Biomedical subjects

Leonardo R Andrade

Publications and source records attributed to Leonardo R Andrade.

12 recordsLinked to original sources

PubMed2006

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma.

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

Animals↗

PubMed2006

Collagen colocalizes with a protein containing a decorin-specific peptide in the tissues of the ascidian Styela plicata.

Decorin is an extracellular matrix dermatan sulfate/chondroitin sulfate proteoglycan found in a variety of vertebrate species. In the extracellular matrix of mammals, decorin interacts with fibrillar collagen and regulates its morphology. We report here the occurrence and distribution of collagen type I and the peptide, CEASGIGPEVPDDRD, which is present in the human decorin proteoglycan, in the extracellular matrix of different tissues of the primitive invertebrate chordate Styela plicata. The content of collagen was estimated by hydroxyproline, and its distribution in the tissues by histochemistry. Collagen was detected biochemically in intestine, heart, pharynx and mantle, occurring in higher amounts in the heart, followed by pharynx, mantle and intestine. Histochemical analysis with Sirius red indicates that collagen is present in the extracellular matrix of intestine and pharynx. Further ultrastructural immuno-gold assays using polyclonal antibodies raised against the decorin-specific peptide CEASGIGPEVPDDRD and collagen type I showed a co-localization of these molecules. These data suggest the occurrence of a protein containing a decorin-like peptide sequence, which may be interacting with fibrillar collagen in this primitive chordate.

Animals↗

PubMed2006

New microencapsulation system for ascorbic acid using pea protein concentrate as coat protector.

Microencapsulation is essential to preserve biological activity of ascorbic acid (AA) and pea protein has not been used as a carrier in such processes. This work aimed to produce microparticles by a spray-drying process using pea protein (PPC) as wall material of AA and evaluate the retention of the core by HPLC, overall morphology SEM, size distribution by light scattering and release kinetics. Carboxymethylcellulose (CMC) and blends with maltodextrin (M) were produced for comparative analyses. The yields were compatible with the applied technology and the retention was above 84% for all materials. The PPC microparticles presented irregular and rough surfaces, CMC produced a regular and smooth surface and agglomeration was more intense in microparticles with M. Mean particle diameters were all below 8 microm. The microparticle release rates were lower than those with free AA, being best correlated to the Higuchi kinetic model. These results support the utilization of PPC for microencapsulation of AA.

Aerosols↗

PubMed2005

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts.

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.

Animals↗

PubMed2005

Localization of specific monosaccharides in cells of the brown alga Padina gymnospora and the relation to heavy-metal accumulation.

The brown alga Padina gymnospora has been studied due to their ecological significance and biochemical characteristics, including its high capability of heavy-metal accumulation. It has been suggested that the fucans are among the main polysaccharides related to metal binding and precipitation in cell walls. The main purpose of this work was to determine the localization of specific monosaccharides in P. gymnospora cells. In this way, the lectins Ulex europaeus agglutinin and Canavalia ensiformis concanavalin A with specificity to alpha-L-fucose and to terminal residues of alpha-D-glucosyl and alpha-D-mannosyl, respectively, were applied in young individuals. These revealed a preferential distribution of alpha-L-fucose at cell walls near the external surface in cortical cells and near the plasmalemma in cortical and medullar cells. The distribution of alpha-L-fucose in cell walls indicates the distribution of sulfated polysaccharides (sulfated fucans) that colocalize with the heavy-metal granules (Zn and Cd) described in previous works. Therefore, our results suggest that alpha-L-fucose participates in the nucleation and immobilization of heavy metals in P. gymnospora cell walls. An intense labeling of U. europaeus agglutinin and a weak labeling of concanavalin A was also observed in physodes. X-ray microanalysis revealed the presence of zinc, sulfur, and calcium in physodes of algae collected in a heavy-metal-contaminated area. Besides the affinity between polyphenolic compounds and heavy metals, it is suggested that the mechanism of metal binding by physodes could be related to the presence of sulfated fucans.

