Temperature differentially affects adenosine triphosphatase activity in Hsc70 orthologs from Antarctic and New Zealand notothenioid fishes

To test the temperature sensitivity of molecular chaperones in poikilothermic animals, we purified the molecular chaperone Hsc70 from 2 closely related notothenioid fishes--the Antarctic species Trematomus bernacchii and the temperate New Zealand species Notothenia angustata--and characterized the effect of temperature on Hsc70 adenosine triphosphatase (ATPase) activity. Hsc70 ATPase activity was measured using [alpha-32P]-adenosine triphosphate (ATP)-based in vitro assays followed by separation of adenylates by thin-layer chromatography. For both species, a significant increase in Hsc70 ATPase activity was observed across a range of temperatures that was ecologically relevant for each respective species. Hsc70 from T bernacchii hydrolyzed 2-fold more ATP than did N angustata Hsc70 at 0 degrees C, suggesting that the Antarctic molecular chaperone may be adapted to function more efficiently at extreme cold temperatures. In addition, Q10 measurements indicate differential temperature sensitivity of the ATPase activity of Hsc70 from these differentially adapted fish that correlates with the temperature niche inhabited by each species. Hsc70 from T bernacchii was relatively temperature insensitive, as indicated by Q10 values calculated near 1.0 across each temperature range measured. In the case of Hsc70 purified from N angustata, Q10 values indicated thermal sensitivity across the temperature range of 0 degrees C to 10 degrees C, with a Q10 of 2.714. However, Hsc70 from both T bernacchii and N angustata exhibited unusually high thermal stabilities with ATPase activity at temperatures that far exceeded temperatures encountered by these fish in nature. Overall, as evidenced by in vitro ATP hydrolysis, Hsc70 from T bernacchii and N angustata displayed biochemical characteristics that were supportive of molecular chaperone function at ecologically relevant temperatures.     

Kinetic and structural analysis of the inhibition of adenosine deaminase by acetaminophen

Kinetic and thermodynamic studies have been made on the effect of acetaminophen on the activity and structure of adenosine deaminase in 50 mM sodium phosphate buffer pH 7.5, at two temperatures of 27 and 37 degrees C using UV spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. Acetaminophen acts as a competitive inhibitor at 27 degrees C (Ki = 126 microM) and an uncompetitive inhibitor at 37 degrees C (Ki = 214 microM). Circular dichroism studies do not show any considerable effect on the secondary structure of adenosine deaminase by increasing the temperature from 27 to 37 degrees C. However, the secondary structure of the protein becomes more compact at 37 degrees C in the presence of acetaminophen. Fluorescence spectroscopy studies show considerable change in the tertiary structure of the protein by increasing the temperature from 27 to 37 degrees C. Also, the fluorescence spectrum of the protein incubated with different concentrations of acetaminophen show different inhibition behaviors by the effector at the two temperatures.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Influence of temperature acclimatization on the ionic activation of goldfish intestinal adenosine triphosphatase

1. An adenosine triphosphatase membrane system, dependent on Mg²⁺ and activated further by Na⁺+K⁺, was prepared from goldfish anterior intestine by differential centrifugation of homogenized intestinal scrapings. 2. The affinity of this preparation for Na⁺ in the presence of K⁺+Mg²⁺, for K⁺ in the presence of Na⁺+Mg²⁺ and for Mg²⁺ alone, measured at 37°, did not depend on the previous environmental temperature of the fish. When Na⁺+K⁺ were added to preparations from 8°-acclimatized fish the affinity for Mg²⁺ increased; this was not seen with preparations from 30°-acclimatized fish. 3. Part of the Mg²⁺-activated adenosine triphosphatase was inhibited by Na⁺ and the amount of inhibition appeared to increase at high acclimatization temperatures. 4. This Na⁺-inhibited adenosine triphosphatase was separated from the (Na⁺+K⁺)-activated enzyme by centrifugation on sucrose density gradients. 5. Preparations from 8°-acclimatized fish contained less Mg²⁺-activated and more (Na⁺+K⁺)-activated adenosine triphosphatase than did similar fractions from 30°-acclimatized fish. 6. Acclimatization to different environmental temperatures might involve one form of adenosine triphosphatase changing to another. The origin of various membranes seen in microsomal fractions must, however, be established before this hypothesis can be tested further.     

