The growing competition in Brazilian science: rites of passage, stress and burnout

Brazil's scientific community is under pressure. Each year there is an increase in its contribution to international science and in the number of students who are trained to do research and teach at an advanced level. Most of these activities are carried out in state and federal universities, but with government funding that has decreased by more than 70 percent since 1996. Interviews with graduate students, post-doctoral fellows and professors in one university department with a strong research tradition illustrate the level of stress engendered by the conflict between increasing competition and diminishing resources, and serve to underscore the negative effects on creativity and on the tendency to choose science as a career

The growing competition in Brazilian science: rites of passage, stress and burnout.

Brazil's scientific community is under pressure. Each year there is an increase in its contribution to international science and in the number of students who are trained to do research and teach at an advanced level. Most of these activities are carried out in state and federal universities, but with government funding that has decreased by more than 70% since 1996. Interviews with graduate students, post-doctoral fellows and professors in one university department with a strong research tradition illustrate the level of stress engendered by the conflict between increasing competition and diminishing resources, and serve to underscore the negative effects on creativity and on the tendency to choose science as a career.

Ca2+-ATPases (SERCA): energy transduction and heat production in transport ATPases.

The sarcoplasmic reticulum Ca2+-ATPase is able to cleave ATP through two different catalytic routes. In one of them, a part of the chemical energy derived from ATP hydrolysis is used to transport Ca2+ across the membrane and part is dissipated as heat. In the second route, the hydrolysis of ATP is completed before Ca2+ transport and all the energy derived from ATP hydrolysis is converted into heat. The second route is activated by the rise of the Ca2+ concentration in the vesicle lumen. In vesicles derived from white skeletal muscle the rate of the uncoupled ATPase is several-fold faster than the rate of the ATPase coupled to Ca2+ transport, and this accounts for both the low Ca2+/ATP ratio usually measured during transport and for the difference of heat produced during the hydrolysis of ATP by intact and leaky vesicles. Different drugs were found to selectively inhibit the uncoupled ATPase activity without modifying the activity coupled to Ca2+ transport. When the vesicles are actively loaded, part of the Ca2+ accumulated leaks to the medium through the ATPase. Heat is either produced or released during the leakage, depending on whether or not the Ca2+ efflux is coupled to the synthesis of ATP from ADP and Pi.

Role of the sarcoplasmic reticulum Ca2+-ATPase on heat production and thermogenesis.

The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca2+-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca2+ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol depending on whether or not a transmembrane Ca2+ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca2+ transport and the fraction which is dissipated in the surrounding medium as heat.

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.

Uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase. Regulation by ADP.

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle accumulate Ca2+ at the expense of ATP hydrolysis. The heat released during the hydrolysis of each ATP molecule varies depending on whether or not a Ca2+ gradient is formed across the vesicle membrane. After Ca2+ accumulation, a part of the Ca2+-ATPase activity is not coupled with Ca2+ transport (Yu, X., and Inesi, G. (1995) J. Biol. Chem. 270, 4361-4367). I now show that both the heat produced during substrate hydrolysis and the uncoupled ATPase activity vary depending on the ADP/ATP ratio in the medium. With a low ratio, the Ca2+ transport is exothermic, and the formation of the gradient increases the amount of heat produced during the hydrolysis of each ATP molecule cleaved. With a high ADP/ATP ratio, the Ca2+ transport is endothermic, and formation of a gradient increased the amount of heat absorbed from the medium. Heat is absorbed from the medium when the Ca2+ efflux is coupled with the synthesis of ATP (5.7 kcal/mol of ATP). When there is no ATP synthesis, the Ca2+ efflux is exothermic (14-16 kcal/Ca2+ mol). It is concluded that in the presence of a low ADP concentration the uncoupled ATPase activity is the dominant route of heat production. With a high ADP/ATP ratio, the uncoupled ATPase activity is abolished, and the Ca2+ transport is endothermic. The possible correlation of these findings with thermogenesis and anoxia is discussed.

ATP synthesis and heat production during Ca(2+) efflux by sarcoplasmic reticulum Ca(2+)-ATPase.

