Influence of temperature on complement-dependent immune damage to liposomes

Maximal release of trapped liposomal glucose, in the presence of saturating amounts of liposomal antigen (galactocerebroside), antiserum (anti-galactocerebroside), and complement, was dependent on temperature. At lower temperatures (20--25 degrees C), maximal glucose release was inversely related to liposomal phospholipid fatty acyl chain length (dimyristoyl phosphatidylcholine > dipalmitoyl phosphatidylcholine > distearoyl phosphatidylcholine > sphingomyelin). At higher temperatures (32--35 degrees C) a limiting plateau of glucose release, at approx. 60%, was reached, or approached, by all preparations. Sphingomyelin liposomes still released less glucose than those prepared from other phospholipids, even at 35 degrees C. The titers of antiserum and complement (ABL50/ml and CL50/ml) were dependent on temperature, and differences based on liposomal phospholipid fatty acyl chain length were observed. Analysis of antiserum and complement-dependence on temperature, and on phospholipid type, revealed that although antibody binding to galactocerebroside undoubtedly was subject to steric hindrance due to interference by surrounding phospholipids at 20--25 degrees C, steric hindrance did not play a major role in blocking antibody binding above 32 degrees C.     

Challenge of psychrophilic anaerobic wastewater treatment

Psychrophilic anaerobic treatment is an attractive option for wastewaters that are discharged at moderate to low temperature. The expanded granular sludge bed (EGSB) reactor has been shown to be a feasible system for anaerobic treatment of mainly soluble and pre-acidified wastewater at temperatures of 5--10 degrees C. An organic loading rate (OLR) of 10--12 kg chemical oxygen demand (COD) per cubic meter reactor per day can be achieved at 10--12 degrees C with a removal efficiency of 90%. Further improvement might be obtained by a two-module system in series. Stabile methanogenesis was observed at temperatures as low as 4--5 degrees C. The specific activity of the mesophilic granular sludge was improved under psychrophilic conditions, which indicates that there was growth and enrichment of methanogens and acetogens in the anaerobic system. Anaerobic sewage treatment is a real challenge in moderate climates because sewage belongs to the 'complex' wastewater category and contains a high fraction of particulate COD. A two-step system consisting of either an anaerobic up-flow sludge bed (UASB) reactor combined with an EGSB reactor or an anaerobic filter (AF) combined with an anaerobic hybrid reactor (AH) is successful for anaerobic treatment of sewage at 13 degrees C with a total COD removal efficiency of 50% and 70%, respectively.     

Hydrogen isotope exchange kinetics of single protons in bovine pancreatic trypsin inhibitor.

The exchange kinetics of the slowest exchanging BPTI beta-sheet protons are complex compared to model peptides; the activation energy, E alpha, and the pH dependence are temperature dependent. We have measured the exchange kinetics in the range pH 1--11, 33--71 degrees C, particularly the temperature dependence. The data are fit to a model in which exchange of each proton is determined by two discrete dynamical processes, one with E alpha approximately 65 kcal/mol and less than first order dependence on catalyst ion, and one with E alpha 20--30 kcal/mol and approaching first order in catalyst ion. The low activation energy process is the mechanism of interest in the native conformation of globular proteins and involves low energy, small amplitude fluctuations; the high activation energy process involves major unfolding. The model is simple, has a precedent in the hydrogen exchange literature, and explains quantitatively the complex feature of the exchange kinetics of single protons in BPTI, including the following. For the slowest exchanging protons, in the range 36 degrees--68 degrees C, E alpha is approximately 65 kcal/mol at pH approximately 4, 20--30 kcal/mol at pH greater than 10, and rises to approximately 65 kcal/mol with increasing temperature at pH 6--10; the Arrhenius plots converge around 70 degrees C; the pH of minimum rate, pHmin, is greater than 1 pH unit higher at 68 degrees C than for model compounds; and at high pH, the pH-rate profiles shift to steeper slope; the exchange rates around pHmin are correlated to the thermal unfolding temperature in BPTI derivatives (Wagner and Wüthrich, 1979, J. Mol. Biol. 130:31). For the more rapidly exchanging protons in BPTI the model accounts for the observation of normal pHmin and E alpha of 20--30 kcal/mol at all pH's. The important results of our analysis are (a) rates for exchange from the folded state of proteins are not correlated to thermal lability, as proposed by Wuthrich et al. (1979, J. Mol. Biol. 134:75); (b) the unfolding rate for the BPTI cooperative thermal transition is equal to the observed exchange rates of the slowest exchanging protons between pH 8.4--9.6, 51 degrees C; (c) the rates for exchange of single protons from folded BPTI are consistent with our previous hydrogen-tritium exchange results and with a penetration model of the dynamic processes limiting hydrogen exchange.     

Man's physiologic response to long-term work during thermal and pollutant stress.

Metabolic, temperature, and cardiorespiratory responses of 19 healthy males, age range 18-30 yr for one group and 40-55 yr for another, were studied during 210 minutes submaximal work at 35% Vo2 max. The subjects were exposed to four different pollutant gas mixtures at two different temperatures, 25 degrees C and 35 degrees C (relative humidity 30%). The four gas mixtures were filtered air (FA), 50 ppm carbon monoxide in filtered air (CO), 0.24 ppm peroxyacetyl nitrate in filtered air (PAN), and a combination of all three mixtures (PANCO). In the CO exposure, the heart rate was significantly greater than that observed during FA conditions (P less than 0.05). Metabolic and thermoregulatory responses to long-term work were not different in the various pollutant environments. Significant decreases in stroke volume and increases in heart rate were observed during the course of the 25 degrees C exposures with no alteration in cardiac output. Heart rates were higher during 35 degrees C exposures while cardiac output remained at the same level with a consequent further reduction in stroke output.     

Isolation,characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degrees C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degrees C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degrees C, with a half-life of about 10 h at 80 degrees C. The activity shows a linear Arrhenius plot at 50-85 degrees C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90 degrees ) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.     

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.     

Differentiation of two locally sympatric Protopolystoma (Monogenea: Polystomatidae) species by temperature-dependent larval development and survival

The developmental response of egg stages to different environmental temperature regimes was studied in Protopolystoma xenopodis and Protopolystoma orientalis (Monogenea: Polystomatidae) isolates from southern Africa. Eggs failed to develop at 10 degrees C, whilst at 15 degrees C only P. xenopodis completed larval development, hatching 49--88 days post-collection. Respective hatching windows were 26--34 (P. xenopodis) and 37--49 (P. orientalis) days at 20 degrees C, and 18--26 and 27--37 days at 25 degrees C. Continuous maintenance at 30 degrees C was lethal for eggs of both species. There were no consistent interspecific differences in the response of egg stages to low and high temperature shocks during early embryonic development.     

High-resolution phosphorus nuclear magnetic resonance spectra of yeast phenylalanine transfer ribonucleic acid. Melting curves and relaxation effects.

In a continuation of our studies on structural effects on the 31P chemical shifts of nucleic acids, we present 31P NMR spectra of yeast phenylalanine tRNA in the presence and absence of Mg2+. Superconducting field (146 MHz) and 32-MHz 31P NMR spectra reveal approximately 15 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+, most scattered and main cluster signals do not shift between 22--66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At 70 degrees C, all of the signals merge into a single random coil conformation signal. Similar effects are observed in the absence of Mg2+ except that the transition melting temperature is approximately 20 degrees C lower. Measured spin-lattice and spin-spin relaxation times reveal another lower temperature transition besides the thermal denaturation process. A number of the scattered peaks are shifted (0.2--1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ as a result of this conformational transition between two intact tertiary structures. The loss of the scattered peaks in the absence of Mg2+ occurs in the temperature range expected for melting of a tertiary structure. An attempt to simulate the 31P spectra of tRNA Phe based upon the X-ray crystallographically determined phosphate ester torsional agles supports the suggestion that the large shifts in the scattered peaks are due to bond angle distortions in the tertiary structure.     

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.     

