature transient from 37 degrees to 42 degrees C over a 3-hr period increased thermal resistance by a factor of about 2 relative to a rapid transient or "shock" to 42 degrees C. However, hsp synthesis was not significant during the slow heat transient and was either reduced (at 70 and 22 kDa) or not increased (at 112 and 90 kDa) compared with the rates of hsp synthesis at the same time intervals after the rapid transient to 42 degrees C. In these mammary tumor cells, no differences in the number of hsp were detected during heating at 42 degrees or after 45 degrees C heating. Other proteins did not appear to change their relative rates of synthesis, with the exception of a clear decrease in proteins with nominal molecular weights of histones H2A, H2B, H3, and H4. Hsp synthesis was not triggered by cold shock to 23 degrees or 0 degree C, or by radiation of from 2.5 to 10 Gy. Thus hypothermic stress did not enhance hsp synthesis nor were hsp nonspecifically associated with eventual cell lethality. In these experiments, the synthesis of hsp was generally correlated with the development of thermal resistance. Determining the quantity, function, and location of stress proteins may identify targets for thermal killing and resistance. However, one paradoxical observation remains to be resolved. Under the slow temperature transient conditions, the tumor cells attained more thermal resistance with generally reduced rates of hsp synthesis. These results have two possible implications: (a) one or more hsp were not directly related to thermal resistance, or (b) hsp were involved in thermal resistance but their relative rate of synthesis depended on the severity of the heat transient. The latter would imply that the cells made other regulatory or metabolic adjustments and either utilized the hsp more effectively or required less additional hsp.

The possible role of the superoxide ion in the induction of heat-shock and specific proteins in aerobic Drosophila cells during return to normoxia after a period of anaerobiosis.

Abstract: In vitro cultured Drosophila melanogaster cells were shown to be aerobic and several kinetic parameters of their respiration were measured. This allowed us to define experimental conditions for a transient period of anaerobiosis followed by a reexposure to normal oxygenation. This treatment, applied without any change of temperature, induced not only the heat-shock proteins, but also a new specific peptide of 27 000 daltons and a twofold increase of the maximal rate of O2 uptake. This evokes a common molecular mechanism activated either by heat or by O2, which could involve the increase of the products of oxygen reduction such as the superoxide ion.

Cloning and analysis of cDNA sequences coding for two 16 kilodalton heat shock proteins (hsps) in Caenorhabditis elegans: homology with the small hsps of Drosophila.

Abstract: The nucleotide sequences of two different cDNAs, CEHS48 and CEHS41, coding for the 16,000 dalton heat shock proteins (hsps) of Caenorhabditis elegans have been determined. CEHS48 codes for a polypeptide of 135 amino acids, approximately 15 fewer than the complete protein while CEHS41 is missing approximately 46 amino acids. From nucleotide 113 to the TAA termination signal the extent of homology between the sequences is 91%. Toward the 5' ends, the homology drops to 20% and results in completely divergent amino acid sequences. The 3' noncoding regions are only 30% homologous. Only CEHS48 contains a poly(A) signal and a poly(A) tail, suggesting that CEHS41 has an incomplete 3' end. The region from amino acid 43 to amino acid 115 shows extensive homology with corresponding regions in the four small hsps of Drosophila melanogaster and in mammalian alpha-crystallin. Two-dimensional gel analysis of in vitro synthesized hsp16 reveals the existence of five distinct components of identical molecular weights, but with different isoelectric points.