Thermal tolerance of contractile function in oxidative skeletal muscle: no protection by antioxidants and reduced tolerance with eicosanoid enzyme inhibition

Mechanisms for the loss of muscle contractile function in hyperthermia are poorly understood. This study identified the critical temperature, resulting in a loss of contractile function in isolated diaphragm (thermal tolerance), and then tested the hypotheses 1) that increased reactive oxygen species (ROS) production contributes to the loss of contractile function at this temperature, and 2) eicosanoid metabolism plays an important role in preservation of contractile function in hyperthermia. Contractile function and passive force were measured in rat diaphragm bundles during and after 30 min of exposure to 40, 41, 42 or 43 degrees C. Between 40 and 42 degrees C, there were no effects of hyperthermia, but at 43 degrees C, a significant loss of active force and an increase in passive force were observed. Inhibition of ROS with the antioxidants, Tiron or Trolox, did not inhibit the loss of contractile force at 43 degrees C. Furthermore, treatment with dithiothreitol, a thiol (-SH) reducing agent, did not reverse the effects of hyperthermia. A variety of global lipoxygenase (LOX) inhibitors further depressed force during 43 degrees C and caused a significant loss of thermal tolerance at 42 degrees C. Cyclooxygenase (COX) inhibitors also caused a loss of thermal tolerance at 42 degrees C. Blockage of phospholipase with phospholipase A(2) inhibitors, bromoenol lactone or arachidonyltrifluoromethyl ketone failed to significantly prevent the loss of force at 43 degrees C. Overall, these data suggest that ROS do not play an apparent role in the loss of contractile function during severe hyperthermia in diaphragm. However, functional LOX and COX enzyme activities appear to be necessary for maintaining normal force production in hyperthermia.     

A GTP-binding protein (Era) has an essential role in growth rate and cell cycle control in Escherichia coli.

Era is a membrane-associated GTP-binding protein which is essential for cell growth in Escherichia coli. In order to examine the physiological role of Era, strains in which Era was expressed at 40 degrees C but completely repressed at 27 degrees C were constructed. The growth of these strains was inhibited at the nonpermissive temperature, and cells became elongated. Under such conditions, no constrictions or septum formation could be detected by phase-contrast microscopy, and DNA segregation was apparently normal as revealed by fluorescence staining. These data demonstrate that Era has an essential function in cell growth rate control in liquid media and that depletion of Era blocks cell division either directly or indirectly. Thus, the role of GTP-binding proteins as important regulators of cell growth and division may be ubiquitous in nature.     

Thermoregulation in the Angolan free-tailed bat Mops condylurus: A small mammal that uses hot roosts.

The Angolan free-tailed bat (Mops condylurus) uses roosts that often exceed 40 degrees C, an ambient temperature (Ta) that is lethal to many microchiropterans. We measured the physiological responses of this species at Ta's from 15 degrees to 45 degrees C. Torpor was commonly employed during the day at the lower Ta, but the bats generally remained euthermic at night, with a mean body temperature (Tb) of 35.2 degrees C. Metabolic rate reflected the pattern of Tb, increasing with falling Ta at night but decreasing during the day. Metabolic rate and evaporative losses were lower in torpid than in euthermic bats. Body temperature increased at each Ta >35 degrees C and was 43 degrees C at Ta of 45 degrees C. At Ta of 40 degrees C bats increased dry thermal conductance and evaporative heat loss compared to lower Ta. At 45 degrees C dry thermal conductance was lower than at 40 degrees C and evaporative heat loss was 132% of metabolic heat production. At high Ta there was only a slight increase in metabolic rate despite the employment of evaporative cooling mechanisms and an increase in Tb. Collectively our results suggest that M. condylurus is well suited to tolerate high Ta, and this may enable it to exploit thermally challenging roost sites and to colonise habitats and exploit food sources where less stressful roosts are limiting.     

The effect of combined heat and ultrasound on multicellular tumour spheroids

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.     

On the chaperonin activity of GroEL at heat-shock temperature

The studies of GroEL, almost exclusively, have been concerned with the function of the chaperonin under non-stress conditions, and little is known about the role of GroEL during heat shock. Being a heat shock protein, GroEL deserves to be studied under heat shock temperature. As a model for heat shock in vitro, we have investigated the interaction of GroEL with the enzyme rhodanese undergoing thermal unfolding at 43 degrees C. GroEL interacted strongly with the unfolding enzyme forming a binary complex. Active rhodanese (82%) could be recovered by releasing the enzyme from GroEL after the addition of several components, e.g. ATP and the co-chaperonin GroES. After evaluating the stability of the GroEL-rhodanese complex, as a function of the percentage of active rhodanese that could be released from GroEL with time, we found that the complex had a half-life of only one and half-hours at 43 degrees C; while, it remained stable at 25 degrees C for more than 2 weeks. Interestingly, the GroEL-rhodanese complex remained intact and only 13% of its ATPase activity was lost during its incubation at 43 degrees C. Further, rhodanese underwent a conformational change over time while it was bound to GroEL at 43 degrees C. Overall, our results indicated that the inability to recover active enzyme at 43 degrees C from the GroEL-rhodanese complex was not due to the disruption of the complex or aggregation of rhodanese, but rather to the partial loss of its ATPase activity and/or to the inability of rhodanese to be released from GroEL due to a conformational change.     

Membrane docking of an aggregation-prone protein improves its solubilization

We used preS2-S'-beta-galactosidase, a three domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli to search for multicopy suppressors of protein aggregation and inclusion bodies formation, and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation at 43 degrees C by selecting pink lactose-positive colonies on a background of white lactose-negative colonies after transformation of bacteria with an E. coli gene bank. We found only two multicopy suppressors of preS2-S'-beta-galactosidase aggregation at 43 degrees C, protein isoaspartate methyltransferase (PIMT) and the membrane components ChbBC of the N,N'-diacetylchitobiose phosphotransferase transporter. We have previously shown that PIMT overexpression reduces the level of isoaspartate in preS2-S'-beta-galactosidase, increases its thermal stability and consequently helps in its solubilization at 43 degrees C (Kern et al., J. Bacteriol. 187, 1377-1383). In the present work, we show that ChbBC overexpression targets a fraction of preS2-S'-beta-galactosidase to the membrane, and decreases its amount in inclusion bodies, which results in its decreased thermodenaturation and in a lactose-positive phenotype at 43 degrees C. Cross-linking experiments show that the inner membrane protein ChbC interacts with preS2-S'-beta-galactosidase. Our results suggest that membrane docking of aggregation-prone proteins might be a useful method for their solubilization.     

Protein isoaspartate methyltransferase is a multicopy suppressor of protein aggregation in Escherichia coli

We used preS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43 degrees C after transformation of bacteria with an E. coli gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S'-beta-galactosidase aggregation at 43 degrees C. Overexpression of PIMT reduces the amount of preS2-S'-beta-galactosidase found in inclusion bodies at 43 degrees C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S'-beta-galactosidase and increases its thermal stability in E. coli crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases.     

Endoglucanase-producing fungi isolated from Cape Evans historic expedition hut on Ross Island, Antarctica

Early explorers of Antarctica's Heroic Era erected wooden buildings and brought large quantities of supplies to survive in Antarctica. The introduction of wood and other organic materials provided nutrient sources for fungi that were indigenous to Antarctica or were brought in with the materials and adapted to the harsh conditions. Seventy-two isolates of filamentous fungi were cultured on selective media from interior structural wood of the Cape Evans historic hut and 27 of these screened positive for the ability to degrade carboxymethyl cellulose (CMC). Four non-CMC-degrading isolates were added to a group of 14 CMC-degrading isolates for further study, and endo-1, 4-beta-glucanase activity was demonstrated in the extracellular supernatant from all of these 18 isolates when grown at 4 degrees C, and also when they were grown at 15 degrees C. Isolates of Penicillium roquefortii and Cadophora malorum showed preference for growth at 15 degrees C rather than 25 degrees C or 4 degrees C indicating psychrotrophic characteristics. These results demonstrate that cellulolytic filamentous fungi found in Antarctica are capable of growth at cold temperatures and possess the ability to produce extracellular endo-1, 4-beta-glucanase when cultured at cold and temperate temperatures.     