Respiratory and behavioral effects of ozone on a lizard and a frog

Ozone at concentrations found in urban air pollution is known to have significant physiological effects on humans and other mammals. Exposure of the lizard, Sceloporus occidentalis, to 0.6 ppm ozone for 4 h at 25 degrees C induced 1.6 degrees C of behavioral hypothermia immediately following exposure, but selected body temperature recovered to control 35.3 degrees C the next day. Lizards exposed at 35 degrees C to 0.6 ppm ozone for 4 h selected body temperatures 1.9 degrees C below controls after exposure, and the behavioral hypothermic response persisted and increased to 3.3 degrees C the following day. Four-hour exposures of the frog, Pseudacris cadaverina, to 0.2 to 0.8 ppm ozone resulted in concentration-dependent alterations of respiration including depression of lung ventilation and oxygen consumption and the adoption of a low profile posture that reduced the exposed body surface. Ozone levels in wilderness habitats downwind of urban sources can potentially have stressful physiological effects on wildlife. Defensive physiological and behavioral reactions to ozone exposure may interfere with routine activities, and oxidant air pollution may be in part responsible for observed wildlife population declines.     

Structure and motion of phospholipids in human plasma lipoproteins. A 31P NMR study

The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31P NMR. Lateral diffusion coefficients, DT, obtained from the viscosity dependence of the 31P NMR line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoproteins (HDL2, HDL3), and egg PC/TO microemulsions at 25 degrees C, for VLDL at 40 degrees C, and for LDL at 45 degrees C. At 25 degrees C, the rate of lateral diffusion in LDL (DT = 1.4 x 10(-9) cm2/s) is an order of magnitude slower than in the HDLs (DT = 2 x 10(-8) cm2/s). At 45 degrees C, DT for LDL increases to 1.1 x 10(-8) cm2/s. In contrast, DT for VLDL increases only slightly going from 25 to 40 degrees C. The large increase in diffusion rate observed in LDL occurs over the same temperature range as the smectic to disordered phase transition of the core cholesteryl esters, and provides evidence for direct interactions between the monolayer and core. In order to prove the orientation and/or order of the phospholipid head-group, estimates of the residual chemical shift anistropy, delta sigma, have been obtained for all the lipoproteins and the microemulsions from the viscosity and field dependence of the 31P NMR line widths. For VLDL and LDL, the anisotropy is 47-50 ppm at 25 degrees C, in agreement with data from phospholipid bilayers. For the HDLs, however, significantly larger values of 69-75 ppm (HDL2) and greater than 120 ppm (HDL3) were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of perkinsosis in the carpet shell clam Tapes decussatus in Galicia (NW Spain). I. Identification of the aetiological agent and in vitro modulation of zoosporulation by temperature and salinity

Morphological characters of zoosporulation stages and DNA sequence of the internal transcribed spacer (ITS) region and the small subunit ribosomal RNA (SSU rRNA) gene confirmed that the aetiological agent of perkinsosis in the clam Tapes decussatus from Galicia (NW Spain) was Perkinsus atlanticus Azevedo, 1989. In vitro modulation by temperature and salinity of the zoosporulation of the parasite was studied. The optimum temperature range for zoosporulation was 19 to 28 degrees C. The temperature range allowing zoosporulation in vitro was 15 to 32 degrees C, which is broader than previously reported (24 to 28 degrees C) for P. atlanticus, and strongly suggests that zoospores can be produced in Galician Rias, where temperature ranges from 10 to 22 degrees C. Prezoosporangia held at 10 degrees C for 2 mo (similar to winter conditions in Galician waters) gave rise to viable zoospores after they were transferred to higher temperatures. This suggests that prezoosporangia could overwinter and zoosporulate in the next spring. Zoospores could survive for up to 22 and 14 d at 28 and 10 degrees C, respectively. The optimum salinity range for zooporulation was 25 to 35 per thousand. Zoospore production was abruptly reduced as salinity decreased. The lowest salinity at which zoosporulation was observed was 10 per thousand. The effectiveness of different chlorine concentrations and exposure lengths to kill prezoosporangia and zoospores was tested. No survival of free zoospores, free prezoosporangia and prezoosporangia included in gill tissue was observed after incubation for 1 h with 50, 200 and 3,000 ppm of chlorine, respectively.     

