Heat-shock response in a yeast tps1 mutant deficient in trehalose synthesis

Exponential cells of the Saccharomyces cerevisiae tps1 mutant underwent a rapid loss of viability upon a non-lethal heat exposure (from 28 to 42°C). However, a further more severe heat stress (52.5°C 5 min) induced an increase in the fraction of viable cells. This mutant can not synthesize trehalose either at 28° C or at 42°C due to the lack of a functional trehalose-6P synthase complex. In control experiments carried out with the wild-type W303-1 B, heat-stressed exponential phase cultures grown on YPgal at 28°C acquired thermotolerance to a higher extent than identical cultures grown on YPD, although in both cultures the level of stored trehalose was negligible. These data suggest that the bulk of trehalose accumulated in yeast upon mild heat treaments is not sufficient to account for the acquisition of thermotolerance.     

Molecular evolution of the HSP70 multigene family

Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups. Correspondence to: E.A. Craig     

The influence of polyalcohols and carbohydrates on the thermostability of alpha-amylase

The influence of polyhydric alcohols and carbohydrates on the thermostability, i.e., the heat inactivation kinetics, of Bacillus licheniformis alpha-amylase was studied in the temperature range 96 degrees to 130 degrees C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration. Statements about stabilization should, however, be specified carefully with respect to temperature, because E(A) is mostly altered likewise. For dissolved enzyme E(A) was almost always decreased in the presence of polyol or carbohydrate, whereas for immobilized enzyme it was augmented in each studied instance. The inactivation of dissolved enzyme can, in all the studied cases, be adequately described as a first-order process. Immobilized enzyme, however, shows biphasic then first-order inactivation kinetics, depending on the additive concentration and temperature.     

Cell Size and the Heat-Shock Response in Rat Brain

Abstract: The expression of mRNAs encoding two members of the heat-shock protein 70 family, the constitutively-expressed heat-shock cognate (hsc70) mRNA and the strictly heat-inducible (hsp70) mRNA, was quantitated in cerebellar and hippocampal cells of rats 3 h after amphetamine-induced or heat-induced hyperthermia. Intracellular heat-shock mRNA levels in specific cell types were compared with those of total polyadenylic acid [poly(A)] mRNA or 18S rRNA in the same cell type. Levels of poly(A) mRNAs, 18S rRNAs, and hsc70 mRNAs were highest in large neurons and lowest in glia. hsp70 mRNAs were also present at highest levels in large neurons, suggesting that hsp70 mRNAs accumulated as rapidly in these cell types as they did in small neurons and glia. However, compared with levels of intracellular poly(A) mRNAs or levels of rRNAs, large neurons contained two- to 12-fold lower levels of hsp70 mRNAs than neurons of intermediate size and five- to 30-fold lower levels than glia. These results suggest that hsp70 mRNAs accumulated as rapidly in large neurons as in small neurons and glia, but that the large size of these neurons precluded intracellular hsp70 mRNA concentrations increasing as quickly. The susceptibility of large neurons to stress-induced cell death could be due, in part, to their inability to synthesize rapidly hsp70 in sufficient amounts to protect these cells from the initial molecular consequences of stress.     

Thyrotropin-Releasing Hormone Release Is Enhanced in Hippocampal Slices After Electroconvulsive Shock

Abstract: Hippocampal thyrotropin-releasing hormone (TRH) release was examined after seizures were induced by electroconvulsive shock (ECS). Rat hippocampal slices taken 12, 24, or 48 h after 3 days of alternate-day ECS treatment or sham-ECS treatment were stimulated with potassium with or without calcium in a superfusion system containing in-line charcoal adsorbent to concentrate TRH. Released TRH and tissue TRH were measured by radioimmunoassay. The TRH content of hippocampal slices was increased fivefold over sham-ECS levels 12, 24, and 48 h after ECS, but this was not associated with an increase in basal TRH release. Potassium-stimulated TRH release was significantly elevated over basal release 12, 24, and 48 h after ECS. Potassium-stimulated calcium-dependent TRH release increased linearly after ECS, reaching its highest level 48 h after seizure. Thus, although enhanced calcium-dependent TRH release was associated with elevated tissue levels, this relationship was not proportional in that tissue TRH was elevated to the same extent at all times after ECS, whereas potassium-evoked calcium-dependent TRH release increased gradually over time after seizure. These results suggest that postictal elevations in TRH are associated with an enhanced capacity for release that develops as a result of a time-dependent shift of TRH from a storage compartment to a readily releasable pool. The observed elevation in stimulated TRH release may be relevant to seizure-induced modulation of TRH receptors in vivo.     

