The effect of heat shock on the growth and mycelial morphology of Streptomyces chrysomallus]

The effect of various conditions of heat shock (1 hour at 35, 38, 40, 42, 45 and 50 degrees C) on the growth and morphological features of Streptomyces chrysomallus, an organism producing actinomycin, was studied. A definite regularity in the mycelium morphological changes at high temperatures was observed. After the shock at 35 and 38 degrees C the biomass volume and morphological features of the streptomycete did not markedly differ from those in the control. The shock at 40 degrees C induced the growth inhibition with decreasing the biomass volume by 50 per cent and appearance of submerged spores. When the shock conditions were more rigid (42, 45 and 50 degrees C) the mycelium growth lacked. It is of interest that the temperature of 42 degrees C induced abundant formation of the spores. With further increasing of the temperature to 45 and 50 degrees C the spore formation was not so abundant. The changes in the growth and development of the streptomycete are discussed in relation to the molecular mechanism of the cell protection from temperature shock.     

Genotoxicity evaluation of heat shock in gold fish (Carassius auratus)

Genotoxicity evaluation of heat shock was carried out in Carassius auratus. The genotoxicity end points studied were nuclear anomalies (micronucleus assay), chromosomal aberrations, DNA damage (comet assay) and cell proliferation. The heat shock temperatures used were 34 degrees C, 36 degrees C and 38 degrees C. The results demonstrated that heat shock causes the induction of micronucleus at all the three temperature studied. Heat shock also inhibited cell proliferation at 38 degrees C and caused aberrations in the metaphase chromosomes at 34 degrees C and 36 degrees C. Comet assay demonstrated single strand DNA damage at all the three temperatures. The results obtained indicate that heat shock is a genotoxicant.     

Heat shock proteins and thermotolerance in a cultured cell line from the Mediterranean fruit fly, Ceratitis capitata.

Heat shock proteins (hsps) were identified in a cell line from the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) exposed to elevated temperatures. Cells produced three hsps (Mr 87,000, 69,000, and 34,000) in response to a temperature shift from 26 degrees C to 37 degrees C (30-60 min) with a concomitant decrease in synthesis of most other cellular proteins. Synthesis of low Mr hsps was not evident. The heat shock response is triggered within 30 min at temperatures from 33 degrees C to 41 degrees C. At temperatures greater than 41 degrees C protein synthesis was shut down. Within 2-3 h after return to 26 degrees C, synthesis of proteins repressed at the higher temperatures resumed production while the major hsps disappear. Heat shock proteins were not produced in the presence of actinomycin D. Evaluations on the role of hsps in conferring thermotolerance to the cells showed an increase in cell viability in heat-shocked cells over non-heat-shocked cells (after 3 and 10 days) when subsequently placed at 45 degrees C for 1 h, a normally lethal temperature. Heat shock alone had little effect on subsequent cell viability or growth at 26 degrees C. These results suggest that hsps produced by these cells may aid in the maintenance of cell integrity and thus play a transitory role in thermotolerance.     

Splicing thermotolerance maintains pre-mRNA transcripts in the splicing pathway during severe heat shock

Thermotolerance, the ability of cells and organisms to withstand severe elevated temperatures after brief exposure to mild elevated temperatures, has been studied in numerous laboratories. Survival thermotolerance is defined as the increase in cell or organism survival at severe elevated temperatures after a pretreatment at mild elevated temperatures. This study examines splicing thermotolerance in Drosophila melanogaster, the ability to splice pre-mRNAs made at the severe temperature (38 degrees C) after a brief pretreatment at a milder temperature (35 degrees C). It is probably one of a number of mechanisms by which cells adapt to heat shock. These experiments demonstrate that pre-mRNAs synthesized at the severe temperatures in splicing thermotolerant cells, although protected in splicing-competent complexes, are not actually processed to mature mRNAs until the cells are returned to their normal temperature. We have also studied the kinetics of acquisition and loss of splicing thermotolerance. As little as 10 min of pretreatment at 35 degrees C was sufficient to provide full splicing thermotolerance to a 30-min severe heat shock of 38 degrees C. Pretreatments of less than 10 min provide partial splicing thermotolerance for a 30-min severe heat shock. Full splicing thermotolerance activity begins to decay about 4 h after the cessation of the 35 degrees C incubation and is completely lost by 8 h after the pretreatment. The kinetics experiments of pre-mRNAs synthesized during the 38 degrees C treatment in splicing thermotolerant cells indicate that one or more splicing thermotolerance factors are synthesized during the 35 degrees C pretreatment which interact with pre-mRNA-containing complexes to keep them in a splicing-competent state. These kinetic experiments also indicate that in cells which are partially splicing thermotolerant, the pre-mRNAs synthesized early during the 38 degrees C incubation are protected, whereas those synthesized late are not. In the absence of splicing thermotolerant factors, the pre-mRNA-containing complexes leave the normal splicing pathway and are allowed to exit to the cytoplasm.     

