Polymerization of Antarctic fish tubulins at low temperatures: energetic aspects

Tubulins were purified from the brain tissues of three Antarctic fishes, Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus, by ion-exchange chromatography and one cycle of temperature-dependent microtubule assembly and disassembly in vitro, and the functional properties of the protein were examined. The preparations contained the alpha- and beta-tubulins and were free of microtubule-associated proteins. At temperatures between 0 and 24 degrees C, the purified tubulins polymerized readily and reversibly to yield both microtubules and microtubule polymorphs (e.g., "hooked" microtubules and protofilament sheets). Critical concentrations for polymerization of the tubulins ranged from 0.87 mg/mL at 0 degrees C to 0.02 mg/mL at 18 degrees C. The van't Hoff plot of the apparent equilibrium constant for microtubule elongation at temperatures between 0 and 18 degrees C was linear and gave a standard enthalpy change (delta H degree) of +26.9 kcal/mol and a standard entropy change (delta S degree) of +123 eu. At 10 degrees C, tubulin from N. gibberifrons polymerized efficiently at high ionic strength; the critical concentration increased monotonically from 0.041 to 0.34 mg/mL as the concentration of NaCl added to the assembly buffer was increased from 0 to 0.4 M. Together, the results indicate that the polymerization of tubulins from the Antarctic fishes is entropically driven and suggest that an increased reliance on hydrophobic interactions underlies the energetics of microtubule formation at low temperatures. Thus, evolutionary modification to increase the proportion of hydrophobic interactions (relative to other bond types) at sites of interdimer contact may be one adaptive mechanism that enables the tubulins of cold-living poikilotherms to polymerize efficiently at low temperatures.     

Formation of microtubules at low temperature by tubulin from antarctic fish

Tubulin was isolated from two species of antarctic fish, Pagothenia borchgrevinki and Dissostichus mawsoni, by cycles of temperature-dependent assembly, centrifugation, disassembly, and centrifugation. The preparations were found to consist almost entirely of tubulin and to contain negligibly small amounts of microtubule-associated proteins. This tubulin polymerized to make microtubules of ordinary dimensions. The formed microtubules appear to be in labile equilibrium with free tubulin dimer at all temperatures observed. In a buffer consisting of 0.1 M 1,4-piperazinediethanesulfonic acid, 2 mM dithioerythritol, 1 mM MgSO4, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1 mM guanosine 5'-triphosphate, pH 6.9, the tubulin of P. borchgrevinki has a critical concentration for assembly of 0.046 (+/- 0.008) mg/mL at 35 degrees C and 0.74 (+/- 0.15) mg/mL at the habitat temperature of the fish, -1.8 degrees C. The critical concentration measured at the lower temperature is quite small relative to the critical concentration for formation of mammalian microtubules from pure tubulin at the same temperature, which must be at least 2 orders of magnitude larger. The antarctic fish microtubules may thus be called "cold stable" by comparison with mammalian microtubules. They do not fully dissociate at temperatures near 0 degree C because they are composed of tubulin that assembles more readily at these temperatures than does mammalian tubulin. There is no evidence for the presence of a cold-stabilizing factor in association with the tubulin. These findings suggest that alteration of tubulin may be a means by which some poikilotherms can adapt to a cold environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stenotherms at sub-zero temperatures: thermal dependence of swimming performance in Antarctic fish

We examined the burst swimming performance of two Antarctic fishes, Trematomus bernacchii and T. centronotus, at five temperatures between -1 degrees C and 10 degrees C. As Antarctic fishes are considered one of the most cold specialised and stenothermal of all ectotherms, we predicted they would possess a narrow thermal performance breadth for burst swimming and a correlative decrease in performance at high temperatures. Burst swimming was assessed by videotaping swimming sequences with a 50-Hz video camera and analysing the sequences frame-by-frame to determine maximum velocity, the distance moved throughout the initial 200 ms, and the time taken to reach maximum velocity. In contrast to our prediction, we found both species possessed a wide thermal performance breadth for burst swimming. Although maximum swimming velocity for both T. bernacchii and T. centronotus was significantly highest at 6 degrees C, maximum velocity at all other test temperatures was less than 20% lower. Thus, it appears that specialisation to a highly stable and cold environment is not necessarily associated with a narrow thermal performance breadth for burst swimming in Antarctic fish. We also examined the ability of the Antarctic fish Pagothenia borchgrevinki to acclimate their burst-swimming performance to different temperatures. We exposed P. borchgrevinki to either -1 degrees C or 4 degrees C for 4 weeks and tested their burst-swimming performance at four temperatures between -1 degrees C and 10 degrees C. Burst-swimming performance of Pagothenia borchgrevinki was unaffected by exposure to either -1 degrees C or 4 degrees C for 4 weeks. Maximum swimming velocity of both acclimation groups was thermally independent over the total temperature range of 1 degrees C to 10 degrees C. Therefore, the loss of any capacity to restructure the phenotype and an inability to thermally acclimate swimming performance appears to be associated with inhabiting a highly stable thermal environment.     