Temperature-induced alanine oxidation in a psychrotrophic Pseudomonas.

A psychrotrophic pseudomonad isolated from iced fish oxidized alanine at temperatures close to 0 degrees C and grew over the range 0 degrees C-35 degrees C. The rate of oxidation of alanine, measured manometrically, by cells grown at 2 degrees C was lower than that of cells grown at 22 degrees C. However, the consumption of oxygen after heat treatment at 35 degrees for 35 min was reduced considerably by 2 degrees C grown cells. Alanine oxidase activity was tested in an extract from cells grown at 2 degrees C and 22 degrees C with alanine as the sole carbon, nitrogen, and energy source. Cells grown at 2 degrees C produced an alanine oxidase with a temperature optimum of 35 degrees C and pH optimum of 8, which lost about 80% activity by heat treatment at 40 degrees C for 30 min. There was no change in activity after dialysis at pH 7, 8, or 9. Extracts from cells grown at 22 degrees C contained an alanine oxidase system with an optimum temperature of 45 degrees C, a pH optimum above 8, and only about 30% reduction of activity after heat treatment. This enzyme activity was concentrated in the 0.5 M elution fraction from a Sephadex column, and dialysis reduced the activity at pH 7 and 8. Mesophilic enzyme synthesis apparently started around a growth temperature of 10 degrees C. The crude alanine oxidase systems of Pseudomonas aeruginosa derived from cells grown at 13 degrees C and 37 degrees C had a common optimum temperature of 45 degrees C. These data suggest that one mechanism of psychrophilic growth by psychrotrophic bacteria may be the induction of enzymes with low optimum temperatures in response to low temperature conditions.     

Adenosine Phosphate Hydrolases in Cell Fractions of Vitreoscilla

Bound, soluble, and whole-cell fractions of two strains of the gliding bacterium Vitreoscilla were found to contain two enzymes capable of hydrolyzing adenosine phosphates: a Mg⁺⁺-activated adenosine triphosphatase with a temperature optimum of 37 C, and a Mg⁺⁺-activated adenosine diphosphatase with a temperature optimum of 55 C. Both enzymes had an optimal pH response between 8.5 and 9.5. Maximal activation was achieved at an ionic strength of 0.2 for the adenosine triphosphatase and at 0.3 to 0.4 for the adenosine diphosphatase. Preliminary studies indicated a molecular weight of approximately 50,000 for the adenosine diphosphatase and a molecular weight greater than 60,000 for the adenosine triphosphatase. Comparisons are made with previously reported characteristics of these enzymes in other bacteria, and a hypothesis is offered as to the role these enzymes have in the gliding mechanism.     

The effect of lipid peroxidation on the calcium-accumulating ability of the microsomal fraction isolated from chicken breast muscle.

The effect of lipid peroxidation on the Ca2+-accumulating and Ca2+-retaining abilities of the microsomal fraction from chicken breast muscle was investigated. At 25 degrees C, enzymic lipid peroxidation did not seriously affect either of these abilities unless ascorbic acid was present, when both were diminished. At 37 degrees C, Ca2+-concentrating ability was decreased further by the effects of heat damage to the membrane. Membrane lipid peroxidation did not affect microsomal adenosine triphosphatase activity unless the microsomal fraction was subsequently washed with albumin. This effect of albumin is possibly due to removal of lipid-breakdown products. Addition of soya-bean phospholipids to the peroxidized vesicles washed with albumin restored adenosine triphosphatase activity, demonstrating a non-specific phospholipid requirement.     

