Ultracold heteronuclear fermi-fermi molecules.

We report on the first creation of ultracold bosonic heteronuclear molecules of two fermionic species, 6Li and 40K, by a magnetic field sweep across an interspecies s-wave Feshbach resonance. This allows us to associate up to 4x10(4) molecules with high efficiencies of up to 50%. Using direct imaging of the molecules, we measure increased lifetimes of the molecules close to resonance of more than 100 ms in the molecule-atom mixture stored in a harmonic trap.

Comparative and medical physiology: a theme with three variations.

The aim of this essay is to show how a comparative approach, by adding new variables, might shed unexpected light on some well-known problems of general physiology. In my first example I describe how a classic study of the energetics of Na reabsorption in canine kidneys leads to new insights if kidney size is introduced as a co-variable. The second example demonstrates that if the maximum rate of ATP production does not suffice to meet the ATP-demand of a particular physiological function, the deficit metabolic fuel could be supplied by the ATP spared by the suppression of another function. In evidence I draw attention to the high metabolic cost of growth in larval fish, although any mammalian organ with high energy requirements could run into the same problem under energy limiting conditions. In the third example, I suggest that the experimentally determined degree of sensitivity of cellular functions to hypoxia could be used to make predictions about the ecology of the biological system studied. This inference would be valid irrespective of whether the term "ecology" refers to the external environment of different species or to the internal environment of different organs in the human body.

Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects.

The original aim of the present study was to deal with two problems that had emerged from a study on hierarchies of ATP-consuming processes in cells [Buttgereit and Brand (1995) Biochem. J. 312, 163-167]. Firstly, we wanted to find out whether the results of that study had been influenced by the method used for the determination of process activity and, secondly, we wondered whether and to what extent the structure of the hierarchy established for cell suspensions under energy-limiting conditions might depend on the type of cell or on the lifestyle, ecology and phylogenetic status of the species from which the cells were derived. We confined our study to the two most prominent ATP consumers of cells: protein synthesis and the Na(+)/K(+)-ATPase, measuring their activity directly by [3H]leucine incorporation and Rb(+)-flux respectively. We found large differences in the sensitivity of protein synthesis to energy limitation between hepatocytes from an anoxia-tolerant fish species and an anoxia-sensitive fish species (goldfish and rainbow trout respectively). On the other hand, Na(+)/K(+)-ATPase activity was hardly affected by energy limitation in the hepatocytes from both fish species. We also studied the response of a human hepatoma cell line, HepG2, to energy limitation and found both protein synthesis and Na(+)/K(+)-ATPase activity to be equally sensitive to energy limitation, but more sensitive than the Na(+)/K(+)-ATPase of the two fish species. A comparison of the indirect and direct methods for measuring protein synthesis revealed the rate of oxygen consumption to be functionally related to the concentration of cycloheximide, the inhibitor used. It was found that at 15 mM cycloheximide [three orders of magnitude higher than the concentration at which the incorporation of free amino acids (FAA) into protein is inhibited] total oxygen consumption was suppressed by 71-75%, whereas the measured rate of [3H]leucine incorporation into protein suggested that the cycloheximide-sensitive fraction should have amounted to not more than approx. 10% of the total oxygen consumption. On the other hand, the amount of oxygen consumption suppressed with the high concentration of cycloheximide corresponded almost exactly to the increase in oxygen consumption of cells incubated in an FAA-enriched medium compared with cells incubated in a standard, FAA-free medium. Our major conclusions are, firstly, that high concentrations of cycloheximide disrupt cellular metabolism, bringing to a standstill all those processes that can be stimulated by incubating starved cells in an FAA-enriched medium, secondly, that the attempt to estimate the metabolic cost of protein synthesis by inhibiting oxygen consumption with cycloheximide leads to spurious results, and, thirdly, that the structure of a 'hierarchy' of ATP-consumers may reflect the lifestyle and physiology of the species studied.

Private and collective interests; conflicts and solutions: the central theme of current thinking in evolutionary biology.

The statement made by the population geneticist Theodosius Dobzhansky (1973): "Nothing in Biology Makes Sense Except in the Light of Evolution", is often quoted as a crucially important generalization on the nature of biology. I am inclined to consider as equally important the statement: "Nothing in Evolutionary Biology Makes Sense Except in the Light of Conflicts between Parts and Systems." This generalization takes account of the dynamic nature of biological phenomena, but also of the fact that the study of transitions from autonomous units to cooperative systems has become one of the most exciting and scientifically rewarding enterprises in all of organismic biology. The problems encountered and the speculations generated in the course of this enterprise will be either of the more unit-centered or of the more system-centered type, most biologists tending to lean towards one or the other. This explains why evolutionary biology is fraught with so many antagonistic attitudes and polarizing points of view. In this essay I want specifically to draw attention to and discuss the following issues which in recent years have polarized biologists: the dual nature of genes; the logic of Hamilton's rule; the relationship between kin selection, signalling networks and systemic manipulation; the semantic problem of progress in evolution; and the evolutionary consequences of the vastly differing time scales over which genotypic and phenotypic information processing occurs in higher animals.

Action of adenosine on energetics, protein synthesis and K(+) homeostasis in teleost hepatocytes.

In a comparative study, we analysed the effects of adenosine on the energetics, protein synthesis and K(+ )homeostasis of hepatocytes from the anoxia-tolerant goldfish Carassius auratus and the anoxia-intolerant trout Oncorhynchus mykiss. The rate of oxygen consumption did not respond immediately to the addition of adenosine to the cells from either species, but showed a significant decrease in trout hepatocytes after 30 min. The anaerobic rate of lactate formation was not significantly affected by adenosine in goldfish hepatocytes, but was increased in trout cells. We also studied the effects of adenosine on the two most prominent ATP consumers in these cells, protein synthesis and Na(+)/K(+)-ATPase activity. Under aerobic conditions, adenosine inhibited protein synthesis of hepatocytes from goldfish by 51% and of hepatocytes from trout by 32%. During anoxia, the rate of protein synthesis decreased by approximately 50% in goldfish hepatocytes and by 90% in trout hepatocytes, and this decrease was not altered by the presence of adenosine. Adenosine inhibited normoxic Na(+)/K(+)-ATPase activity and K(+ )efflux by 20-35% in the cells of both species. An investigation into the mechanism underlying the inhibition of protein synthesis by adenosine indicated that, in the goldfish cells, adenosine acts via a membrane receptor-mediated pathway, i.e. the effect of adenosine was abolished by applying the A1 receptor antagonist 8-phenyltheophylline. In the trout, however, the uptake of adenosine into hepatocytes seems to be required for an effect on protein synthesis. [Ca(2+)](i) does not seem to be involved in the inhibition of protein synthesis by adenosine.

Oxygen-dependent energetics of anoxia-tolerant and anoxia-intolerant hepatocytes.

The oxygen-dependence of cellular energetics was investigated in hepatocytes from goldfish Carassius auratus (anoxia-tolerant) and rainbow trout Oncorhynchus mykiss (anoxia-intolerant). In goldfish hepatocytes, an approximately 50 % reduction in the rate of oxygen consumption was observed in response to both acute and prolonged hypoxia, the latter treatment shifting the threshold for this reduction to a higher oxygen level. A concomitant increase in the rate of lactate production did not compensate for the decreased aerobic ATP supply, resulting in an overall metabolic depression of 26 % during acute hypoxia and of 42 % during prolonged hypoxia. Trout hepatocytes showed a similar suppression of cellular respiration after prolonged hypoxia but were unresponsive to acute hypoxia. Similarly, the rate of lactate production was unaltered during acute hypoxia but was increased during prolonged hypoxia, metabolic depression amounting to 7 % during acute hypoxia and 30 % during prolonged hypoxia. In both species, the affinity of hepatocytes for oxygen decreased during hypoxia, but this alteration was not sufficient in absolute terms to account for the observed decrease in aerobic ATP supply. Protein synthesis was suppressed in both cell types under hypoxia, whereas Na(+)/K(+)-ATPase activity decreased in trout but not in goldfish hepatocytes, emphasising the importance of membrane function in these cells during conditions of limited energy supply.

Energetics of trout hepatocytes during A23187-induced disruption of Ca2+ homeostasis.

The impact of an increase of intracellular Ca2+ i on the energy metabolism of trout hepatocytes was assessed by applying the Ca2+ ionophore A23187 and studying the consequences of the ensuing elevation of Ca2+ i on various metabolic parameters. After application of A23187 no loss of viability occurred for 2 h, but glutathione content decreased by 46%. A concomitant decrease of [ATP] as well as of Na,K-ATPase activity by over 50% could be prevented by incubating the cells in a Ca2+-free medium. Upon addition of the ionophore cellular oxygen consumption more than doubled in a strictly Ca2+-dependent manner, with half of this increase being sensitive to ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter. This increase in oxygen consumption was transient in nature and at its peak it was similar in magnitude to that induced by 2,4-dinitrophenol. Similarly, oxygen consumption sensitive to the protein synthesis inhibitor cycloheximide was transiently increased by A23187, but returned to control levels within 30 min of incubation. These results suggest that elevation of intracellular Ca2+ leads to an energetic imbalance not related to stimulation of ATP consuming processes, but mainly due to impairment of mitochondrial function, possibly by the decoupling of oxidative phosphorylation and by inducing dissipative Ca2+ cycling.

Loss of K+ homeostasis in trout hepatocytes during chemical anoxia: a screening study for potential causes and mechanisms.

In isolated trout hepatocytes intoxication with CN- (chemical anoxia) leads to a rapid breakdown of K+ homeostasis. In the present study an attempt has been made to identify the causes and mechanisms underlying this phenomenon. Our results indicate that neither Ca2+ elevation nor cell swelling, both of which occurred during chemical anoxia and could be prevented by exposure to Ca2+ chelating agents or to hyperosmotic conditions, respectively, is solely responsible for the breakdown of K+ homeostasis. From a number of inhibitors of dissipative K+ fluxes tested, only BaCl2, an inhibitor of voltage-gated K+ channels, proved to be effective in significantly reducing K+ efflux during chemical anoxia. The KCl cotransporter known to be involved in regulatory volume decrease after hypoosmotic shock does not seem to be activated during CN(-)-induced cell swelling.

Functional role of ecto-ATPase activity in goldfish hepatocytes.

Extracellular [gamma-32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus gamma-32Pi due to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 +/- 0.4 nmol P(i).(10(6) cells)-1.min-1 and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 +/- 123 microM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 microM [alpha-32P]ATP to the hepatocyte suspension induced the extracellular release of alpha-32P(i) [8.2 pmol.(10(6) cells)-1.min-1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 microM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol.(10(6) cells)-1.min-1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+ (Ca2+i). This effect could be partially mimicked by adenosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of Ca2+i and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

A note on interactions between temperature, viscosity, body size and swimming energetics in fish larvae

In a previous study, it was shown that at a given speed the larvae of a species of freshwater fish, the Danube bleak Chalcalburnus chalcoides, expended considerably more metabolic energy at 15 degreesC than at 20 degreesC. We applied hydromechanical arguments to our previous data in order to determine whether the higher cost of swimming at the lower temperature might be due to the effects of viscous forces. However, even under the unrealistic assumption of the larvae swimming in the viscous regime at Reynolds numbers as high as 2000, we show here that hydromechanical forces cannot explain the high energy cost of swimming at 15 degreesC. Instead, we offer a new hypothesis that the 'two-gear system' of the swimming muscles operating in juvenile and adult fish is not yet functional in the larvae, with the consequence that, when these fish are swimming at high speeds in cold water, the muscle fibres have to operate over an increasingly inefficient range of shortening velocities.

Effects of energy limitation on Ca2+ and K+ homeostasis in anoxia-tolerant and anoxia-intolerant hepatocytes.

To gain more insight into the mechanistic basis of anoxia tolerance and intolerance, a comparative study was conducted on calcium homeostasis in goldfish and trout hepatocytes subjected to different forms of energy limitation. Using the fluorescent Ca2+ indicator fura 2, we observed that both chemical anoxia and true anoxia led to an increase of the concentration of cytosolic free calcium (Ca2+i) in the anoxia-sensitive hepatocytes of rainbow trout, whereas Ca2+i was maintained at control levels in the anoxia-tolerant hepatocytes of goldfish. Various lines of evidence suggest an intracellular origin of the Ca2+ increase observed in trout cells. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition pore in mammalian cells, was ineffective in preventing the Ca2+ increase, whereas a high dose of fructose depressed the Ca2+ surge by approximately 50%. The latter effect was not accompanied by improvement of the energetic state of the cells. A comparison of chemical anoxia with true (physiological) anoxia revealed that both treatments affected energy metabolism to a similar degree in trout hepatocytes, whereas the decrease of ATP seen in goldfish hepatocytes during chemical anoxia was absent during true anoxia. Elevation of Ca2+i with the calcium ionophore A-23187 led to a decoupling of unidirectional K+ fluxes in both normoxic and anoxic trout cells, whereas in goldfish hepatocytes the coupling of K+ fluxes was not affected by the rise of Ca2+i.

Acute and chronic effects of temperature, and of nutritional state, on ion homeostasis and energy metabolism in teleost hepatocytes.

Short- and long-term effects of temperature on ion flux and energy turnover were studied in hepatocytes from thermally acclimated trout and roach. In trout hepatocytes K+ efflux was insensitive towards acute exposure to low temperature but was downregulated during cold acclimation of the fish so as to balance the uncompensated decreased K+ (Rb+) uptake of the cells. In contrast, both K+ (Rb+) uptake and K+ efflux of roach hepatocytes were temperature sensitive in the short term. These acute effects, however, were offset during cold acclimation by a near perfect compensation of both fluxes leading to re-establishment of ion flux homeostasis at the original level. Our findings, based on a new method permitting the simultaneous monitoring of K+ efflux and uptake in the same cell population, provide experimental verification of two of the three possible strategies, recently discussed by Cossins et al. (1995), by which the ionic steady state of fish cells may adjust to acute and chronic temperature change. By comparing hepatocytes from two groups of trout, one kept on a maintenance diet (ration I), the other fed ad libitum (ration II), we discovered striking effects of nutritional state on the absolute levels as well as on the temperature relationships of K+ uptake and protein synthetic activity. Both of these functions in the hepatocytes increased in the ration II fed as compared to the ration I fed trouts, but the increase of protein synthetic activity was greater and more uniform at the three experimental temperatures than that of K+ uptake. Moreover, protein synthetic activity proved to be considerably more temperature sensitive than K+ uptake and, in contrast to the latter, showed a compensatory response after cold acclimation.

A major transition in Darwinism.

Many lines of evidence indicate that in recent: years the focus of evolutionary biology has begun to shift from explaining the 'origin of species' to the modelling of processes by which autonomous entities cooperate (or are coerced) to form systems of greater complexity. If such a major shift is indeed occurring, then it should be more widely promulgated to counteract the public image of evolutionary theory, which appears to be as dogmatically simplistic today as it was a century ago.

Membrane-metabolic coupling and ion homeostasis in anoxia-tolerant and anoxia-intolerant hepatocytes.

The relationship between membrane function and energy metabolism was studied in rainbow trout hepatocytes, an anoxia-intolerant cell system, and compared with the situation in hepatocytes from the goldfish, a typical anoxia-tolerant species. In trout hepatocytes, under normoxia and under chemical anoxia, inhibition of ATP consumption by the Na+ pump induced a decrease in ATP production of the same magnitude. In response to chemical anoxia, total ATP production was reduced to 15% and Na+ pump activity to 22% of the control rate under normoxia. Measurement of the cellular ATP content under these conditions revealed that, despite the reduction in Na+ pump activity, the cells became rapidly depleted of ATP, with the time course of this process resembling that observed in the anoxic rat hepatocyte. This is in contrast to the responses of goldfish hepatocytes, where, during chemical anoxia, 1) inhibition of the Na+ pump did not lead to a corresponding reduction in ATP production and 2) ATP levels, after a transient decrease, stabilized at a new steady state. To investigate the consequences of chemical anoxia on ion homeostasis, efflux and uptake rates of K+ were determined simultaneously. In the trout cells, chemical anoxia led to a decoupling of influx and efflux rates, the latter exceeding the former three- to eightfold. In contrast, goldfish hepatocytes were able to preserve ion homeostasis by a concerted decrease in Rb+ uptake and K+ efflux, so that the net flux of K+ was always close to zero. In neither species did chemical anoxia induce a change in pump density. Other potential control mechanisms are briefly discussed.

Energetics of fish larvae, the smallest vertebrates.