Temperature and chemical shocks induce chilling tolerance in germinating Cucumis sativus (cv. Poinsett 76) seeds

Roots of 24-h-old germinated cucumber ( Cucumis sativus cv. Poinsett 76) seeds were subjected to thermal and chemical stresses, equilibrated at 25°C for 2 h and chilled at 2.5°C for 96 h. The germinated seeds were then held at 25°C for 72 h after they were chilled and the elongation of the primary root was used as a measure of chilling tolerance. Control roots elongated from an initial length of 0.2 cm to a final length of 6.3 cm at the end of 72 h. while chilled roots elongated to a final length of only 0.4 to 0.6 cm. Exposure to 0.4 M ethanol for 4 h or to 40°C for 1 h induced substantial chilling tolerance and the roots had a final length of 4.1 and 3.1 cm. respectively. Exposure to 7.5°C for 3 h conferred less chilling tolerance (elongation to 1.4 cm). while exposure to other chemicals (i.e. aqueous solutions of Ca(NO₃)₂, mannitol. methanol and NaCl) produced less, though still significant increases in chilling tolerance. A more severe chilling treatment of 144 h at 2.5°C was required to consistently induce elevated rates of ion leakage. Only the heat and the ethanol shock treatments significantly reduced chilling-induced ion leakage. Inclusion of the protein synthesis inhibitor cycloheximide negated the protective effects of these shock treatments. It appears that de novo protein synthesis is required for induction of chilling tolerance by a variety of chemical and thermal shock treatments.     

Selection for higher yields and heat resistance in Pima cotton has caused genetically determined changes in stomatal conductances

Gas exchange analysis was used to characterize photosynthetic and stomatal responses to key environmental stimuli in five commercial lines of Pima cotton ( Gossypium barbadense L.) which represent a gradient of selection for higher yields and heat resistance, and a primitive, uncultivated G. barbadense. At constant light and vapor pressure deficit, stomatal conductance increased linearly with air temperature in the 23 to 36$C range in all five commercial lines, and conductance at each temperature increased as a function of selection. In contrast, photosynthetic rates had a low sensitivity to temperature in the 23 to 36$C range, particularly in the advanced lines. In a segregating F₂ population from a cross between the advanced line, Pima S-6, and the primitive cotton, B368, the slope of the stomatal response to temperature in each F₂ plant was positively correlated with the stomatal conductance measured at 40$C. An analysis of the frequency distribution of stomatal conductance in F₁ and F₂ progeny of the cross showed that the differences in stomatal conductance between lines were genetically determined. These data indicate that selection for higher yield and heat resistance in Pima cotton has caused genetically determined changes in stomatal properties. Characterization of the relationship between the altered stomatal properties and the attained increases in heat resistance and yields could make it possible to use these physiological traits as selection criteria in future breeding programs.     

Transpiration measurements on apple trees with an improved stem heat balance method

Since the late eighties a handy and user-friendly sap flow meter (Dynagage®) is on the market which can quantify 0205 the sap flow through intact plant stems, based on the stem heat balance method. The documentation about its accuracy and reliability, however, is still too limited to use it as a standard method in field experiments with apple trees. We therefore tested this commercial system on potted apple trees (Malus domestica L.; cv. Red Elstar and Jonagold; on rootstock M9 vf) with stem diameters of 1.8 to 4 cm. The measured sap flow was compared with mass loss measured by an automated balance, supposing the total mass loss of the trees was equal to the water loss by transpiration. The results revealed three major problems:

Effect of shock-loading of heavy metals on total organic carbon and phosphate removal in an anaerobic-aerobic activated sludge process

The effect of shock-loading of zinc, copper and cadmium ions on the removal of total organic carbon (TOC) and phosphate in an anaerobic-aerobic activated sludge process was investigated. TOC removal was not sensitive to shock-loading of Zn²⁺ and Cd²⁺ ions, and complete removal was achieved even at 20 mg Zn²⁺/l and 20 mg Cd²⁺/l. However, with over 1 mg Cu²⁺/1 TOC removal efficiency decreased. PO _inf4 ^sup3- removal, in contrast, was extremely sensitive to these metal ions, with the threshold being 1 mg Zn²⁺/l and 1 mg Cd²⁺/l. Higher concentrations adversely affected PO _inf4 ^sup3- removal. Copper again proved detrimental; no PO _inf4 ^sup3- removal was achieved even at 1 mg Cu/l. These results highlight the sensitivity of the removal efficiencies of TOC and PO _inf4 ^sup3- to shock loadings of these heavy metals.Y.P. Ting is with the Department of Chemical Engineering, National University of Singapore, Kent Ridge, 0511, Singapore; H. Imai and S. Kinoshita are with the Department of Chemical Process Engineering, Hokkaido University, Sapporo 060, Japan.     

Inhibition of Bacillus spores by combinations of heat,potassium sorbate,NaCl and pH

Viability of spores of Bacillus cereus was totally inhibited at 85°C over 30 min by adding 0.4% (w/v) potassium sorbate with 6% (w/v) NaCl at pH 4.5. Viability of B. stearothermophilus spores was totally inhibited at 95°C for 45 min in a buffer at pH 4.2 containing 0.8% (w/v) potassium sorbate and 8% (w/v) NaCl. A synergistic inhibitory effect was demonstrated in some of the combinations. The inhibition may be due to interference with the heat-resistance apparatus of the spores.O.B. Oloyede was and J. Scholefield is with the Department of Bioscience & Biotechnology, Food Science Division, University of Strathclyde, Glasgow G1 1SD, UK. O.B. Oloyede is now with the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria.