Heat Stress Protein Synthesis Research Articles

The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium.

Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni.

Heat stress response and myocardial protection.

Prior whole-body hyperthermia is able to protect the myocardium against ischaemia-reperfusion injury by reducing cellular necrosis, preserving the ventricular function and preventing the occurrence of arrhythmias. These cardioprotective effects are associated with reduction of oxidative stress, preservation of the high-energy phosphate levels and synthesis of heat stress proteins. A better understanding of this powerful protective adaptation of the myocytes would be of interest for potential clinical application, and rational design of specific agents that activate this mechanism will hopefully follow soon.

Synthesis of heat stress proteins in lymphocytes from livestock.

Cultured bovine, equine, ovine and chicken lymphocytes responded to heat stress by the increased synthesis of a specific set of proteins known as heat stress proteins (HSP). Proteins with molecular weights of 70 and 90 kDa were synthesized in all species. Additional proteins were found in bovine, ovine and chicken lymphocytes. A time course of induction showed an increased synthesis of some of these proteins with only 30 min of heat stress and of several proteins with 60 min of heat stress. A specific monoclonal antibody was used to identify HSP70 as one of the stress proteins in bovine lymphocytes. Cells from these animals are capable of responding to heat stress by the production of heat stress proteins. These may be of physiological importance.

The effect of calmodulin antagonists on hyperthermic cell killing and the development of thermotolerance

The role of calmodulin (CaM) in hyperthermic cell killing, and the development of thermotolerance in rat 13762NF mammary adenocarcinoma cells, was investigated by using the CaM antagonists W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and W-13 [N-(4-aminobutyl)-2-chloro-naphthalenesulphonamide] and their less active analogues W-5 [N-(6-aminohexyl)-1-naphthalenesulphonamide] and W-12 [N-(4-aminobutyl)-2-naphthalenesulphonamide]. The CaM antagonists W-7 and W-13 potentiated 43 degrees C cell killing (and the less active analogues did not) at a concentration compatible with CaM inhibition, thus hyperthermic perturbation of CaM-regulated processes may contribute to cellular lethality. The potentiation of hyperthermic killing by antagonists appeared to be temperature-dependent, sensitizing much more effectively to 43 degrees C than to 42 degrees C killing. The effect may be related to differing primary mechanisms of hyperthermic killing activated at the two temperatures, or simply to differences in incorporation or localization of the antagonists. The presence of the CaM antagonists throughout fractionated 42 degrees C or 43 degrees C heating, or during continuous 42 degrees C heating, did not significantly inhibit or potentiate the triggering and development of thermotolerance or alter the rates of heat stress protein (HSP) synthesis. Studies using CaM-agarose isolation of CaM-binding proteins indicated that binding of some HSP to CaM-agarose occurred and was Ca2+-dependent. The specificity and physiological relevance of these HSP binding to CaM was not clear, since their affinity was not high in these cells. Presumably W-7 would perturb any physiologically relevant CaM-protein interactions in cells but W-7 concentrations that reduced HSP and other protein binding to CaM-agarose columns by 50 per cent or more, had no effect on thermotolerance development in cells. These observations, combined with the studies that showed little effect of CaM antagonists on HSP synthesis at concentrations which potentiated cell killing, suggested that events leading to triggering or developing thermotolerance were not strongly dependent on any putative HSP binding to CaM. These studies also suggest some targets of hyperthermic cell killing at 43 degrees C are different from those that lead to the triggering and development of thermotolerance.

Relationship between cell survival and heat-stress protein synthesis in a Drosophila cell line.

Heat-stress protein (hsp) kinetics and clonogenic survival were studied at 33, 37 and 42 degrees C in a continuous Drosophila cell line, WR69-DM-1. Induction and repression of hsp were temperature-dependent and independently modulated. The subsequent cell-survival curves were complex; however, survival generally decreased in a time- and temperature-dependent manner during continuous heating at 33, 37 or 42 degrees C. Constant 33 degrees C heating induced five hsp at 90, 72, 70, 24 and 19 kilodaltons (kDa). A 30 min 33 degrees C heat dose led to thermotolerance after 1, 3 or 6 h incubations at 28 degrees C. The hsp synthesized after this dose were quickly repressed, suggesting the cells were able to respond to this stress. Increasing the challenge temperature to 37 degrees C induced three additional hsp at 34, 22 and 14 kDa, but hsp synthesis did not lead to thermotolerance over the 6 h interval. The number and intensity of hsp synthesized was higher and repression was much slower than at 33 degrees C. Heating at 42 degrees C inhibited all protein synthesis, and thermotolerance was not observed. Direct survival data are critical to understanding the role and function of hsp in Drosophila thermotolerance since the relevance of information on number and kinetics of hsp synthesis and their subsequent localization is dubious without it.

The effects of glutathione depletion on thermotolerance and heat stress protein synthesis.

The effects of cellular glutathione depletion by buthionine sulfoximine on the development of thermotolerance and synthesis of heat stress protein was studied. Cellular glutathione levels were found to increase rapidly following an acute heat treatment of either 12 min at 45.5 degrees C or 1 h at 43 degrees C and remain elevated for prolonged periods. Glutathione depletion and prevention of glutathione synthesis by buthionine sulfoximine resulted in inhibition of the development of thermotolerance and a decrease in total protein as well as specific heat stress proteins. While the degree of inhibition of thermotolerance was similar for both glutathione depletion protocols, inhibition in heat stress protein synthesis was greater when glutathione was depleted to low levels prior to heating. The possible role of glutathione and the cellular redox state to thermotolerance and synthesis of heat stress protein is discussed.