Successful achievement after heterotopic transplantations of long-term stored ovarian tissue in Hodgkin's lymphoma survivor.

In this case report, we describe the outcomes of two heterotopic transplantations of cryopreserved ovarian tissue performed in a patient with HL, after 11 and 15 years of storage. At the age of 30, the patient underwent laparoscopy to collect ovarian tissue for cryopreservation before chemotherapy and radiotherapy. Eleven years later she experienced premature ovarian failure (POF). As the patient was only interested in endocrine function recovery, two heterotopic ovarian tissue transplantations were performed in the abdominal wall above the rectus muscle respectively 11 and 15 years after cryopreservation. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. The analysis on thawed ovarian tissue did not reveal micrometastasis and it showed well-preserved follicles and stroma. After both ovarian tissue grafting, menopausal symptoms ceased. The patient had periods approximately every 30-days and hormonal levels were within the premenopausal range. The endocrine function lasted 3-years after the first heterotopic transplantation and is still ongoing after second transplantation. Cryopreservation of ovarian tissue should be proposed to HL patients, as the incidence of POF as a long-term complication is not negligible. In these patients heterotopic transplantation is a useful tool to eliminate menopausal symptoms, preventing osteoporosis and reducing cardiovascular risks.

Imaging in gynecological disease. 10: Clinical and ultrasound characteristics of decidualized endometriomas surgically removed during pregnancy.

OBJECTIVES: To describe the clinical history and ultrasound findings in women with decidualized endometriomas surgically removed during pregnancy. METHODS: In this retrospective study, women with a histological diagnosis of decidualized endometrioma during pregnancy who had undergone preoperative ultrasound examination were identified from the databases of seven ultrasound centers. The ultrasound appearance of the tumors was described on the basis of ultrasound images, ultrasound reports and research protocols (when applicable) by one author from each center using the terms and definitions of the International Ovarian Tumor Analysis (IOTA) group. In addition, two authors reviewed together available digital ultrasound images and used pattern recognition to describe the typical ultrasound appearance of decidualized endometriomas. RESULTS: Eighteen eligible women were identified. Median age was 34 (range, 20-43) years. Median gestational age at surgical removal of the decidualized endometrioma was 18 (range, 11-41) weeks. Seventeen women (94%) were asymptomatic and one presented with pelvic pain. In three of the 18 women an ultrasound diagnosis of endometrioma had been made before pregnancy. The original ultrasound examiner was uncertain whether the mass was benign or malignant in 10 (56%) women and suggested a diagnosis of benignity in nine (50%) women, borderline in eight women (44%), and invasive malignancy in one (6%) woman. Seventeen decidualized endometriomas contained a papillary projection, and in 16 of these at least one of the papillary projections was vascularized at power or color Doppler examination. The number of cyst locules varied between one (n = 11) and four. No woman had ascites. When using pattern recognition, most decidualized endometriomas (14/17, 82%) were described as manifesting vascularized rounded papillary projections with a smooth contour in an ovarian cyst with one or a few cyst locules and ground-glass or low-level echogenicity of the cyst fluid. CONCLUSIONS: Rounded vascularized papillary projections with smooth contours within an ovarian cyst with cyst contents of ground-glass or low-level echogenicity are typical of surgically removed decidualized endometriomas in pregnant women, most of whom are asymptomatic.

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.

Catalytic activity and heat production by the Ca(2+)-ATPase from sea cucumber (Ludwigothurea grisea) longitudinal smooth muscle: modulation by monovalent cations.

In muscle cells, excitation-contraction coupling involves the translocation of Ca(2+) between intracellular compartments and the cytosol. Heat derived from the hydrolysis of ATP by the sarcoplasmic reticulum Ca(2+)-ATPase of skeletal muscle plays an important role in the thermoregulation and energy balance of the cell. Although several Ca(2+)-ATPase isoforms have been described in vertebrates, little is known about Ca(2+) transport in invertebrates. In this report, a Ca(2+)-ATPase is identified in the microsomal fraction obtained from sea cucumber (Ludwigothurea grisea) smooth muscle. The activity of this enzyme is enhanced three- to fivefold by K(+) and Na(+). During Ca(2+) transport, the ATPase can synthesise ATP from ADP and inorganic phosphate (P(i)) using the energy derived from the Ca(2+) gradient formed across the microsomal membrane (ATP<->P(i) exchange). The apparent affinity of the enzyme for P(i) is increased by more than one order of magnitude by K(+). In the presence of K(+), the fraction of ATP synthesised during the exchange reaction by sea cucumber microsomes was found to be larger than that measured in microsomes derived from either rabbit or trout muscle. Like the isoforms found in skeletal muscle, the sea cucumber Ca(2+)-ATPase can convert osmotic energy into heat. The amount of heat produced after the hydrolysis of each ATP molecule increases two- to threefold when a Ca(2+) gradient is formed across the microsomal membrane.

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.

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.

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.