Vesicles derived from the sarcoplasmic reticulum of rabbit white skeletal muscle were loaded with Ca(2+) and used to measure the rates of Ca(2+) efflux, heat production, and ATP synthesis from ADP and P(i). It was found that the Ca(2+)-ATPase can function in three different forms: (i) it absorbs heat from the medium (5 Kcal/mol Ca(2+)) when the efflux was coupled with ATP synthesis; (ii) it converts the energy derived from the gradient into heat (30 Kcal/mol Ca(2+)) when Mg(2+) is removed from the medium and the synthesis of ATP is impaired; and (iii) the ATPase becomes uncoupled when the different ligands of the enzyme are removed from the medium. As a result, there is no ATP synthesis and no heat production or absorption during Ca(2+) efflux. The Ca(2+) efflux, ATP synthesis, and heat production were inhibited by thapsigargin, a specific inhibitor of the Ca(2+)-ATPase.

Sarcoplasmic reticulum Ca(2+)-ATPase of sea cucumber smooth muscle: regulation by K(+) and ATP.

Although several Ca(2+)-ATPase isoforms have been described in vertebrates, little is known about Ca(2+)-transport in the muscle of invertebrates. In the microsomal fraction obtained from the sea cucumber (Ludwigothurea grisea) longitudinal body wall smooth muscle, we identified a Ca(2+)-transport ATPase that is able to transport Ca(2+) at the expense of ATP hydrolysis. This enzyme has a high affinity for both Ca(2+) and ATP, an optimum pH around 7.0, and - different from the vertebrate sarcoplasmic reticulum Ca(2+)-ATPases isoforms so far described - is activated 3- to 5-fold by K(+) but not by Li(+), at all temperatures, Ca(2+) and ATP concentrations tested. Calcium accumulation by the sea cucumber microsomes is inhibited by Mg/ATP concentrations >1 mM and the accumulated Ca(2+) is released to the medium when the ATP concentration is raised from 0.1 to 4.0 mM.

Ca(2+) release and heat production by the endoplasmic reticulum Ca(2+)-ATPase of blood platelets. Effect of the platelet activating factor.

Different sarco/endoplasmic reticulum Ca(2+)-ATPases isoforms are found in blood platelets and in skeletal muscle. The amount of heat produced during ATP hydrolysis by vesicles derived from the endoplasmic reticulum of blood platelets was the same in the absence and presence of a transmembrane Ca(2+) gradient. Addition of platelets activating factor (PAF) to the medium promoted both a Ca(2+) efflux that was arrested by thapsigargin and an increase of the yield of heat produced during ATP hydrolysis. The calorimetric enthalpy of ATP hydrolysis (DeltaH(cal)) measured during Ca(2+) transport varied between -10 and -12 kcal/mol without PAF and between -20 and -24 kcal/mol with 4 microM PAF. Different from platelets, in skeletal muscle vesicles a thapsigargin-sensitive Ca(2+) efflux and a high heat production during ATP hydrolysis were measured without PAF and the DeltaH(cal) varied between -10 and -12 kcal/mol in the absence of Ca(2+) and between -22 up to -32 kcal/mol after formation of a transmembrane Ca(2+) gradient. PAF did not enhance the rate of thapsigargin-sensitive Ca(2+) efflux nor increase the yield of heat produced during ATP hydrolysis. These findings indicate that the platelets of Ca(2+)-ATPase isoforms are only able to convert osmotic energy into heat in the presence of PAF.

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid.

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP<-->Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.

Glucose 6-phosphate and fructose 1,6-bisphosphate can be used as ATP-regenerating systems by cerebellum Ca2+-transport ATPase.