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.     

Effects of several factors on the heat-shock-induced thermotolerance of Listeria monocytogenes.

The influence of the temperature at which Listeria monocytogenes had been grown (4 or 37 degrees C) on the response to heat shocks of different durations at different temperatures was investigated. For cells grown at 4 degrees C, the effect of storage, prior to and after heat shock, on the induced thermotolerance was also studied. Death kinetics of heat-shocked cells is also discussed. For L. monocytogenes grown at 37 degrees C, the greatest response to heat shock was a fourfold increase in thermotolerance. For L. monocytogenes grown at 4 degrees C, the greatest response to heat shock was a sevenfold increase in thermotolerance. The only survival curves of cells to have shoulders were those for cells that had been heat shocked. A 3% concentration of sodium chloride added to the recovery medium made these shoulders disappear and decreased decimal reduction times. The percentage of cells for which thermotolerance increased after a heat shock was smaller the milder the heat shock and the longer the prior storage.     

Evidence for a role of heat-shock proteins in proliferation after heat treatment of synchronized mouse neuroblastoma cells

A role for heat-shock proteins (HSPs) in proliferation after heat treatment was considered in synchronized mouse neuroblastoma cells. For this purpose enhancement of HSP synthesis after heat treatment was inhibited by actinomycin D and the effect of this on cell cycle progression into mitosis and on cell survival was studied both in thermoresistant G1- and in thermosensitive late S/G2-phase cells. In G1-phase cells expression of basal and heat-induced HSP synthesis was the same as that in late S/G2-phase cells, which suggests that regulation of thermoresistance throughout the cell cycle is not directly linked with HSP synthesis. The synthesis of HSP36, HSP68, and HSP70 was enhanced after a 30-min treatment at 41-43 degrees C. Increase of HSP synthesis after heat shock was partly suppressed by the presence of 0.1 microgram/ml actinomycin D during heat treatment, while 0.2 micrograms/ml prevented enhancement of HSP synthesis completely. Suppression of heat-induced HSP synthesis by actinomycin D had the same concentration dependency in G1- and late S/G2-phase cells. Actinomycin D potentiated induction of mitotic delay by heat treatment (30 min, 42.5 degrees C) but only under conditions where it actually inhibited heat-induced enhancement of HSP synthesis. Heat-induced cell killing was also potentiated by actinomycin D. The potentiating effect of actinomycin D on heat-induced mitotic delay and on heat-induced cell killing was more pronounced in G1-phase cells than in late S/G2-phase cells. These results give evidence for a role of HSPs in the resumption of proliferation after heat treatment and suggest that heated G1-phase cells are more dependent on HSP synthesis for recovery of proliferation after heat treatment than heated late S/G2-phase cells.     

Changes of sensitivity to heat shock during logarithmic growth of Bacillus subtilis

The effect of heat shock on B. subtilis was found to vary within the logarithmic growth phase. Depending on the age of the culture, all cells, or as little as less than 1% of the population, may survive heating for 6 min at 54 degrees C. These characteristic changes in sensitivity to heat shock were observed with B. subtilis grown on various media, as well as with E. coli. The increased sensitivity of B. subtilis to heat shock was observed within a rather narrow time span in the log phase. Preheating at 45 degrees C had a protective effect on the samples collected at the time of greatest heat sensitivity. It is suggested that besides heat shock proteins other factors are also involved in the processes leading to survival after heat shock.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 degrees C) and low (20 degrees C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T(m) (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 degrees C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T(m) was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 degrees C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T(m) by 10.5 degrees C. In mineral media at 20 degrees C the corresponding changes of T(m) were almost negligible. After a temperature shift from 40 to 20 degrees C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.     

HeLa cells synthesize a specific heat shock protein upon exposure to heat shock at 42 degrees C but not at 45 degrees C

Upon exposure to heat shock, HeLa cells synthesize a small set of proteins having the molecular weights of 70,000, 73,000, 78,000, 85,000, 92,000, and 105,000. In addition to these proteins, we found an unusual heat shock protein induced by heat shock at 42 degrees C, but not at 45 degrees C. The 42 degrees C-specific protein, the molecular weight of which was 90,000, was not produced in control cells and the induction of the protein was completely inhibited by actinomycin D. The protein was not induced by other treatments that induced most heat shock proteins. Thus, this 42 degrees C-specific protein seems to have a peculiar induction mechanism and a specific function in the cells.     

Inactivation of splicing factors in HeLa cells subjected to heat shock

The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.     