Post-mortem changes in cytochemical localization and enzymological measurement of marker enzymes of the mitochondria, SDH and Mg-ATPase, of porcine muscle stored at 4 degrees C, -18 degrees C, or -80 degrees C

As a series of studies on postmortem changes in the fine structure of porcine muscle, activity of two mitochondrial marker enzymes, succinate dehydrogenase (SDH) and magnesium dependent adenosine triphosphatase (Mg-ATPase), was measured and localized in cardiac, red and white muscles stored at 4 degrees C, -18 degrees C or -80 degrees C. The postmortem loss of SDH activity was most remarkable in cardiac muscle. The variation of SDH activity was proportional to the amount of absolute activity. The postmortem change of Mg-ATPase was more variable than SFH, though the activity was well preserved up to 15 weeks in all three types of porcine muscle stored at -80 degrees C. The loss of Mg-ATPase was most remarkable in red muscle stored at -18 degrees C or -80 degrees C. Cytochemical localization of SDH was between the outer and the inner mitochondrial membranes while that of Mg-ATPase was on the inner surface or matrix side of the inner membrane. Those localization was not altered by the difference in temperature and the duration of storage.     

Anisakis simplex: uncoupling of oxidative phosphorylation in the muscle mitochondria of infected fish

Adenosine triphosphatase activity stimulated by Mg2+ was greater in muscle mitochondria of fish infected with larval Anisakis simplex nematodes than in uninfected fish. When muscle mitochondria were isolated in a sucrose ethylene-glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid medium from fresh uninfected fish, they were loosely coupled, and their adenosine triphosphatase activity was comparable to that of mitochondria from rat tissue. Activity in infected fish was dose dependent, increasing with the number of worms per fish. Excretory secretory products or a cytoplasmic fraction of anisakines, when incubated with coupled rat mitochondria, also caused adenosine triphosphatase activity to increase. Storage of fish flesh caused an increase in adenosine triphosphatase activity, but such aging was not significant until 5 and 10 days after death in refrigerated and frozen samples, respectively. The Mg2+ stimulated adenosine triphosphatase activity of muscle mitochondria can be used to estimate the number of nematodes per market fish. The type of medium used to isolate the mitochondria is crucial in such studies; an ionic medium with Nagarse proteinase was optimal for fish muscle mitochondria.     

The effect of incubation temperature on the maintenance of rat palatal mucosa in organ culture

The effect of incubation temperature on the behavior of neonatal rat palatal mucosa maintained in a chemically defined medium in organ culture for periods up to 7 days was investigated. Explant survival was optimal at 37 degrees C with increasing mortality at temperatures of 34 degrees C and 30 degrees C. There was a transient increase in the epithelial mitotic activity at all temperatures, but at all time intervals mitotic activity was greatest at 37 degrees C. While the mitotic activity at 37 degrees C after 5 hr in vitro was comparable with previously described in vivo values, it was subsequently increased, only returning to values approximating those at the start of the experiment at 6 days. At 30 degrees and 34 degrees C the epithelial mitotic activity increased more slowly than at 37 degrees C; then it followed a similar pattern with time and after 5 days in vitro had fallen to values approximating initial values. At the cut edges of the explants, the rate of epithelial migration and subsequent keratinization increased with increasing temperature. It is suggested that survival of neonatal rat palatal mucosa is optimal in this organ culture system when maintained at 37 degrees C.     

Temperature dependent behavior of the canine medial collateral ligament

The temperature dependent tensile behavior of ligament was investigated from 2 degrees C to 37 degrees C. Nondestructive cyclic tests were performed on ten canine femur-medial collateral ligament-tibia (FMT) complexes at sequential temperatures of 22 degrees C, 22 degrees C, 27 degrees C, 32 degrees C, 37 degrees C, and again at 22 degrees C. The samples were rested at zero load between tests for sufficient time periods to allow for full recovery from the ligament's time and history dependent viscoelastic properties. Ten additional FMT complexes were sequentially tested in a similar fashion, but at temperatures of 22 degrees C, 22 degrees C, 2 degrees C, 6 degrees C, 14 degrees C, and 22 degrees C. All canine FMT complexes showed temperature dependent viscoelastic properties: the measured area of hysteresis decreased with increasing temperature; the cyclic load relaxation behavior plateaued to a higher value at lower temperatures; and the tensile load at a predetermined ligament substance strain level had an inversely proportional relationship with respect to temperature.     