Dynamic light scattering studies on hydrodynamic properties of fibrinogen-fibronectin complex.

A high molecular weight 'cryogel' was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37 degrees C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20 degrees C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20 degrees C in 0.05 M TrisHCl(pII7.4) containing 0.5 M NaCl was estimated as 8.5 x 10(-8) cm2s-1. The complex did not dissociate over the temperature range from 20 to 37 degrees C. The diffusion coefficients of the complex decreased significantly at 12 degrees C and 40 degrees C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40 degrees C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15 degrees C, but not at near-freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Disruption of the gene for Hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in import of proteins into mitochondria

The gene for Hsp30, the only known alpha-crystallin-related heat shock protein of Neurospora crassa, was disrupted by repeat-induced point mutagenesis, leading to loss of cell survival at high temperature. Hsp30, which is not synthesized at 30 degrees C, associates reversibly with the mitochondria at high temperature (45 degrees C). In this study, we found that import of selected proteins into internal compartments of mitochondria, following their synthesis in the cytosol, was severely impaired at high temperature in a strain mutant in Hsp30. After 70 min of cell incubation at 45 degrees C, most matrix, inner membrane, and intermembrane-space proteins tested were reduced in import by about 50-70% in the mutant, as compared to wild-type cells. In contrast, assembly of selected proteins into the outer mitochondrial membrane was not reduced, except for one component of the preprotein translocase complex of the mitochondrial outer membrane. Three proteins of this complex co-immunoprecipitated with Hsp30 of wild-type cells incubated at 45 degrees C. We propose that Hsp30 interacts with the preprotein translocase of the mitochondrial outer membrane and that it chaperones the activity of one or more components of this translocase complex at high temperature.     

Transient-phase studies on the creatine kinase reaction. The analysis of a reaction pathway with three intermediates.

1. The initial formation of creatine phosphate by creatine kinase was studied in the millisecond range and the effect of temperature on the transient and steady-state phases exploited. 2. At 25 degrees C and 35 degrees C there was no transient phase. This is in agreement with the results of Gutfreund [Engelborghs, Y., Marsh, A., and Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 3. At 4 degrees C the time course of creatine phosphate formation was complex and consisted of three transient phases: a lag phase, a burst phase and a steady-state phase. Based on this result a reaction scheme for creatine kinase which includes three intermediates was proposed. Despite the completeness of the time course, the extraction of estimates for the rate constants was difficult and computer simulation and iterative methods had to be resorted to. 4. Attempts were made to provide evidence for the complex enzyme.ADP.metaphosphate.creatine on the creatine kinase reaction pathway [cf. Milner-White, E.J. and Watts, D.C. (1971) Biochem. J. 122, 727--740]. Under the conditions used these attempts were unsuccessful at times down to 2.5 ms, at 4 degrees C or 35 degrees C.     

Transient-phase studies on the arginine kinase reaction.

1. The initial formation of arginine phosphate by arginine kinase was studied in the time range 2.8--50 ms by the quenched-flow method. 2. A transient burst phase of product formation was obtained, the amplitude of which was temperature-dependent. At 35 degrees C it was 0.64 mol arginine phosphate/mol arginine kinase and at 12 degrees C, 0.25 mol/mol. 3. These results show that for the reaction pathway of arginine kinase the rate-limiting step follows the formation of arginine phosphate on the enzyme. This is in contrast to the creatine kinase reaction where no transient phase was observed [Engelborghs, Y., Marsh, A. & Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 4. The rate-limiting step on the arginine kinase reaction pathway is only slightly affected by temperature: the change in Kcat with temperature is due to a change of an equilibrium constant pertaining to at least two previous steps.     

The effect of short term thermal stress on the heart and respiration rates of conscious and anaesthetized chickens

Heart and respiration rates were measured in eight 6-week-old, White Rock chicks at different ambient temperatures: 24--26 degrees C (neutral), 6.5--8.5 degrees C (low) and 3,95--43.5 degrees C (high). The animals were exposed to these temperatures for 10 min. In both groups the low ambient temperature did not influence the respiration rate, whereas the high temperature caused a significant increase of the respiration rates both in the conscious and anaesthetized birds. In both groups no significant changes in the heart rate at different temperatures were found. Statistically significant differences in the heart and respiration rates between the conscious and anaesthetized chickens were noted only at the low environmental temperature.     