In this review recent findings on the energetics of fish larvae are presented, highlighting some of the physiological problems linked to small body size. The existence of a mass-independent phase of specific metabolic rate is confirmed but it is pointed out that in young fish ontogenetic transitions of metabolic scaling have so far been documented only for the routine level of activity. Maximum metabolic rate is limited by mitochondrial density in the swimming muscles which scales with a mass exponent of approximately 0.9. Mitochondrial density in the swimming muscles of a species of fish, from larva to adult, covers about the same range as mitochondrial density in the skeletal muscles of mammals. However, the aerobic capacity (power density) of mitochondria is one order of magnitude lower in fish than in mammals. Energy metabolism in embryos and early larvae of fish is almost entirely aerobic. Anaerobic power in the fast muscle fibres is low after hatching but increases during the transition from larva to juvenile with a mass exponent greater than one. In hypoxic water fish larvae swim more economically (i.e. their cost of transport is lower) than in normoxic water. If the rate of growth exceeds a critical threshold (about 10% d-1) fish larvae are capable of increasing the apparent efficiency of growth, probably by reducing the costs of other energy-consuming functions of maintenance.

Cost of growth in cells and organisms: general rules and comparative aspects.

In a crude fashion it can be said that metabolizable energy (M) is partitioned into metabolic work, paid for by 'oxidations' (R), and 'assimilation', i.e. production (P), so that M = R+P. However, a fraction of R is required to meet the expenses of production and if these expenses represent, Joule for Joule, a constant proportion of the amount produced, then Rt = Rm+cP, where Rt = total metabolic expenditures, Rm = metabolic expenditures for maintaining the non-producing organism, and cP = Rp = metabolic expenditures connected with the processes of production. The partitioning of metabolizable energy into R and P as well as into Rm and Rp may vary depending on the phylogeny and life-history of the species concerned and on ecological circumstances. Thus selection is expected to act on both ratios, R/P and Rm/Rp. By comparing the ratios P/(P+Rp) (the apparent efficiency of production) and Rp/P (the apparent metabolic cost of production) in different types of organisms, one finds that a value of P/(P+Rp) = 0.75, equal to 75% efficiency, 10 mgdbm/mmol ATP, and 16 mumolO2/mg dbm (when I mg identical to 22 J), can be used as a 'consensus value' for the average efficiency, or cost, of the transformation of metabolizable energy into production in a wide range of organisms, from bacteria to mammals. This value corresponds to about three times the theoretical cost of synthesizing the same amount of tissue on the basis of known biochemical principles. The reasons why the empirical costs of production are higher than the theoretical costs of synthesis by what appears to be a common factor may be quite different in bacteria, small ectothermic and large endothermic organisms. Deviations from the consensus value may be due to differences in energy density of the nutrients assimilated and the tissues synthesized. Further complications arise because of interactions between P, Rp, and Rm. In microorganisms the existence of a constant and a variable component of maintenance metabolism has been postulated, the latter decreasing with increasing rate of production. In small ectothermic metazoans, on the other hand, the nonlinear relationship between growth metabolism and growth rate has led to the speculation that above a critical value of Pg certain energy consuming functions of maintenance are suppressed and the energy thus gained used for fuelling growth processes. There is some evidence that, at least in ectothermic metazoans, the apparent cost of growth decreases with the rate of growth, reaching a low plateau of about 10 mumolO2/mgdbm at growth rates exceeding about 8 mgdbm/g/h.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolic rate and cost of growth in juvenile pike (Esox lucius L.) and perch (Perca fluviatilis L.): the use of energy budgets as indicators of environmental change.

Energy budgets of juvenile pike and perch (weighing approximately 3 g) were determined in experiments lasting up to 4 days, by simultaneously measuring oxygen consumption, food consumption, and growth of individual fish. Although the basic pattern of energy allocation was identical in the two species, perch subjected to constant light (PEL) showed faster growth, higher assimilation and conversion efficiency, and higher oxygen consumption than perch subjected to a short daylength regime (PED). The efficiency with which food energy was converted into body mass was 39±5% in PED but 49±4% in PEL. However, the "work coefficient" (increment of body mass/post-prandial increase of oxygen consumption: mg · µmol O inf2sup-1 ) differed only insignificantly between the two groups of perch, indicating that the metabolic cost of growth was unaffected by the manipulation of experimental conditions. This identifies the higher assimilation efficiency, i.e. the increased flow of food energy into the tissues as being the cause of accelerated growth of perch under the constant-light regime. In both species the maximum feeding-induced metabolic rate was 4 times higher than the lowest preprandial rate. In perch (which were kept on low rations before the experiments) the post-prandial metabolic rate increased steadily from day to day during the 4-day experiments, so that on the last day the rate of oxygen consumption exceeded the rate on the first day by about 41%. This investigation provides further evidence that the allocation of metabolic energy in fish is based on a flexible strategy which responds sensitively to changes in both internal and external conditions.