Factors affecting the cold inactivation of an acetyl-coenzyme-A hydrolase purified from the supernatant fraction of rat liver

An extramitochondrial acetyl-coenzyme-A hydrolase from rat liver is shown to be a cold-labile oligomeric enzyme that undergoes a reversible conformational transition between a dimeric and a tetrameric form in the presence of adenosine 5'-triphosphate or adenosine 5'-diphosphate at 25-37 degrees C, and between a dimeric and a monomeric form at low temperature. The enzymatically active dimer is fairly stable at 25-37 degrees C, but much less stable at low temperature, dissociating into monomer with no activity. At 37 degrees C and low concentrations of enzyme protein (less than or equal to 14 micrograms/ml), the activity decreased rapidly and only 10% of the initial activity remaining after 60 min. Addition of bovine serum albumin or immunoglobulin G to the medium completely prevented inactivation of the dimeric enzyme at low concentration at 37 degrees C, but had little effect on cold inactivation of the enzyme. Cold inactivation of the dimeric enzyme was partially prevented by the presence of various CoA derivatives. The order of potency was acetyl-CoA (substrate) greater than or equal to butyryl-CoA greater than octanoyl-CoA greater than CoA (product) greater than acetoacetyl-CoA. Another enzyme product, acetate, had little effect on cold inactivation. Polyols, such as sucrose, glycerol, and ethylene glycol, and high concentrations of NaCl, KCl, pyrophosphate and phosphate also greatly prevented cold inactivation. Cold inactivation was scarcely affected by pH within the pH range at which the enzyme was stable at 37 degrees C.     

Transient regulation of protein synthesis in Escherichia coli upon shift-up of growth temperature.

Synthesis of total cellular proteins of Escherichia coli was studied upon transfer of a log-phase culture from 30 (or 37) to 42 degrees C. Cells were pulse-labeled with [3H]leucine, and the labeled proteins were analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate. The rates of synthesis of at least five protein chains were found to increase markedly (5- to 10-fold) within 5 min after temperature shift-up and gradually decrease to the new steady-state levels, in contrast to the majority of proteins which gradually increase to the steady-state levels (about 1.5-fold the rate at 30 degrees C). Temperature shift-down did not cause any appreciable changes in the pattern of protein synthesis as detected by the present method. Among the proteins greatly affected by the temperature shift-up were those with apparent molecular weights fo 87,000 (87K), 76K, 73K, 64K, and 61K. Two of them (64K and 61K) were found to be precipitated with specific antiserum against proteins that had previously been shown to have an adenosine triphosphatase activity. The bearings of these findings on bacterial adaptation to variation in growth temperature are discussed.     

Adenylate energy charge in Escherichia coli CR341T28 and properties of heat-sensitive adenylate kinase

