The Effect of Sodium Malonate on Yeast Thermotolerance

The study of the effect of malonate (an inhibitor of the succinate dehydrogenase complex of the respiratory chain of mitochondria) on the thermotolerance of the fermentative Saccharomyces cerevisiae and nonfermentative Rhodotorula rubra yeasts showed that malonate augmented the damaging effect of heat shock on the yeasts utilizing glucose (or other sugars) by means of oxidative phosphorylation. At the same time, malonate did not influence, and sometimes even improved, the thermotolerance of the yeasts utilizing glucose through fermentation. The suggestion is made that cell tolerance to heat shock depends on the normal functioning of mitochondria. On the other hand, their increased activity at elevated temperatures may accelerate the formation of cytotoxic reactive oxygen species and, hence, is not beneficial to cells.     

Effect of cytochrome oxidase inhibitors on the yeast thermotolerance

The investigation of the effect of the cytochrome oxidase inhibitors sodium cyanide and sodium azide on the thermotolerance of the yeasts Rhodotorula rubra, Debaryomyces vanriji, and Saccharomyces cerevisiae showed that these inhibitors diminish the thermotolerance of R. rubra and D. vanriji, but do not affect the thermotolerance of S. cerevisiae. Taking into account the fact that, unlike the latter yeast, R. rubra and D. vanriji are nonfermentative yeasts, the difference in the effects of the inhibitors on the yeast thermotolerance can be readily explained by the different types of glucose utilization (either oxidative or fermentative) in these yeasts. The data obtained also provide evidence that there is a correlation between the functional activity of mitochondria and the thermotolerance of yeast cells.     

The effect of sodium azide on the thermotolerance of the yeast Saccharomyces cerevisiae and Candida albicans

The addition of sodium azide (a mitochondrial inhibitor) at a concentration of 0.15 mM to glucosegrown Saccharomyces cerevisiae or Candida albicans cells before exposing them to heat shock increased cell survival. At higher concentrations of azide, its protective effect on glucose-grown cells decreased. Furthermore, azide, even at low concentrations, diminished the thermotolerance of galactose-grown yeast cells. It is suggested that azide exerts a protective effect on the thermotolerance of yeast cells when their energy requirements are met by the fermentation of glucose. However, when cells obtain energy through respiratory metabolism, the azide inhibition of mitochondria enhances damage inflicted on the cells by heat shock.     

The absence of direct relationship between the ability of yeasts to grow at elevated temperatures and their survival after lethal heat shock

The study of the growth of the yeasts Rhodotorula rubra, Saccharomyces cerevisiae, and Debaryomyces vanriji at elevated temperatures and their survival after transient lethal heat shock showed that the ability of these yeasts to grow at supraoptimal temperatures (i.e., their thermoresistance) and their ability to tolerate lethal heat shocks (i.e., their thermotolerance) are determined by different mechanisms. The thermotolerance of the yeasts is suggested to be mainly determined by the division rate of cells before their exposure to heat shock.     

The Effect of Sodium Azide on the Thermotolerance of the Yeasts Saccharomyces cerevisiae and Candida albicans

The addition of sodium azide (a mitochondrial inhibitor) at a concentration of 0.15 mM to glucose-grown Saccharomyces cerevisiae or Candida albicans cells before exposing them to heat shock increased cell survival. At higher concentrations of azide, its protective effect on glucose-grown cells decreased. Furthermore, azide, even at low concentrations, diminished the thermotolerance of galactose-grown yeast cells. It is suggested that azide exerts a protective effect on the thermotolerance of yeast cells when their energy requirements are met by the fermentation of glucose. However, when cells obtain energy through respiratory metabolism, the azide inhibition of mitochondria enhances the damage inflicted on the cells by heat shock.     

The Absence of a Direct Relationship between the Ability of Yeasts to Grow at Elevated Temperatures and Their Survival after Lethal Heat Shock

The study of the growth of the yeasts Rhodotorula rubra, Saccharomyces cerevisiae, and Debaryomyces vanriji at elevated temperatures and their survival after transient lethal heat shock showed that the ability of these yeasts to grow at supraoptimal temperatures (i.e., their thermoresistance) and their ability to tolerate lethal heat shocks (i.e., their thermotolerance) are determined by different mechanisms. It is suggested that the thermotolerance of the yeasts is mainly determined by the division rate of cells before their exposure to heat shock.     