Immobilization of Streptomyces flavochromogenes pullulanase on tannic acid and TEAE--cellulose.

Pullulanase was immobilized successfully by simple, inexpensive methods that may be useful for industrial application of this enzyme. A tannin--pullulanase(TP) complex was obtained by addition of tannic acid to the culture filtrate of thermophilic Streptomyces flavochromogenes. TP could be bound to TEAE--cellulose (TTCP). Immobilization in this manner took place with quantitative retention of activity. The immobilized enzymes were stable for more than six months. The optimum temperatures of the native enzyme and TP were both 50 degrees C; that of TTCP was 45 degrees C. In the presence of 5mM Ca2+, the activity of TTCP was increased approximately twofold and the optimum temperature was raised to 50--60 degrees C. Pullulanase was not significantly eluted from TP or TTCP by NaCl solution (0.1--0.5M).     

13C NMR studies of the thermal properties of a model high density lipoprotein. Apolipoprotein A-I-dimyristoylphosphatidylcholine complex

The most abundant lipid and protein components of human plasma high density lipoproteins are phosphatidylcholine and apolipoprotein A-I (A-I). Under appropriate conditions, A-I spontaneously associates with dimyristoylphosphatidylcholine (DMPC) to quantitatively form a lipid-protein complex with a DMPC/A-I molar ratio of 100:1. Differential scanning calorimetry of this complex reveals two broad thermal transitions centered at approximately 27 and 72 degrees C. 13C NMR spectra of the complex have been obtained above, at, and below the lower transition temperature. The 13C resonance arising from the 3' carbon of the fatty acyl chains is a doublet, split by approximately 0.2 ppm, suggesting that the 3' carbon nuclei occupy two magnetically inequivalent sites. By replacing the sn-2 fatty acyl chain with myristate selectively 13C-enriched at carbon 3', we have shown that the splitting is, in fact, a result of magnetic inequivalence of the two sites and have assigned the lower field resonance to the 3' carbon nucleus of the sn-2 chain. The temperature dependence of the NMR relaxation rates indicates that the endothermic transition at 27 degrees C is associated with increased motional freedom for the phospholipids within this complex. The temperature dependence of the fatty acyl chain methylene 13C chemical shifts suggests that the population of gauche conformers increases above the transition temperature. These dynamic and conformational changes are characteristic of gel----liquid crystalline phase transitions observed in pure phospholipid systems. For the DMPC-A-I complex at 37 degrees C, the chemical shifts of the fatty acyl C 4'- 11' methylene envelope and of the C 7' and C 13' resonances occur significantly downfield from the corresponding chemical shifts for the DMPC vesicle. These results suggest that the apoprotein rigidifies the acyl chains by increasing their number of trans conformers.     

Effect of increased temperatures on RNA and protein synthesis in the cells of a synchronous Candida utilis culture

The rate of incorporation of labeled precursors for RNA ([14C]uracil) and protein ([14C]DL-leucine) into the cells of the synchronous culture of Candida utilis VKMY-1668 (the optimum temperature of growth, 31--32 degrees C) was studied as a function of different temperatures (28, 31, 32, 34, 36, 38, and 41 decrees C). The yeast was grown on a simple mineral medium containing glycerol. RNA synthesis was found to be more susceptible to elevated temperature than protein synthesis: the maximum rate of incorporation was registered at 32--34 degrees C for [14C]DL-leucine and only at 32 degrees C for [14C]uracil (the rate of its incorporation at 34 degrees C decreased by 50% as compared to that at 32 degrees C). The rate of incorporation of [14C]uracil at 34 degrees C reached 100% (the rate at 32 degrees C) when yeast autolysate was added to the medium, and 75 and 70%, respectively, upon the addition of DL-methionine or Mg2+ (as compared to 50% without them).