Diagnóstico perinatal de rabdomiomas intracardiacos y esclerosis tuberosa / Perinatal diagnosis of cardiac rhabdomyomas and tuberous sclerosis

Los tumores cardiacos son poco comunes, y los más frecuentes son los rabdomiomas. Su diagnóstico puede realizarse durante la época fetal. La evolución es variable: algunos son asintomáticos y otros se manifiestan por una obstrucción al flujo o en forma de arritmia. Este tipo de tumores puede ser la clave diagnóstica de una esclerosis tuberosa. Dado el amplio espectro en su presentación, mostramos dos casos, uno de diagnóstico prenatal y otro a partir de un hallazgo en la ecocardiografía(AU)

Primary cardiac tumors are not very common. Rhabdomyomas are those most frequently reported during childhood. They can be diagnosed as early as the fetal period. The course varies; in some cases, there are no symptoms, while in others, the diagnosis is based on the presence of an obstruction to the flow or an arrhythmia. Tumors of this type can be the clue to the diagnosis of tuberous sclerosis. Because of the wide variety of presenting features, we describe two cases, one diagnosed prenatally and the other through a finding on echocardiography(AU)

Ethanol affects calmodulin and the calmodulin-binding proteins neuronal nitric oxide synthase and alphaII-spectrin (alpha-fodrin) in the nucleus of growing and differentiated rat astrocytes in primary culture.

The distribution of calmodulin (CaM) and the CaM-binding proteins neuronal nitric oxide synthase (nNOS) and alphaII-spectrin (alpha-fodrin) in the nucleus of growing and differentiated astrocytes was analysed using immunogold electronmicroscopy. We also analysed the effect of moderate ethanol exposure on these proteins. For this, female Wistar rat were fed with an alcoholic liquid diet and exposed to males after several weeks. Pregnant rats were fed with this diet and, after birth, the foetuses brains were used to establish primary cultures of astrocytes. Astrocytes from control and ethanol-exposed rats foetuses were cultured in the absence or presence of ethanol (30 mM) for 7 days (growing cells) and 21 days (differentiated astrocytes). Our results indicate that all the proteins studied appeared mainly on the condensed chromatin of both control- and alcohol-exposed cells and that there are significant variations in the amount of these proteins between quiescent and dividing astrocytes. Altogether, we have not found a co-localisation between CaM and the CaM-binding proteins.

Ethanol impairs monosaccharide uptake and glycosylation in cultured rat astrocytes.

Astrocyte and glial-neuron interactions have a critical role in brain development, which is partially mediated by glycoproteins, including adhesion molecules and growth factors. Ethanol affects the synthesis, intracellular transport, subcellular distribution and secretion of these glycoproteins, suggesting alterations in glycosylation. We analyzed the effect of long-term exposure to low doses of ethanol (30 mm) on glycosylation process in growing cultured astrocytes in vitro. Cells were incubated for short (5 min) and long (90 min) periods with several radioactively labeled carbohydrate precursors. The uptake, kinetics and metabolism of these precursors, as well as the radioactivity distribution in protein gels were analyzed. The levels of GLUT1 and mannosidase II were also determined. Ethanol increased the uptake of monosaccharides and the protein levels of GLUT1 but decreased those of mannosidase II. It altered the carbohydrate moiety of proteins and increased cell surface glycoproteins containing terminal non-reduced mannose. These results indicate that ethanol impairs glycosylation in rat astrocytes, thus disrupting brain development.

Endocytosis and transcytosis in growing astrocytes in primary culture. Possible implications in neural development.