Effect of sodium azide on heat-shock resistance in Saccharomyces cerevisiae and Debaryomyces vanriji yeasts]

The pretreatment of Saccharomyces cerevisiae and Debaryomyces vanriji with sodium azide was found to induce thermotolerance in both yeasts, whereas sodium azide used in combination with heat shock enhanced the thermotolerance of S. cerevisiae and substantially decreased the thermotolerance of D. vanriji. It is suggested that the different responses of the yeasts to sodium azide during heat shock are due to the different functional organizations of their mitochondrial apparatus.     

The Effect of Cytochrome Oxidase Inhibitors on the Thermotolerance of Yeasts

The investigation of the effect of the cytochrome oxidase inhibitors sodium cyanide and sodium azide on the thermotolerance of the yeasts Rhodotorula rubra, Debaryomyces vanriji, and Saccharomyces cerevisiae showed that these inhibitors diminish the thermotolerance of R. rubraand D. vanriji, but do not affect the thermotolerance of S. cerevisiae. Taking into account the fact that, unlike the latter yeast, R. rubra and D. vanriji are nonfermentative yeasts, the difference in the effects of the inhibitors on the yeast thermotolerance can be readily explained by the different types of glucose utilization (either oxidative or fermentative) in these yeasts. The data obtained also provide evidence that there is a correlation between the functional activity of mitochondria and the thermotolerance of yeast cells.     

Effect of Sodium Azide on the Thermotolerance of the Yeasts Saccharomyces cerevisiaeand Debaryomyces vanriji

The pretreatment of Saccharomyces cerevisiaeand Debaryomyces vanrijiwith sodium azide was found to induce thermotolerance in both yeasts, whereas sodium azide used in combination with heat shock enhanced the thermotolerance of S. cerevisiaeand substantially decreased the thermotolerance of D. vanriji.It is suggested that the different responses of the yeasts to sodium azide during heat shock are due to the different functional organizations of their mitochondrial apparatus.     

Interleukin-6 induces thermotolerance in cultured Caco-2 cells independent of the heat shock response

In recent studies, induction of the heat shock response increased IL-6 production in gut mucosa in vivo and in cultured Caco-2 cells in vitro. The heat shock response is associated with increased survival of cells exposed to otherwise lethal hyperthermia, so called thermotolerance, but the role of IL-6 in the induction of thermotolerance is not known. We tested the hypothesis that treatment of cultured Caco-2 cells with IL-6 results in the development of thermotolerance. Cells were treated with human recombinant IL-6 for 1h followed by 3 h recovery in cytokine-free medium whereafter cells were exposed to heat stress (48 degrees C for 2 h). In untreated cells, the heat stress resulted in an approximately 80% cell death. In cells treated with IL-6, cell viability after heat stress was significantly improved and was doubled at an IL-6 concentration of 20 ng/ml. Treatment of the cells with other cytokines (IL-4, IL-10, IL-1beta, or TNFalpha) did not induce thermotolerance, suggesting that the effect of IL-6 may be specific for this cytokine. The induction of thermotolerance by IL-6 was blocked by an IL-6 receptor antibody, suggesting that the development of thermotolerance was receptor-mediated. Treatment of cells with IL-6 did not induce an heat shock response as suggested by unaltered heat shock protein 70 and 90 levels and unaffected heat shock factor DNA binding activity. In addition, the IL-6-induced thermotolerance was not inhibited by quercetin. The present study provides the first evidence of IL-6-induced thermotolerance and suggests that this effect of IL-6 is independent of the heat shock response.     

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

The Effect of Sodium Azide on Basic and Induced Thermotolerance in Saccharomyces cerevisiae

The action mechanism of the mitochondrial inhibitor sodium azide on thermotolerance in Saccharomyces cerevisiae was studied. At ambient growth temperature, pretreatment with sodium azide was shown to improve the thermotolerance of parent cells and the hsp104 mutant. Treating with the inhibitor during a mild heat shock suppressed the development of induced thermotolerance due to the inhibition of heat shock protein (Hsp104) synthesis. Treating with the inhibitor immediately before lethal heat shock produced a variety of effects on thermotolerance depending on whether the yeast metabolism was oxidative or fermentative. The conclusions are: (1) the protective effect of sodium azide on the thermotolerance of S. cerevisiae cells grown on glucose-containing medium is not related to Hsp104 functioning, and (2) the mechanisms of basic and induced thermotolerance differ considerably.     