Escherichia coli strain CR341T28 will not grow at temperatures above 34 degrees C in liquid medium, and the adenylate kinase of this strain is heat sensitive. When a culture was shifted from a permissive (30 degrees C) to a nonpermissive (36 degrees C) temperature, the adenylate energy charge fell from 0.9 to 0.2, with a concurrent decrease in the number of viable cells and in the specific activity of adenylate kinase. When cultures of the temperature-sensitive strain were grown at temperatures above 30 degrees C, the adenylate energy charge, the specific activity of adenylate kinase, and the growth rate were lower than the corresponding parameters for the parental strain. By isotopic labeling of the adenine nucleotides in vivo, it was determined that increasing growth temperatures between 30 and 34 degrees C for the heat-sensitive strain resulted in a decrease in the adenosine triphosphate-to-adenosine monophosphate and adenosine triphosphate-to-adenosine diphosphate ratios. Between 26 and 30 degrees C the adenosine triphosphate-to-adenosine diphosphate ratio was essentially normal in the temperature-sensitive strain, but the adenosine triphosphate-to-adenosine diphosphate ratio was decreased. The adenylate ratios in the parental strain did not change between 30 and 34 degrees C. The adenylate kinase mass action ratio for each strain was essentially constant under all growth conditions. When assayed at 30 degrees C, the affinities of the enzyme from the mutant strain were somewhat lower than those of the parent adenylate kinase. The mutant enzyme also did not exhibit the substrate inhibition that was observed at high adenosine monophosphate concentrations with the parental enzyme. An increase in the assay temperature from 30 degrees to 40 degrees C had little or no effect on the Km values determined for the parental adenylate kinase, but caused the Km values determined for the mutant adenylate kinase to increase by a factor of two or more.     

Exhaustive exercise does not affect the preferred temperature for recovery in juvenile rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that juvenile rainbow trout (Oncorhynchus mykiss) would select a temperature colder than their acclimation temperature (16 deg +/-1 deg C) to minimize postexhaustive exercise metabolic demands and enhance oxygen availability. After an initial 3-h exploratory period in a thermal gradient (6 degrees -25 degrees C), fish selected a temperature of approximately 14 degrees C and had a baseline exploratory swimming activity of approximately 60 cm min(-1). Subsequently, experimental (chased) fish were individually removed, exhaustively exercised for 1.5 min, and replaced. Both control (unchased) and experimental fish were allowed to explore the thermal gradient for another 2 h. Immediately after being chased, trout had a metabolic profile that was consistent with being exhausted; levels of plasma and muscle lactate were 4.38+/-0.25 mmol L(-1) and 28.0+/-2.0 mmol kg(-1), respectively, and levels of muscle glycogen, adenosine triphosphate, and phosphocreatine were 3.89+/-0.95, 4.23+/-0.62, and 3.07+/-0.73 mmol kg(-1), respectively. Although exploratory swimming activity of the chased fish was significantly lower (by 81%) as compared with control fish during the first 5 min postchase, differences in the mean, median, and mode values for selected temperatures during the next 2 h were neither large (<1 degrees C) nor significant (P>0.05). Contrary to our initial hypothesis, these findings suggest that juvenile rainbow trout do not select a colder temperature to decrease metabolic rate following exhaustive exercise. Instead, rainbow trout selected a temperature marginally cooler than their acclimation temperature (16 degrees C) regardless of whether they had been previously exhausted.     

Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation.

1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5'-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7--8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only.     

Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.     

Temperature effect on the immune defense functions of Arctic charr Salvelinus alpinus

The Arctic charr Salvelinus alpinus is an endangered fish species in Finland, and thus farming is carried out mainly for stocking purposes. Farmed charr are susceptible to infection with atypical Aeromonas salmonicida (aAS). Losses of valuable brood stock will severely reduce the genetic diversity of stocked charr. No commercial vaccines are available to prevent aAS infection, and vaccines against furunculosis (caused by typical A. salmonicida, tAS) do not protect the charr against aAS infection. The effects of a metabolizable oil-adjuvanted, bivalent vaccine (containing killed aAS and A. salmonicida salmonicida bacteria) on the immune system of 1 yr old hatchery-reared charr originating from Lake Inari in Northern Finland were examined. Fish vaccination in Finland generally takes place either from October to November or from February to April, when the water temperature is low (1 to 3degrees C). The water temperature starts to increase in mid-May. Therefore, we also investigated whether post-vaccination (p.v.) temperature had an influence on the immune system of this cold-water fish species. The fish were immunized intraperitoneally at 2.9 degrees C at the end of April. After 52 d, during which the water temperature increased from 2.9 to 10.0 degrees C, the charr were exposed to 1 of 3 test temperatures: 10.3, 14.1 or 18.1 degrees C. Prior to vaccination, and 49, 75 and 103 d p.v., several immune parameters were measured in both unvaccinated and vaccinated charr. Vaccination induced a significant anti-aAS-specific antibody response, and increased plasma lysozyme activity at all p.v. temperatures. The haemolytic activity of the complement system was unaffected either by vaccination or p.v. temperatures. There was a slight positive correlation between p.v. temperature and lysozyme activity of the charr. The significant increase in lysozyme activity took place in vaccinated charr in the first 49 d p.v. as water temperatures increased from 2.9 to 10 degrees C. Furthermore, the highest activity of lysozyme in the plasma was observed 49 d p.v. Our results indicate that a rise in water temperature above 10 degrees C does not significantly enhance the vaccination response of charr. This could be one reason why farmed Arctic charr, which are well adapted to a cold climate, are highly susceptible to aAS infection in the summer.     