Endocytosis constitutes an essential process in the regulation of the expression of cell surface molecules and receptors and, therefore, could participate in the neural-glial interactions occurring during brain development. However, the relationship between endocytic pathways in astroglial cells under physiological and pathological conditions remains poorly understood. We analyzed the endocytosis and transcytosis processes in growing astrocytes and the possible effect of ethanol on these processes. Evidence demonstrates that ethanol affects endocytosis in the liver and we showed that ethanol exposure during brain development alters astroglial development changing plasma membrane receptors and surface glycoprotein composition. To study these processes we use several markers for receptor-mediated endocytosis, fluid phase endocytosis and non-specific endocytosis. These markers were labeled for fluorescence microscopy and electron microscopy. 125I-BSA was used to study the effect of ethanol on the internalization and recycling of this macromolecule. The distribution of several proteins involved in endocytosis (caveolin, clathrin, rab5 and beta-COP) was analyzed using immunofluorescence, immunoelectron microscopy and immunoblotting. Our results indicate that growing astrocytes have a developed endocytic system mainly composed of caveolae, clathrin coated pits and vesicles, tubulo-vesicular and spheric endosomes, multivesicular bodies and lysosomes. Ethanol exposure induces a fragmentation of tubular endosomes, decreases the internalization of 125I-BSA, alters the processing of internalized BSA, and decreases the levels of caveolin, clathrin, rab5 and beta-COP. These results indicate that ethanol alters the endocytosis and transcytosis processes and impairs protein trafficking in astrocytes, which could perturb astrocyte surface expression of molecules involved in neuronal migration and maturation during brain development.

Determination of proteins in the presence of imidazole buffers.

Imidazole buffers were found to interfere with the determination of soluble proteins using Lowry's classical method. The influence of the constituent elements of the buffer on the calibration line was studied statistically. By combining the data corresponding to different experimental sequences, interserial calibration curves for different concentrations of imidazole buffer (10-30 mM) were obtained. The absorbance-buffer volume dependence curves produced a good fit to second-order polynomials. The accuracy of protein determination in a medium with imidazole buffer, using appropriate calibration curves, were tested by comparison with the technique of multiple standard addition and by means of recovery studies. These experiments were performed on chick brain homogenate samples. Other important aspects of validation, such as sensitivity and accuracy, were also studied.

Cytochemical and stereological analysis of rat cortical astrocytes during development in primary culture. Effect of prenatal exposure to ethanol.

This study has investigated the effect of prenatal alcohol exposure on the qualitative and quantitative ultrastructure of proliferating and differentiated astrocytes in primary cultures as well as on the cytochemical activity of several subcellular phosphatase markers, including acid phosphatase, uridine diphosphatase, thiamine pyrophosphatase, 5'-nucleotidase and glucose-6-phosphatase. The astrocytes were obtained from 21-day-fetuses of both control and alcohol-fed rats. Our results show that several cell components, such as mitochondria, rough endoplasmic reticulum and lysosomes, exhibit qualitative and/or quantitative ultrastructural changes during the process of astrocyte maturation. In some cases these morphological changes are accompanied by variations in the cytochemical activity of enzymes located in these and other cell components, suggesting that these enzymes, and therefore the functional state of these organelles, are modulated during astrocyte development. When prenatally exposed to ethanol, both proliferating and differentiated astrocytes showed striking ultrastructural alterations compared with controls, including an increment of lysosomes as well as a decrease in the values of stereological parameters relative to mitochondria, rough endoplasmic reticulum and Golgi apparatus. Cytochemical analysis of these cells indicates that prenatal exposure to ethanol decreased the activities of all the enzymes tested, except for acid phosphatase, which was increased in both groups of treated astrocytes. These results suggest that prenatal exposure to ethanol could affect astrocytes during development in two different but probably complementary ways: a) by causing a delay in astrocyte maturation and, b) by inducing a direct toxic effect on these cells.

Combined effects of ethanol and manganese on cultured neurons and glia.

Manganese is essential for normal development and activity of the nervous tissue. Mn2+ ions are involved in protein synthesis and may prevent free radical damage. Since it is now established that alcohol degradation may produce free radicals, we studied the effect of Mn2+ on ethanol induced alterations using cultured nerve cells as an experimental model of the central nervous system. Neurons and glial cells were cultured from rat brain cortex; a tumoral rat glial cell line (C6) was also examined. We measured enzymatic markers of nerve cell maturation (enolase, glutamine synthetase) and superoxide dismutase, a scavenger of free radicals; all these enzymes being activated by Mn2+ ions. Only for the glial cell types an alcohol antagonizing effect was found when Mn2+ was combined with ethanol. Neurons were not sensitive to that Mn2+ effect.