Blood rheology of Antarctic fishes: viscosity adaptations at very low temperatures

Viscosity of whole blood and plasma from Antarctic fishes were compared over a temperature range of −1.5 to 5°C; human samples and water provided reference values. Blood viscosity of nototheniids was greater than that of the haemoglobinless icefish, reflecting differences in packed cell volume, being 5.27 v. 3.27 cP at 0°C for Notothenia coriiceps and Chaenocephalus aceratus , respectively. The reduction in MCHC, rather than haematocrit, in nototheniids suggests that selection pressure has not acted at the level of oxygen transport. However, icefish plasma viscosity was similar to human, but greater than that of Notothenia spp., suggesting that viscometric influences on cardiac afterload may be adaptive for the latter. Indeed, handling stress induced a significant increase in viscosity of both whole blood and plasma which may impair cardiovascular performance. Such a response was not observed in icefish, and in view of the large blood vessels it is unlikely that viscosity plays any significant role in limiting activity of this species.     

Antarctic fish can survive prolonged exposure to elevated temperatures

The Antarctic notothenioid Pagothenia borchgrevinki was collected from the stenothermal waters of McMurdo Sound in the summers of 2004, 2005 and 2006. Acclimation ability at 4° C was tested in healthy P. borchgrevinki and in individuals infected with x‐cell gill disease. All healthy fish successfully acclimated to 4° C, establishing compensatory changes in resting oxygen consumption rate (R_rest) and critical swimming speed (U_crit) during a 1 month acclimation period, which were maintained during a longer, 6 month acclimation period. In contrast, individuals infected with x‐cell disease were unable to acclimate to 4° C, demonstrating significantly reduced survival rates compared with healthy individuals at 4° C. Measurements of R_rest suggest that limitations in the ability of x‐cell fish to uptake oxygen from the external milieu may have a negative effect on their survival at 4° C.     

Characteristics of leucine transport by isolated hepatocytes of antarctic fish at low temperatures

Hepatocytes prepared by collagenase perfusion from Antarctic nototheniid fish of genus Trematomus are active in uptake of [¹⁴C]leucine at 0, 5, and 10°C. The system is saturable with apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 1.0 mM. Isoleucine and phenylalanine were major competitors, valine was about one-half as effective, while alanine, glycine and histidine had no effect. Temperature dependency of rates in the 0–10°C range yielded $\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{= 65}\text{kJ/mol}$ ($\text{Q}{}_{10}\text{= 2.7}$). The average first-order rate constant at 0°C was 0.1 min^?1, one-third the value of 0.3 min^?1 estimated for clearance of [¹⁴C]leucine by liver of these species in vivo. Affinity and specificity agreed well with in vivo data on liver clearance of leucine, both in Antarctic fish at 0°C and in temperate fish acclimated to 10°C and 20°C. The results indicate similar modifications of leucine transport associated with evolutionary cold adaptation and seasonal acclimation in fish.     

Dynamics of lipid oxidation and antioxidant depletion in Mediterranean fish stored at different temperatures

Numerous changes occur post-mortem in fish, affecting its chemical composition and nutritional quality. In the present paper we describe the effect of storage on ice or at -30 degrees C or -80 degrees C on 10 species of Mediterranean fish. Water and lipid soluble antioxidants, lipid pattern and products of oxidative attack on lipids, proteins and DNA were quantified for 7 consecutive days on homogenates of fish light muscle. The earliest events were oxidation of ubiquinol and vitamin C, which disappeared almost completely within 48 hours. Ubiquinol oxidation gave rise to an initial increase of ubiquinone, which peaked at the second day: thereafter ubiquinone itslef decreased, more rapidly and to a greater extent than vitamin E. The decrease in antioxidants was accompanied by significant oxidative damage to lipids, proteins and DNA. TBARS significantly increased beginning from the third day of storage in all species and were linked to a significant reduction in the n-3 PUFA of triglycerides (TG) and phospholipid fractions (PL). A remarkable elevation of protein carbonyls and 8OHdG occurred approximately 24 hours later than PUFA oxidation. For SOD, GPX and GSH significant depletions occurred for all species only at 6th or 7th day, but the final values were always higher than 50% compared to the initial ones. Deep-freezing of the same species at -30 degrees C and -80 degrees C for up to 12 months did not significantly affect the levels of enzymatic antioxidants, the redox couple GSH/GS-SG, n-3 and n-6 PUFA of TG and PL fractions of the light muscle. The only antioxidants, which at -30 degrees C and -80 degrees C appeared to be degraded after 6 and 12 months were ubiquinol and vitamin C. As expected their degradation was higher at -30 degrees C than at -80 degrees C. In fact the average decrease for ubiquinol at -80 degrees C was 42% at 6 and 12 months respectively, whereas at -30 degrees C the decrease was 61% and 87% For vitamin C the average decrease at -80 degrees C was 36% and 67% at 6 and 12 months respectively, and at -30 degrees C it was 61% and 82%. Vitamin E was considerably more stable than ubiquinol and vitamin C. The relative stability of the antioxidants, with the exceptions of ubiquionols, vitamin C and, to a certain extent, vitamin E, was accompanied by a very limited increase in oxidation products. In addition no significant hydrolysis of TG and PL fractions were observed throughout the storage time. The dynamics of lipid, protein and DNA oxidation is discussed in the light of depletion of the various antioxidant systems.     