Effect of high temperature and water stress on in vitro germination and growth in isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin

In non-irrigated agricultural fields in tropical zones, high temperature and water stress prevail during the main cropping season. Natural epizootics of Beauveria bassiana on lepidopteran pests occur during winter. Application of B. bassiana during hot months when pest populations are at their climax may prove an effective management strategy. Therefore, 29 isolates of B. bassiana were tested for their ability to germinate and grow in temperature and water availability conditions prevailing during the pest season in these fields. The effect of temperature cycles with 8 h duration of high temperature fluctuating with 16 h duration of lower temperature (similar to field conditions); low water availability; and a combination of these two stress conditions was studied. Germination and growth assays were done at fluctuating temperature cycles of 32, 35, 38, and 42+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and in media with water stress created by 10, 20, 30, and 40% polyethylene glycol (PEG 6000). Assays set at a continuous temperature of 25+/-1 degrees C with no PEG in the medium served as controls. Stress was assessed as percentage germination or as growth relative to control. Isolates showing 90% growth relative to the control at temperature cycles including high temperatures of 35 and 38+/-1 degrees C were identified. One isolate (ARSEF 2860) had a thermal threshold above 43 degrees C. At 25 degrees C, all but one isolate of B. bassiana showed >90% growth relative to the control in 10% PEG (-0.45 MPa). Some isolates were found with >90% growth relative to control in medium having 30% PEG with water availability (1.33 MPa), nearly equivalent to that in soils which induce permanent wilting point of plants. When isolates that showed >90% growth relative to the control at both stress conditions, were stressed simultaneously, a decrease in growth was observed. Growth was reduced by approximately 20% at 35+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG and was affected to a greater degree in combinations of harsher stress conditions. The isolate ARSEF 2860 with a thermal threshold of >43 degrees C showed approximately 80% relative growth at a combined stress of 38+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG. These findings will aid the selection of isolates for use in field trials in hot or dry agricultural climates.     

Up-regulation of yggG promotes the survival of Escherichia coli cells containing Era-1 mutant protein

Era is a highly conserved GTPase essential for bacterial growth. Using a digoxigenin-labeled Era protein to screen a phage expression library of Escherichia coli genomic DNA, yggG, a gene that encodes a putative zinc metalloprotease was isolated and characterized. The deduced amino acid sequence of YggG showed high degrees of similarity to some reported heat shock proteins. In this study, the direct interaction between Era and YggG was confirmed, and it was found that the yggG gene, encoding a 25 kDa heat shock protein, was up-regulated at the mRNA level in partially defective Era GTPase mutants (era-1) and in E. coli cells overproducing Era-1. The delta yggG strain displayed the same growth rate as wild-type strain under normal growth conditions and after heat shock. Overexpression of Era-1 in the delta yggG strain resulted in a stronger growth-inhibitory effect than that in the wild-type strain, while coexpression of YggG partially restored the bacterial growth rate. The results indicated that YggG expression is significantly increased in response to stress caused by the Era-1 mutant protein in E. coli, thus promoting the growth of E. coli.     

Thermal properties of enzymes from Bacillus flavothermus,grown between 34 and 70°C

The activity and stability of several enzymes from the facultative thermophile Bacillus flavothermus, grown within the mesophilic and thermophilic region at 34 degrees C, 43 degrees C, 52 degrees C and 70 degrees C, have been examined. While the temperature optima and maxima of all enzymes tested were found to remain unchanged at all growth temperatures, it was demonstrated that the heat stability of the proteins increased with ten perature, however, not uniformly for all enzymes. One exception was acetate kinase and the intrinsic stability of pyruvate kinase was found to increase only slightly. With all other proteins tested (alanine dehydrogenase, isocitric dehydrogenase and glucose-6-phosphate dehydrogenase, glutamate-oxalacetate and glutamate-pyruvate transaminase and myokinase) the intrinsic stability was found to increase to about 55 degrees C, but stayed unaltered at higher growth temperatures. Except for acetate kinase and myokinase, the enzymes could be stabilized by their respective substrates and the heat stability of the ES-complexes was found also to depend on the growth temperature of the cells. These data lead to the conclusion that the enzymes undergo a transition from heat-labile to thermostable within the growth temperature range between 44 degrees C and 51 degrees C while the thermal characteristics are not changed below and beyond this crucial region.     