Effect of maternal alcohol consumption on nerve cell development in the offspring.

The effect of prenatal alcohol exposure on nerve cell development was investigated in neurons and glial cells cultured from fetal rat brain. Neurons were grown for one week from two week-old cortical brain cells and glial cells were cultured during four weeks from new born cortical brain cells. Two situations were examined: maternal alcohol treatment before and during pregnancy and alcohol exposure only until the beginning of pregnancy. In both situations we found a delayed nerve cell maturation assessed by biochemical markers like enolase and glutamine synthetase. Alterations of lactate dehydrogenase activity and reduced superoxide dismutase activity, involved in free radicals elimination were also observed. "In vitro" addition of alcohol to the culture medium produced an additional effect showing a lower response in the prenatal alcohol exposed brain cells than in the controls. The results confirm our previous "in vivo" experiments showing long lasting effects of maternal alcohol exposure in the offspring.

Maternal alcohol exposure before and during pregnancy: effect on development of neurons and glial cells in culture.

The effect of maternal alcohol exposure on nerve cell development was investigated in neurons and glial cells cultured from foetal rat brain. Neurons were grown for one week from two-week-old cortical brain cells and glial cells were cultured for four weeks from newborn cortical brain cells. Two types of maternal alcohol treatment were performed; either before and during pregnancy or only until the beginning of pregnancy. In both situations, we found a delayed nerve cell maturation assessed by microscopic observations and determination of enzymatic markers of nerve cell development (non-neuronal and neuron-specific enolase for the neuronal cells, non-neuronal enolase and glutamine synthetase for the glial cells). The results confirmed our previous in vivo experiments pointing out long-lasting effects of maternal alcohol exposure in the offspring.

Modulation of Mn2+ accumulation in cultured rat neuronal and astroglial cells.

The effects of physiological concentrations of K+ on Mn2+ accumulation were compared in rat glial cells and neurons in culture. Increasing the K+ concentration in growth medium increased significantly the Mn2+ level of the cultivated cells, with glial cells more affected than neurons. Ethanol markedly increased the Mn2+ accumulation within glia but not within neurons while ouabain caused inhibition of Mn2+ uptake with neurons and glial cells. A modulation of the total protein synthesis by Mn2+ and ethanol level in the growth medium was observed with glial cells. These data suggest that the mechanisms involved in Mn2+ accumulation in glial cells are different from those present in neurons. Moreover, the results are consistent with the hypothesis that Mn2+ plays a regulatory role in glial cell metabolism.

Effects of prolonged ethanol exposure on the glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study.

We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.

Lipid biosynthesis, oxidative enzyme activities and cellular changes in growing olive fruit.

Fruit of Olea europea L. was examined by light and electron microscopy to determine whether commencement of lipid accumulation depended upon the fruit achieving structural maturity. Maturation of fruit develops progressively from the smallest changes towards the largest in cellular structures. Important metabolic and structural changes have been observed: oil body formation, changes in the structural and reserve lipid biosynthesis and in the fatty acid of total lipid content, as well as in G6PDH and LOX activities. The labelling of fruit lipids by previously incubating the leaves with (1-14C)-acetate and (1,5-14C)-citrate or by putting the labelled substrates directly on the fruit surface, shows a 14C assimilate derived from acetate greater than that from citrate; the incorporation of the latter is higher in the methanol-water fractions. At the beginning of fruit development the lipid biosynthesis with both substrates is greater in polar lipids; on the contrary, the incorporation of 14C into neutral lipids increases during fruit maturation. Additionally, a maximum of substrate export from leaves to fruit coincides with an increase in the lipoxygenase and, above all, in the glucose-6-phosphate dehydrogenase activities. The transported 14C from leaves begins its activity before the small oil bodies close to the tonoplast can be observed in the fruit, and well before the beginning of maturation. The results suggest that structural development and some other rate controlling metabolic steps can govern the initiation of lipid accumulation in olive fruit.