The molecular chaperone Hsp78 confers compartment-specific thermotolerance to mitochondria

Hsp78, a member of the family of Clp/Hsp100 proteins, exerts chaperone functions in mitochondria of S. cerevisiae which overlap with those of mitochondrial Hsp70. In the present study, the role of Hsp78 under extreme stress was analyzed. Whereas deletion of HSP78 does not affect cell growth at temperatures up to 39 decrees C and cellular thermotolerance at 50 degrees C, Hsp78 is crucial for maintenance of respiratory competence and for mitochondrial genome integrity under severe temperature stress (mitochondrial thermotolerance). Mitochondrial protein synthesis is identified as a thermosensitive process. Reactivation of mitochondrial protein synthesis after heat stress depends on the presence of Hsp78, though Hsp78 does not confer protection against heat-inactivation to this process. Hsp78 appears to act in concert with other mitochondrial chaperone proteins since a conditioning pretreatment of the cells to induce the cellular heat shock response is required to maintain mitochondrial functions under severe temperature stress. When expressed in the cytosol, Hsp78 can substitute for the homologous heat shock protein Hsp104 in mediating cellular thermotolerance, suggesting a conserved mode of action of the two proteins. Thus, proteins of the Clp/Hsp100-family located in the cytosol and within mitochondria confer compartment-specific protection against heat damage to the cell.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

The effects of glucose on protein synthesis and thermosensitivity in Chinese hamster ovary cells

Glucose deprivation induces the major glucose regulated proteins (GRPs) in Chinese hamster ovary cells. When these cells are then returned to a glucose containing environment, GRP synthesis is repressed while concurrently other proteins, identified as heat shock proteins, are induced. The induction of the GRPs is found to mark precisely the onset of a decline in the cell's ability to survive a thermal stress while the expression of heat shock proteins, when glucose is restored, is paralleled by significant increases in survival protection or thermotolerance.     

Impact of mild heat treatments on induction of thermotolerance in the biocontrol yeast Candida sake CPA-1 and viability after spray-drying

Aims: The objective of this study was to examine the induction of thermotolerance in the biocontrol agent Candida sake CPA‐1 cells by mild heat treatments to enhanced survival of formulations using spray‐drying. The possible role of heat‐shock proteins (HSPs) biosynthesis in induced thermotolerance and the role of sugars and sugar alcohols were also determined. Methods and Results: Studies were conducted on C. sake cells grown in molasses medium and exposed to mild temperatures of 30 and 33°C during mid‐ (16 h), late‐exponential (24 h), early‐ (30 h) and mid‐stationary (36 h) growth phases. The effect on viability was determined both before and after spray‐drying. Cycloheximide and chloramphenicol were used to examine the role of HSPs and HPLC was used to analyse the accumulation of sugar and sugar alcohols. The results indicate that both temperatures induced thermotolerance in cells of C. sake. Mild heat‐adapted cells at 33°C in the early‐ or mid‐stationary phases had survival values after spray‐drying significantly higher (P ≤ 0·05) than nonadapted cells. However, viabilities were not high enough to be considered for commercial use with values up to 17%. HSPs were not implicated in thermotolerance acquired by mild heat‐adapted cells as similar viabilities were obtained in the presence of protein inhibitors. Little change was observed in sugar and sugar alcohols with an increase in glucose and arabitol in some treatments. Conclusions: This study suggests that it is possible to induce thermotolerance in biocontrol yeasts such as C. sake. However, this does not improve survival of cells exposed to spray‐drying sufficiently to consider this a suitable formulation method for this biocontrol agent. HSPs, sugars and sugar polyols were not directly responsible for induced thermotolerance in yeast cells. Significance and Impact of the Study: This type of information can be effectively applied to improve the viability of cells in the process of formulation.