Cell Wall↗

PubMed2004

Occurrence of sulfated galactans in marine angiosperms: evolutionary implications.

We report for the first time that marine angiosperms (seagrasses) possess sulfated polysaccharides, which are absent in terrestrial and freshwater plants. The structure of the sulfated polysaccharide from the seagrass Ruppia maritima was determined. It is a sulfated D-galactan composed of the following regular tetrasaccharide repeating unit: [3-beta-D-Gal-2(OSO3)-1-->4-alpha-D-Gal-1-->4-alpha-D-Gal-1-->3-beta-D-Gal-4(OSO3)-1-->]. Sulfated galactans have been described previously in red algae and in marine invertebrates (ascidians and sea urchins). The sulfated galactan from the marine angiosperm has an intermediate structure when compared with the polysaccharides from these two other groups of organisms. Like marine invertebrate galactan, it expresses a regular repeating unit with a homogenous sulfation pattern. However, seagrass galactan contains the D-enantiomer of galactose instead of the L-isomer found in marine invertebrates. Like red algae, the marine angiosperm polysaccharide contains both alpha and beta units of D-galactose; however, these units are not distributed in an alternating order, as in algal galactan. Sulfated galactan is localized in the plant cell walls, mostly in rhizomes and roots, indicative of a relationship with the absorption of nutrients and of a possible structural function. The occurrence of sulfated galactans in marine organisms may be the result of physiological adaptations, which are not correlated with phylogenetic proximity. We suggest that convergent adaptation, due to environment pressure, may explain the occurrence of sulfated galactans in many marine organisms.

Alismatales↗

PubMed2004

Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development.

We developed a simple methodology for labeling sulfated glycosaminoglycans (GAGs) in adult Drosophila melanogaster and studied some aspects of the biosynthesis and metabolism of these polymers during development. Adult D. melanogaster flies were fed with Na(2)(35)SO(4) for 72 h. During this period, (35)S-sulfate was incorporated into males and females and used to synthesize (35)S-sulfate-heparan sulfate (HS) and (35)S-sulfate-chondroitin sulfate (CS). The incorporation of (35)S-sulfate into HS was higher when compared to CS. In a pulse-chase experiment, we observed that (35)S-sulfate incorporated into adult female was recovered in embryos and used for the synthesis of new (35)S-sulfate-GAGs after 2 h of embryonic development. The synthesis of CS was higher than that of HS, indicating a change in the metabolism of these glycans from adult to embryonic and larval stages. Analysis of the CS in embryonic and larval tissues revealed the occurrence of nonsulfated and 4-sulfated disaccharide units in embryos, L1 and L2. In L3, in addition to these disaccharides, we also detected significant amount of 6-sulfated units that are reported here for the first time. Immunohistochemical analysis indicated that HS and CS were present in nonequivalent structures in adult and larval stages of the fly. Overall, these results indicate that (35)S-sulfate-precursors are transferred from adult to embryonic and larval tissues and used to assemble different morphological structures during development.

Animals↗

PubMed2004

Ultrastructure of acidic polysaccharides from the cell walls of brown algae.

We have studied the ultrastructure of acidic polysaccharides from the cell walls of brown algae using a variety of electron microscopy techniques. Polysaccharides from Padina gymnospora present self assembled structures, forming trabecular patterns. Purified fractions constituted by alginic acid and sulfated fucan also form well-organized ultrastructures, but the pattern of organization varies depending on the polysaccharide species. Alginic acid presents sponge-like structures. Sulfated fucan exhibits particles with polygonal forms with a polycrystalline structure. These particles are in fact constituted by sulfated fucan molecules since they are recognized by a lectin specific for alpha-l-fucosyl residues. X-ray microanalysis reveal that S is a constituent element, as expected for sulfated groups. Finally, an exhaustive purified sulfated fucan shows the same ultrastructure formed by polygonal forms. Furthermore, elemental analyses of acidic polysaccharides indicate that they retain Zn, when algae were collected from a contaminated area. This observation is supported by direct quantification of heavy metal in the biomass and also in the solubilized polysaccharides compared with the algae from a non-contaminated site. We conclude that these molecules have specific ultrastructure and elemental composition; and act as metal binder for the nucleation and precipitation of heavy metals when the algae are exposed to a metal contaminated environment.