In this work, it is shown that the Ca2+-transport ATPase found in the microsomal fraction of the cerebellum can use both glucose 6-phosphate/hexokinase and fructose 1,6-bisphosphate/phosphofructokinase as ATP-regenerating systems. The vesicles derived from the cerebellum were able to accumulate Ca2+ in a medium containing ADP when either glucose 6-phosphate and hexokinase or fructose 1,6-bisphosphate and phosphofructokinase were added to the medium. There was no Ca2+ uptake if one of these components was omitted from the medium. The transport of Ca2+ was associated with the cleavage of sugar phosphate. The maximal amount of Ca2+ accumulated by the vesicles with the fructose 1,6-bisphosphate system was larger than that measured either with glucose 6-phosphate or with a low ATP concentration and phosphoenolpyruvate/pyruvate kinase. The Ca2+ uptake supported by glucose 6-phosphate was inhibited by glucose, but not by fructose 6-phosphate. In contrast, the Ca2+ uptake supported by fructose 1,6-bisphosphate was inhibited by fructose 6-phosphate, but not by glucose. Thapsigargin, a specific SERCA inhibitor, impaired the transport of Ca2+ sustained by either glucose 6-phosphate or fructose 1,6-bisphosphate. It is proposed that the use of glucose 6-phosphate and fructose 1,6-bisphosphate as an ATP-regenerating system by the cerebellum Ca2+-ATPase may represent a salvage route used at early stages of ischemia; this could be used to energize the Ca2+ transport, avoiding the deleterious effects derived from the cellular acidosis promoted by lactic acid.

Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum.

The sarcoplasmic reticulum Ca2+-ATPase of rabbit skeletal muscle can convert the energy derived from a Ca2+ gradient into heat (L. de Meis, M. L. Bianconi, and V. A. Suzano. FEBS Lett. 406: 201-204, 1997). In this report, it is shown that this conversion varies depending on the temperature and on whether rabbit (endotherm) or trout (poikilotherm) sarcoplasmic reticulum vesicles are used. The gradient doubled the yield of heat produced during ATP hydrolysis and the calorimetric enthalpy of ATP hydrolysis (DeltaHcal) value found with both rabbit and trout varied between -10 and -12 kcal/mol in leaky vesicles (no gradient) and between -20 and -22 kcal/mol with intact vesicles (gradient). For the rabbit, the difference of DeltaHcal measured with and without gradient was detected in the range of 30-35 degrees C and disappeared when the temperature was decreased below 30 degrees C. For the trout, the difference was detected between 20 and 25 degrees C and disappeared below 20 degrees C. The effect of the gradient on the DeltaHcal for ATP hydrolysis was modified by DMSO, trifluoperazine, and heparin sodium.

Reversibility of H+-ATPase and H+-pyrophosphatase in tonoplast vesicles from maize coleoptiles and seeds

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H+-ATPase and H+-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to 32Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H+ pumps showed that the H+-ATPase was more active than H+-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H+-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.

Modification of the pH dependence of animal and plant transport ATPases by sulfated polysaccharides.

The effects of heparin and dextran sulfate 8,000 on two isoforms of the sarco/endoplasmic reticulum Ca(2+)-ATPase of different animal tissues and on the corn root H(+)-ATPase were examined. In the absence of sulfated polysaccharides the pH profile's of the three transport ATPases were quite different, but after the addition of heparin or dextran sulfate 8,000 the pH profiles of the three enzymes became similar, all showed maximal activity at pH 7.0. Potassium and sodium antagonized the effects of sulfated polysaccharides on the three transport ATPases, but the antagonism was considerably reduced at acidic pH values.

Control of heat produced during ATP hydrolysis by the sarcoplasmic reticulum Ca(2+)-ATPase in the absence of a Ca2+ gradient.

The amount of heat produced during the hydrolysis of ATP by the sarcoplasmic reticulum Ca(2+)-ATPase was found to vary depending on the Ca2+ concentration in the medium. When the CaCl2 concentration is raised from 0.1 to 2.0 mM a part of the energy derived from ATP hydrolysis is not dissipated as heat but it is used by the enzyme to resenthesize a small fraction of the ATP previously cleaved. Thus, Ca2+ seems to regulate the ATPase in such a way as to vary the fraction of energy derived from ATP hydrolysis which is converted into heat and that which is conserved as chemical energy.

Control of energy fluxes by the sarcoplasmic reticulum Ca2+-ATPase: ATP hydrolysis, ATP synthesis and heat production.

The experiments described indicate that heat is released when Ca2+ leaks through the Ca2+-ATPase of sarcoplasmic reticulum vesicles. In the presence of a transmembrane Ca2+ concentration gradient, agents that modify the amount of ATP synthesized from ADP and Pi also modify the amount of heat produced by the hydrolysis of each ATP molecule. Thus, in the presence of heparin, less ATP is synthesized and more heat is produced. Conversely, with dimethyl sulfoxide more ATP is synthesized and less heat is produced. The data indicate that between limits (-10 to -30 kcal/mol) the Ca2+-ATPase can regulate the interconversion of energy in such a way as to vary the fraction of energy derived from ATP hydrolysis which is converted into heat and that which is converted into other forms of energy.

