Vera Maria Treis Trindade

ACL-I, a lectin from the marine sponge Axinella corrugata: isolation, characterization and chemotactic activity.

The lectin from the marine sponge Axinella corrugata (ACL-I) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-I is a hexameric glycoprotein with a Mr of 82.3 kDa estimated by SDS-PAGE and 78.5 kDa by FPLC on Superose 12 HR column. The pI of lectin is 6.3 and ACL-I is constituted of 13.9 kDa similar subunits some of them linked by disulphide bridges. This lectin agglutinates native rabbit, goat and dog erythrocytes and in less extent human erythrocytes. The hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but it is strongly inhibited by carbohydrates containing N-acetyl groups. ACL-I is stable up to 70 degrees C for 30 min, with optimum pH between 7 and 8, and it is also resistant to enzymatic proteolysis in vitro. In the presence of reducing or denaturant agents, the lectin activity decreases. ACL-I displays chemotactic effect on rat neutrophil in vitro which is inhibited by N-acetyl-d-glucosamine.

Amyloid-β induced toxicity involves ganglioside expression and is sensitive to GM1 neuroprotective action.

The effect of Aβ25-35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C(14)] galactose and results showed that Aβ25-35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aβ25-35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 μM was able to prevent the toxicity triggered by the fibrillar Aβ25-35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24h) upon the Aβ-induced alterations on GSK3β dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aβ-induced dephosphorylation (activation) of GSK3β, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimers disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimers disease experimental models and suggest a protective role for GM1 in Aβ-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimers treatment.

Neonatal hypoxia-ischemia reduces ganglioside, phospholipid and cholesterol contents in the rat hippocampus

Hypoxia-ischemia is a common cause of neonatal brain damage producing serious impact on cerebral maturation. This report demonstrates that rats submitted to hypoxia-ischemia present a marked decrease in hippocampal gangliosides, phospholipids and cholesterol contents as from 7 days after the injury. Although chromatographic profiles of the different ganglioside species (GM1, GD1a, GD1b, and GT1b) from the hippocampus of hypoxic-ischemic hippocampi groups (HI) were apparently unaffected, as compared with controls, there were quantitative absolute reductions in HI. The phospholipid patterns were altered in HI as from the 14th to the 30th day after the injury, where phosphatidylcholine (PC) quantities were higher than phosphatidylethanolamine (PE); additionally, the cardiolipin band was detected only in hippocampi of control adult rats. In general, the absolute quantities of phospholipids were lower in HI than in correspondent controls since 7th day after the injury. Considering that reported effects were maintained, we suggest they express a late biochemical response triggered by the neonatal hypoxic-ischemic episode; the consequences would be cell death and a delay on brain development, expressed by a reduction on synaptogenesis and myelinogenesis processes.

Diminished concentrations of ganglioside N-acetylneuraminic acid (G-NeuAc) in cerebellum of young rats receiving chronic administration of methylmalonic acid.

Sustained levels of methylmalonate comparable to those of human methylmalonic acidemia were achieved in the blood of young rats from the 5th till the 25th day of life by injecting them subcutaneously with buffered methylmalonic acid (MMA) twice a day at 8-h intervals. A matched group of rats (controls) was treated with saline. The animals were weighed and killed by decapitation at 25 days of age. Cerebellum and cerebrum were weighed and their contents of protein, DNA and ganglioside N-acetylneuraminic acid (G-NeuAc), as well as the protein/DNA ratio determined. Body weight, cerebral and cerebellar weight did not differ in both groups. The concentrations of protein, DNA and the protein/DNA ratio were also similar in the experimental and control groups. The results indicate that MMA per se does not interfere with the appetite of the animals and does not affect cellular proliferation and growth in cerebrum and cerebellum. We also found that G-NeuAc concentration is significantly reduced in the cerebellum. Therefore, since a deficit of an important component of brain closely related to the dendritic surface (synaptogenesis) occurs in MMA-treated rats, it is tempting to speculate whether this alteration may be associated or even partly responsible for the mental retardation in patients affected by methylmalonic acidemia.