Photodynamic hemolysis at low temperatures

It is shown that photodynamic hemolysis may occur at –79°C. if the erythrocytes are suspended in a solution containing 70 per cent glycerol which prevents hemolysis by freezing; but that there is no hemolysis under the same conditions at –210°C. At the higher temperature the viscosity of the solution is still low enough to permit appreciable movement of molecules, whereas at the lower temperature the molecules must be virtually immobile. The findings are compatible with the idea that the dye molecule acts in a cycle, bringing about successive oxidations by O₂ molecules, as has been shown for photodynamic hemolysis at room temperature. The assumption of a combination between dye, O₂, and substrate does not explain photosensitized hemolysis in the semi-solid state. The mechanism of photosensitized oxidation by O₂ is discussed.     

Flow cytometric analysis from fish samples stored at low,ultra-low and cryogenic temperatures

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (−20 °C), ultra-low (−80 °C) and cryogenic temperatures (−196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (−20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (−80 °C) and cryogenic (−196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at −80 °C and −196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation.     

High-pressure stopped-flow spectrometry at low temperatures

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.     

Bacterial gene expression at low temperatures

Under suboptimal environmental conditions such as low temperatures, many bacteria have an extended lag phase, altered cell structures, and composition such as a less fluid (more rigid) and leaky cytoplasmic membrane. As a result, cells may die, enter into a starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. In the latter state, the amount of gene expression per cell is virtually undetectable. In this article, gene expression under (suboptimal) low temperature conditions in non-psychrophilic environmental bacteria is examined. The pros and cons of some of the molecular methodologies for gene expression analysis are also discussed.     

Microbial lipolysis at low temperatures.

It was found that lipase production during the growth of Pseudomonas fluorescens was not a function of the total number of bacteria. The optimal temperatures for bacterial growth and lipase production were determined as 20 and 8 degrees C, respectively. The lipolytic activity was studied in emulsions of olive oil at temperatures ranging from +8 to -30 degrees C. After an initially rapid lipolysis, the reactions retarded at different levels depending on storage temperature. Transference to a higher temperature resulted in a resumed lipolysis. Also, at low temperatures, lipolysis was studied as a function of water activity and was found to occur in dehydrated substrates.     

Minnows become nocturnal at low temperatures

Juveniles of several species of salmonid become almost exclusively nocturnal in winter. Their diel activity schedule is determined mainly by temperature, with the fish seeking shelter increasingly during the day as the temperature drops. Through controlled laboratory experiments, it is demonstrated that a comparable response is found in another stream-dwelling species, the minnow Phoxinus phoxinus . Daytime observations showed that fish were found hiding in refuges on only 20% of occasions at 13.2° C but on over 75% of occasions at 6.6° C. In contrast, the minnows remained in the water column and used the refuges rarely at night irrespective of water temperature. As a consequence their activity became increasingly nocturnal as temperature dropped. It is suggested that this may be an adaptation to avoid diurnal predators.     

The feeling of dampness at low temperatures

The widely held view that in cold weather one feels colder when the atmosphere is damp than when it is dry was investigated on clothed subjects. Subjective coldness and dampness, determined by a panel of six people who walked about half a mile in the open in winter, were compared with measurements of temperature, relative humidity, and other meteorological variables. The panellists comments showed that when the weather is cold, people think it is damp when the sky is overcast, irrespective of relative humidity. However, when this factor has been allowed for there still appears to be some correlation between subjective dampness and relative humidity. Relative humidity does appear to have an effect on how cold one feels at low temperatures, but only to a limited extent, and in the opposite direction to popular belief.