Sarco/endoplasmic reticulum Ca2+-ATPase isoforms: diverse responses to acidosis.

The effects of acidic pH on the kinetics of Ca2+-ATPase isoforms from intracellular membranes of skeletal muscle, cardiac muscle, cerebellum and blood platelets were studied. At neutral pH, all four Ca2+-ATPase isoforms exhibited similar Ca2+-concentration requirements for half-maximal rates of Ca2+ uptake and ATP hydrolysis. A decrease in the pH from 7.0 to 6.0 promoted a decrease in both the apparent affinity for Ca2+ [increasing half-maximal activation (K0.5)] and the maximal velocity (Vmax) of Ca2+ uptake. With skeletal muscle vesicles these effect were 5 to 10 times smaller than those observed with all the other isoforms. Acidification of the medium from pH 7.0 to 6.5 caused the release of Ca2+ from loaded vesicles and a decrease in the amount of Ca2+ retained by the vesicles at the steady state. With the vesicles derived from skeletal muscle these effects were smaller than for vesicles derived from other tissues. The rate of passive Ca2+ efflux from skeletal and cardiac muscle vesicles, loaded with Ca2+ and diluted in a medium containing none of the ligands of Ca2+-ATPase, was the same at pH 7.0 and 6.0. In contrast, the rate of Ca2+ efflux from cerebellar and platelet vesicles increased 2-fold after acidification of the medium. The effects of DMSO, Mg2+ with Pi and arsenate on the rate of Ca2+ efflux varied among the different preparations tested. The differences became more pronounced when the pH of the medium was decreased from 7.0 to 6.0. It is proposed that the kinetic differences among the Ca2+-ATPase isoforms may reflect different adaptations to cellular acidosis, such as that which occurs during ischaemia.

Uncoupling of Ca2+ transport ATPase in muscle and blood platelets by diacylglycerol analogues and cyclosporin A antagonism.

The possibility that diacylglycerol analogues might have a wider spectrum of intracellular targets than the well-known protein kinase C was investigated with vesicles containing the Ca2+-ATPase derived from the dense tubular system in platelets and from the sarcoplasmic reticulum of skeletal muscle. The diacylglycerol analogues PMA and 1-oleoyl-2-acetyl-rac-glycerol (OAG) inhibited Ca2+ accumulation by these vesicles, an effect that was antagonized by cyclosporin A. The inhibitory activity of PMA and OAG resulted from the uncoupling of the Ca2+-ATPase, characterized by a pronounced inhibition of Ca2+ uptake accompanied by a discrete decrease in ATPase activity and by the inhibition of the enzyme's phosphorylation by Pi, leading to both a decrease in ATP synthesis and an enhancement of Ca2+ efflux. The inhibition of Ca2+ uptake by PMA was found to decrease as the Ca2+ concentration in the medium was raised from 0.1 to 10.0 microM. This was observed with muscle, but not with platelet vesicles. In contrast, the ability of cyclosporin A to antagonize the inhibition of Ca2+ uptake by PMA also increased when the Ca2+ concentration in the medium was raised from 0.1 to 10.0 microM, but this was observed with both muscle and platelet vesicles. The fact that phospholipase C activity and products from the inositol metabolism have been described as localized in regions of the sarcoplasmic reticulum where Ca2+-ATPase and Ca2+ channels are found suggests a possible physiological role for these products in the regulation of cytosolic Ca2+ levels.

Modern science and the explosion of new knowledge.

The technological evolution of mankind accelerated enormously after the institutionalization of science in the 19th century. In parallel with the vast number of beneficial effects derived from the scientific revolution, the explosion of new knowledge and its centralization in only a few countries has generated a number of complex situations that present major challenges for the modern science. These include the asymmetrical distribution in the planet of young people and science, the super-specialization derived from the information overload and the difficulties in teaching the vast amount of new knowledge generated each year by science.