Thermosensation and pain

We feel a wide range of temperatures spanning from cold to heat. Within this range, temperatures over about 43 degrees C and below about 15 degrees C evoke not only a thermal sensation, but also a feeling of pain. In mammals, six thermosensitive ion channels have been reported, all of which belong to the TRP (transient receptor potential) superfamily. These include TRPV1 (VR1), TRPV2 (VRL-1), TRPV3, TRPV4, TRPM8 (CMR1), and TRPA1 (ANKTM1). These channels exhibit distinct thermal activation thresholds (>43 degrees C for TRPV1, >52 degrees C for TRPV2, > approximately 34-38 degrees C for TRPV3, > approximately 27-35 degrees C for TRPV4, < approximately 25-28 degrees C for TRPM8 and <17 degrees C for TRPA1), and are expressed in primary sensory neurons as well as other tissues. The involvement of TRPV1 in thermal nociception has been demonstrated by multiple methods, including the analysis of TRPV1-deficient mice. TRPV2, TRPM8, and TRPA1 are also very likely to be involved in thermal nociception, because their activation thresholds are within the noxious range of temperatures.     

Temperature effects on cell-functioning--a critical role for vicinal water.

Interfacially modified water ('vicinal water') appears to extend over distances of several molecular diameters from an adjacent interface and possesses highly unusual thermal properties. Specifically, the vicinal water undergoes relatively abrupt changes over narrow temperature intervals centered around 15 degrees, 30 degrees, 45 degrees and 60 degrees C and these changes most likely reflect higher order (structural) phase transitions. Vicinal water occurs at all interfaces (independent of specific surface characteristics) and will therefore also be present in all biological cells. As a result, the effects of vicinal water ramify through all of cell biology and this explains many unexpected thermal anomalies seen in the temperature responses of a great variety of living systems. Thus, the concept of the thermal anomalies may explain such observations as abrupt changes in enzyme functioning; anomalous temperature effects in membrane processes; unusual cell volume responses to temperature; dramatic changes in erythrocyte sedimentation rates; anomalies in chromosome aberration rates; pasteurization temperature; upper and lower thermal limits for microbial growth; multiple growth optima and minima and body temperatures of mammals and birds.     

Relationship between thermal tolerance and protein degradation in temperature-sensitive mouse cells.

The induction of thermotolerance was studied in a temperature sensitive mouse cell line, ts85, and results were compared with those for the wild-type FM3A cells. At the nonpermissive temperature of 39 degrees C, ts85 cells are defective in the degradation of short-lived abnormal proteins, apparently because of loss of activity of a ubiquitin-activating enzyme. The failure of the ts85 cells to develop thermotolerance to 41-43 degrees C after incubation at the nonpermissive temperature of 39 degrees C correlated with the failure of the cells to degrade short-lived abnormal proteins at 39 degrees C. However, the failure of the ts85 cells to develop thermotolerance to 43 degrees C during incubation at 33 degrees C after either arsenite treatment or heating at 45.5 degrees C for 6 or 10 min did not correlate with protein degradation rates. Although the rate of degrading abnormal protein was reduced after heating at 45.5 degrees C for 10 min, the rates were normal after arsenite treatment or heating at 45.5 degrees C for 6 min. In addition, when protein synthesis was inhibited with cycloheximide both during incubation at 33 degrees C or 39 degrees C and during heating at 41-43 degrees C, resistance to heating was observed, but protein degradation rates at 39 degrees C or 43 degrees C were not altered by the cycloheximide treatment. Therefore, there is apparently no consistent relationship between rates of degrading abnormal proteins and the ability of cells to develop thermotolerance and resistance to heating in the presence of cycloheximide.     

Thermotolerance in preirradiated intestine and its influence on time-temperature relationships

The crypt compartment of mouse jejunum showed a transient increase in thermal susceptibility approximately 10 days after moderate X-ray doses to the abdomen (9-10 Gy). The increase in response was manifest as an increase in slope of the crypt dose-response curve but was limited to temperatures below 43 degrees C. As a result, the 43 degrees C inflexion in the Arrhenius plot (the relationship between treatment time and temperature) for thermal sensitivity of crypts was eliminated in preirradiated tissue, and the curve became monophasic over the range 42.0-44.5 degrees C. At temperatures below 42 degrees C, the curve again deviated. At supranormal temperatures of 42 degrees C and below, the durations of hyperthermia needed for measurable effect were sufficient to allow thermotolerance to be expressed within the heating period. Neither the threshold heating times nor this thermotolerance were affected by prior irradiation. In the temperature range 42-43 degrees C, an earlier development of thermotolerance could be demonstrated in control tissue by challenging with an acute high-temperature heat treatment. This thermotolerance was eliminated in preirradiated tissue, resulting in the apparent increase in sensitivity. The findings support the view that the complex nature of the time-temperature relationship seen in normal tissue in vivo is a manifestation of the ability of the tissue to progressively acquire a thermotolerant state during treatment at temperatures below approximately 43 degrees C, so that the "intrinsic" sensitivity is modulated while being assessed.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Thermal dosimetry of normal porcine tissue