Changes of sphingolipid species in the phenotype conversion from myofibroblasts to lipocytes in hepatic stellate cells

Sphingolipids play a relevant role in cell–cell interaction, communication, and migration. We studied the sphingolipid content in the murine hepatic stellate cell line GRX, which expresses the myofibroblast phenotype, and can be induced in vitro to display the fat‐storing phenotype. Lipid modifications along this induction were investigated by labeling sphingolipids with [14C]galactose, [14C]serine, or [14C]choline, and determination of fatty acid composition of sphingomyelin. The total ganglioside content and the GM2 synthase activity were lower in myofibroblasts. Both phenotypes presented similar gangliosides of the a‐pathway: GM2, GM1, and GD1a as well as their precursor GM3. Sphingomyelin and all the gangliosides were expressed as doublets; the upper/lower band ratio increased in lipocytes, containing more long‐chain fatty acids in retinol‐induced lipocytes as compared to the insulin/indomethacin induced ones. Time‐course experiments indicated a transfer of metabolic precursors from phosphatidylcholine to sphingomyelin in the two phenotypes. Taken together, these results indicate that myofibroblast and lipocytes can use distinct ceramide pools for sphingolipid synthesis. Differential ganglioside expression and presence of the long‐chain saturated fatty acids suggested that they may participate in formation of distinct membrane microdomains or rafts with specific functions on the two phenotypes of GRX‐cells.

Effect of Protein Malnutrition on Glycoprotein, Protein and Lipid Synthesis in the Rat Cerebellum during the Period of Brain Growth Spurt

Female Wistar rats were fed a normal-protein diet (25% casein) or a low-protein diet (8% casein) during pregnancy and lactation. The two diets were isocaloric and contained appropriate amounts of mineral salts and vitamins. Pups from dams submitted to the low-protein diet had a lower body weight than normally fed controls as early as on the day of birth, but a difference in cerebellar weight between the two groups was observed only on the 15th postnatal day. Malnutrition had no effect on cerebellar protein concentration, which increased with age in both groups. The cerebellar DNA concentration was higher at 7 and 15 days of age in normally fed rats than in malnourished rats, whereas at 21 days of age it was higher in the malnourished animals. [U-14C]Leucine and [2-3H]mannose incorporation into proteins and lipid synthesis from acetyl coenzyme A (CoA) derived from [U-14C]leucine markedly decreased with age in the cerebellum of rats fed both diets. [2-3H]Mannose incorporation into cerebellar glycoproteins was greater in malnourished rats during the period of brain growth spurt than in normally fed rats at all ages studied. Prenatal and postnatal protein malnutrition had no effect on [U-14C]leucine incorporation into cerebellar proteins or on cerebellar lipid synthesis from acetyl-CoA derived from [U-14C]leucine during the period of brain growth spurt.

Gangliosides of myelosupportive stroma cells are transferred to myeloid progenitors and are required for their survival and proliferation

In previous studies, we have shown that the myelopoiesis dependent upon myelosupportive stroma required production of growth factors and heparan-sulphate proteoglycans, as well as generation of a negatively charged sialidase-sensitive intercellular environment between the stroma and the myeloid progenitors. In the present study, we have investigated the production, distribution and role of gangliosides in an experimental model of in vitro myelopoiesis dependent upon AFT-024 murine liver-derived stroma. We used the FDC-P1 cell line, which is dependent upon GM-CSF (granulocyte/macrophage colony-stimulating factor) for both survival and proliferation, as a reporter system to monitor bioavailability and local activity of GM-CSF. G(M3) was the major ganglioside produced by stroma, but not by myeloid cells, and it was required for optimal stroma myelosupportive function. It was released into the supernatant and selectively incorporated into the myeloid progenitor cells, where it segregated into rafts in which it co-localized with the GM-CSF-receptor alpha chain. This ganglioside was also metabolized further by myeloid cells into gangliosides of the a and b series, similar to endogenous G(M3). In these cells, G(M1) was the major ganglioside and it was segregated at the interface by stroma and myeloid cells, partially co-localizing with the GM-CSF-receptor alpha chain. We conclude that myelosupportive stroma cells produce and secrete the required growth factors, the cofactors such as heparan sulphate proteoglycans, and also supply gangliosides that are transferred from stroma to target cells, generating on the latter ones specific membrane domains with molecular complexes that include growth factor receptors.