Is fluid-phase endocytosis conserved in hepatocytes of species acclimated and adapted to different temperatures?

Our primary objective was to determine if rates of fluid-phase endocytosis (FPE) were conserved in hepatocytes from organisms acclimated and adapted to different temperatures. To this aim, the fluorescent dye Lucifer yellow was employed to measure FPE at different assay temperatures (AT) in hepatocytes from 5 degrees C- and 20 degrees C-acclimated trout, Oncorhynchus mykiss (at 5 and 20 degrees C AT), 22 degrees C- and 35 degrees C-acclimated tilapia, Oreochromis nilotica (at 22 and 35 degrees C AT), and the Sprague-Dawley rat (at 10, 20, and 37 degrees C AT). FPE was also studied in rats fed a long-chain polyunsaturated fatty acid (PUFA)-enriched diet (at 10 degrees C AT). Despite being temperature dependent, endocytic rates (values in pl. cell(-1). h(-1)) in both species of fish were compensated after a period of acclimation. For example, in 20 degrees C-acclimated trout, the rate of endocytosis declined from 1.84 to 1.07 when the AT was reduced from 20 to 5 degrees C; however, after a period of acclimation at 5 degrees C, the rate (at 5 degrees C AT) was largely restored (1.80) and almost perfectly compensated (95%). In tilapia, endocytic rates were also temperature compensated, although only partially (36%). Relatively similar rates obtained at 5 degrees C in 5 degrees C-acclimated trout (1.8), at 20 degrees C in 20 degrees C-acclimated trout (1.84), and at 22 degrees C in 22 degrees C-acclimated tilapia (2.2) suggest that endocytic rates are somewhat conserved in these two species of fish. In contrast, the rate in rat measured at 37 degrees C (16.83) was severalfold greater than in fish at their respective body temperatures. A role for lipids in determining rates of endocytosis was supported by data obtained at 10 degrees C in hepatocytes isolated from rats fed a long-chain PUFA-enriched diet: endocytic rates were higher (5.35 pl. cell(-1). h(-1)) than those of rats fed a standard chow diet (2.33 pl. cell(-1). h(-1)). The conservation of endocytic rates in fish may be related to their ability to conserve other membrane characteristics (i.e., order or phase behavior) by restructuring their membrane lipid composition or by modulating the activities of proteins that regulate endocytosis and membrane traffic, whereas the lack of conservation between fish and rat may be due to differences in metabolic rate.     

Effect of temperature on the seasonal production of testicular androgens, in vitro, by the lizard Tiliqua rugosa

The effect of temperature on the seasonal production of testicular androgens, in vitro, was examined in the scincid lizard Tiliqua (Trachydosaurus) rugosa. Testicular tissue was incubated, in vitro, at various temperatures (18, 25, 32 and 37 degrees C). Endogenous androgens, testosterone and epitestosterone, were measured by radioimmunoassay. Epitestosterone production was maximal at 37 degrees C and minimal at 18 degrees C. There was no consistent effect of incubation temperature on testosterone production. Incubation temperature had no effect on the seasonal pattern of androgen production. The results suggest that temperature may play a part in the regulation of androgen biosynthesis in T. rugosa.