The response of normal porcine fat and muscle to graduated doses of hyperthermia provided by an annularly focused acoustic source was measured. Temperatures and exposure times were varied between 43 degrees C (20-90 min), 45 and 47 degrees C (20-60 min), and 49 degrees C (20 min). Response, based on histologic grading of the treated sites 30 days after exposure, was found to correlate well when mapped against several methods of estimating thermal energy deposition. The threshold for damage production was at or near 43 degrees C. For a given temperature, a nearly exponential increase in relative tissue damage as a function of increased exposure time was found. A twofold increase in tissue damage was produced in fat relative to muscle at any given thermal dose.     

A minor arginine tRNA mutant limits translation preferentially of a protein dependent on the cognate codon.

The Escherichia coli argU gene encodes a rare arginine tRNA (anticodon UCU) that translates the similarly rare AGA codon. The argU10(Ts) mutation is a transition that changes the first nucleotide of the mature tRNA from G to A, presumably destabilizing the acceptor stem. This mutation, when present in haploid condition in the chromosome, reduces the growth rate at 30 degrees C and results in cessation of growth after 60 to 90 min at 43 degrees C. The mutation also preferentially limits (compared with total protein synthesis) translation of an induced gene that depends on five AGA codons, i.e., the lambda cI repressor gene. Translation of another inducible protein, beta-galactosidase, which does not involve AGA codons, was inhibited to a much lesser extent. The chromosomal argU(Ts) mutation also confers the Pin phenotype, that is, loss of ability of the host, as a P2 lysogen, to inhibit growth of bacteriophage lambda, probably the result of reduced translation of the P2 old gene, which contains five AGA codons (E. Hagg?rd-Ljungquist, V. Barreiro, R. Calendar, D. M. Kurnit, and H. Cheng, Gene 85:25-33, 1989).     

Regulation of capsular polysialic acid biosynthesis by temperature in Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40 degrees C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP-Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43 degrees C (at least 25 times lower than those observed when the growth temperature was 37 degrees C). The synthesis of these enzymes increased rapidly when bacteria grown at 43 degrees C were transferred to 37 degrees C and decreased dramatically when cells grown at 37 degrees C were transferred to 43 degrees C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.     

The effects of temperature upon the electrophysiological properties of Tetrahymena pyriformis-NT1

Cells of Tetrahymena pyriformis--NT1 were cultured at 38 degrees C (Tg 38 degrees C) and 20 degrees C (Tg 20 degrees C) and their properties investigated over the range 0-40 degrees C. Tg 20 degrees C cells were viable in the range 3-33 degrees C and changes in their properties were readily reversible between 10 degrees C and 30 degrees C. Tg 38 degrees cells were viable in the range 40-10 degrees C and their property changes were immediately reversible in the range 40-23 degrees C. The I-V relations of Tg 38 degrees C cells showed increased excitability as the cells were cooled from 40 degrees C. At 10 degrees C there was a considerable loss of excitability and slope resistance. Cooling Tg 20 degrees C cells from 20 degrees C gave a similar pattern, although over a narrower temperature range. Warming Tg 20 degrees C Tetrahymena above 20 degrees C led to a progressive loss of excitability and the cells were markedly less viable above 35 degrees C. Within physiological limits the regenerative spike magnitude, repolarization time, time to peak and input resistance increased as temperature was lowered, whereas resting potential was diminished. When compared at their growth temperatures and most intermediate temperatures, the value of the various parameters monitored were generally different for the two cultures. The Q10 value for resting potential changes of Tg 20 degrees C cells about 20 degrees C was 1.20. As in T. vorax this was significantly (P less than 0.01) greater than that predicted for a diffusion potential and suggested that T. pyriformis--NT1 may have an electrogenic pump component in its membrane potential.