Ganglioside alterations in the central nervous system of rats chronically injected with methylmalonic and propionic acids.

Neurological dysfunction and structural cerebral abnormalities are commonly found in patients with methylmalonic and propionic acidemia. However, the mechanisms underlying the neuropathology of these disorders are poorly understood. We have previously demonstrated that methylmalonic and propionic acids induce a significant reduction of ganglioside N-acetylneuraminic acid in the brain of rats subjected to chronic administration of these metabolites. In the present study, we investigated the in vivo effects of chronic administration of methylmalonic (MMA) and propionic (PA) acids (from the 6th to the 28th day of life) on the distribution and composition of gangliosides in the cerebellum and cerebral cortex of rats. Control rats were treated with the same volumes of saline. It was first verified that MMA and PA treatment did not modify body, cerebellum, or cortical weight, nor the ganglioside concentration in the cerebral cortex of the animals. In contrast, a significant reduction in total ganglioside content in the cerebellum of approximately 20–30% and 50% of control levels occurred in rats injected with MMA and PA, respectively. Moreover, chronic MMA and PA administration did not interfere with the ganglioside pattern in the cerebral cortex, whereas the distribution of individual gangliosides was altered in the cerebellum of MMA- and PA-treated animals. Rats injected with MMA demonstrated a marked decrease in GM1 and GD3, whereas chronic PA treatment provoked a significant reduction of all ganglioside species, with the exception of an increase in GM2. Since gangliosides are closely related to the dendritic surface and other neural membranes, indirectly reflecting synaptogenesis, these ganglioside abnormalities may be associated with the brain damage found in methylmalonic and propionic acidemias.

Biological activities of ACL-I and physicochemical properties of ACL-II, lectins isolated from the marine sponge Axinella corrugata

Lectin II from the marine sponge Axinella corrugata (ACL-II) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel, followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-II is a lectin with an Mr of 80 kDa and 78 kDa, estimated by SDS-PAGE and by FPLC on Superose 12 HR column, respectively. ACL-II mainly agglutinates native rabbit erythrocytes and this hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but is inhibited by d-galactose, chitin and N-acetyl derivatives, with the exception of GalNAc. ACL-II is stable for up to 65 °C for 30 min, with a better stability at a pH range of 2 to 6. In contrast, ACL-I displays a strong mitogenic and cytotoxic effect.

Reduction of gangliosides, phospholipids and cholesterol content in cerebral cortex of rats caused by chronic hypermethioninemia

Neurological dysfunction is observed in patients with severe hypermethioninemia, whose physiopathology is still poorly understood. In the current study we investigated the effect of chronic administration of methionine on the content and species of gangliosides and phospholipids, as well as on the concentration of cholesterol in rat cerebral cortex. Wistar rats received subcutaneous injections of methionine (1.34–2.68 μmol/g of body weight), twice a day, from the 6th to the 28th day of age and controls received saline. Animals were killed 12 h after the last injection. Results showed that methionine administration significantly decreased the total content of lipids in cerebral cortex of rats. We also observed that this amino acid significantly reduced the absolute quantity of the major brain gangliosides (GM1, GD1a, GD1b and GT1b) and phospholipids (sphingomyelin, phosphatidylcholine and phosphatidylethanolamine). We also showed that Na+,K+‐ATPase activity and TBARS were changed in cerebral cortex of rats subjected to hypermethioninemia. If confirmed in human beings, these data could suggest that the alteration in lipid composition, Na+,K+‐ATPase activity and TBARS caused by methionine might contribute to the neurophysiopathology observed in hypermethioninemic patients.