Suppression of S-adenosylmethionine decarboxylase activity is a major cause for high-temperature inhibition of pollen germination and tube growth in tomato (Lycopersicon esculentum Mill.)

Possible involvement of impaired polyamine biosynthesis in the poor performance of tomato pollen (Lycopersicon esculentum Mill.) at high temperatures was investigated. Incubation of pollen at 38 degrees C suppressed the increase of S-adenosylmethionine decarboxylase (SAMDC) activity in germinating pollen with little influence on arginine decarboxylase activity. Consequently, spermidine and spermine content in the pollen did not increase at 38 degrees C, while putrescine content increased at both 25 degrees C and 38 degrees C. High-temperature inhibition of pollen germination was alleviated by the addition of spermidine or spermine but not of putrescine to the germination medium. Cycloheximide inhibited SAMDC activity in parallel with pollen germination at 25 degrees C, whereas actinomycin D had no effect on either of them, indicating that enhanced SAMDC activity is associated with de novo protein synthesis. Incubation of crude enzyme extracts at 40 degrees C for 1 h did not affect SAMDC. In addition, high temperatures did not enhance protease activity in germinating pollen. These results indicate that low activity of SAMDC, probably due to impaired protein synthesis or functional enzyme formation, is a major cause for the poor performance of tomato pollen at high temperatures.     

Identification of a putative ribosomal protein mRNA in Chironomus riparius and its response to cadmium, heat shock, and actinomycin D

A putative ribosomal protein (rp) mRNA in Chironomus riparius has been found using differential display (DD). Its sequence has 84.8% identity with mosquito rp L8, Aedes albopictus, and is approximately 0.9 kb. Studies were undertaken in order to evaluate rp as a control for environmentally relevant genes. Responses of Drosophila heat shock 70 gene (hsp70) were used to establish heat shock temperatures and cadmium (Cd) concentrations for Chironomus experiments and to validate DD. Expression of hsp70 was induced over control by 28 degrees C at 30 minutes and 1 mM Cd at 24 hours (p< or =0.05). For Chironomus, DD, Northern blot, and nuclease sensitivity were used to measure responses to two stressors: heat shock for 30 minutes and Cd for 24 or 48 hours. Differential display and nuclease sensitivity assays found expression of rp mRNA at 37 degrees C and 16 mM Cd to be similar to controls. Northern blots indicated statistically significant effects for heat shock (p = 0.046) but not Cd (p = 0.406). However, mRNA levels at 37 degrees C were increased only 1.72-fold over controls. A concentration of 24 nM actinomycin D suppressed rp expression as measured by nuclease sensitivity assays. Stressors should not affect rp mRNA levels below their LC-50s.     

Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect,Panstrongylus megistus (Burmeister) (Hemiptera,Reduviidae)

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40 degrees C, 1 h) or cold (5 or 0 degrees C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0 degrees C, respectively, after 8, 18, 24 and 72 h at 28 degrees C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0 degrees C were more effective than the temperatures of 35 and 5 degrees C in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.     

Influence of heat transmission mode on heating rates and on the selection of patches for heating in a mediterranean lizard

Heliothermy (heat gain by radiation) has been given a prominent role in basking lizards. However, thigmothermy (heat gain by conduction) could be relevant for heating in small lizards. To ascertain the importance of the different heat transmission modes to the thermoregulatory processes, we conducted an experimental study where we analyzed the role of heat transmission modes on heating rates and on the selection of sites for heating in the Mediterranean lizard Acanthodactylus erythrurus (Lacertidae). The study was conducted under laboratory conditions, where two situations of different operative temperatures (38 degrees and 50 degrees C) were simulated in a terrarium. In a first experiment, individuals were allowed to heat up during 2 min at both temperatures and under both heat transmission modes. In a second experiment, individuals were allowed to select between patches differing in the main transmission mode, at both temperatures, to heat up. Experiences were conducted with live, nontethered lizards with a starting body temperature of 27 degrees C. Temperature had a significant effect on the heating rate, with heat gain per unit of time being faster at the higher operative temperature (50 degrees C). The effect of the mode of heat transmission on the heating rate was also significant: at 50 degrees C, heating rate was greater when the main heat transmission mode was conduction from the substrate (thigmothermy) than when heating was mainly due to heat gain by radiation (heliothermy); at 38 degrees C, heating rates did not significantly differ between transmission modes. At 38 degrees C, selection of the site for heating was not significantly different from that expected by chance. However, at 50 degrees C, the heating site offering the slowest heating rate (heliothermic patch) was selected. These results show that heating rates vary not only with environmental temperature but also with different predominant heat transmission modes. Lizards are able to identify and exploit this heterogeneity, selecting the source of heat gain (radiation) that minimizes the risk of overheating when temperature is high.     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)