Alginates↗

PubMed2004

Effects of copper on Enteromorpha flexuosa (Chlorophyta) in vitro.

Enteromorpha species are widely used as biomonitors of copper (Cu) contamination in coastal waters, but the effects of Cu at the subcellular level and possible mechanisms of metal resistance are scarcely known. To contribute to the understanding of the Cu accumulation process in macroalgae species, we exposed adult individuals of Enteromorpha flexuosa to 50, 250, and 500 microg Cu/L in seawater for 5 days under controlled conditions. Thereafter, gross photosynthesis rate (GPR) and metal accumulation were measured. Conventional transmission electron microscopy (CTEM) and energy-dispersive X-ray analysis (EDXA) were used to study the metal accumulation process at the cellular level. Treatments with 250 and 500 microg Cu/L were observed to cause an inhibition of the GPR in the algae. Cu accumulation in samples exposed to 500 microgCu/L was 5284+/-561 microg Cu/g (dry wt), whereas in control samples (no Cu addition), accumulation was 9+/-1 microg/g. In cells of plants undergoing the 50 microg Cu/L treatment, the cytoplasm was clearly vacuolated. Thickening of cell walls and increase of relative number of starch granules and of lipid bodies were the main cellular changes observed in plants exposed to 250 and 500 microg Cu/L. EDXA of algae cells after 250 and 500 microg Cu/L exposure detected Cu mainly in vacuole precipitates. Cu was also detected in chloroplasts and in some epiphytic bacteria. It was concluded that E. flexuosa did not avoid penetration of Cu into the cytoplasm and consequently its toxic effects in concentrations of 250 and 500 microg Cu/L. Precipitates containing Cu in vacuoles should be related to a metal immobilization mechanism, minimizing the Cu toxicity for cells. The epiphytic bacteria can act as a biofilter diminishing the availability of free Cu(+2) for algae accumulation.

Biodegradation, Environmental↗

PubMed2003

Fibrillar aggregates of the tumor suppressor p53 core domain.

Alzheimer's disease, Parkinson's disease, cystic fibrosis, prion diseases, and many types of cancer are considered to be protein conformation diseases. Most of them are also known as amyloidogenic diseases due to the occurrence of pathological accumulation of insoluble aggregates with fibrillar conformation. Some neuroblastomas, carcinomas, and myelomas show an abnormal accumulation of the wild-type tumor suppressor protein p53 either in the cytoplasm or in the nucleus of the cell. Here we show that the wild-type p53 core domain (p53C) can form fibrillar aggregates after mild perturbation. Gentle denaturation of p53C by pressure induces fibrillar aggregates, as shown by electron and atomic force microscopies, by binding of thioflavin T, and by circular dichroism. On the other hand, heat denaturation produced granular-shaped aggregates. Annular aggregates similar to those found in the early aggregation stages of alpha-synuclein and amyloid-beta were also observed by atomic force microscopy immediately after pressure treatment. Annular and fibrillar aggregates of p53C were toxic to cells, as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay. Interestingly, the hot-spot mutant R248Q underwent similar aggregation behavior when perturbed by pressure or high temperature. Fibrillar aggregates of p53C contribute to the loss of function of p53 and seed the accumulation of conformationally altered protein in some cancerous cells.

Animals↗

PubMed2003

Dissociation of amyloid fibrils of alpha-synuclein and transthyretin by pressure reveals their reversible nature and the formation of water-excluded cavities.

Protein misfolding and aggregation have been linked to several human diseases, including Alzheimer's disease, Parkinson's disease, and systemic amyloidosis, by mechanisms that are not yet completely understood. The hallmark of most of these diseases is the formation of highly ordered and beta-sheet-rich aggregates referred to as amyloid fibrils. Fibril formation by WT transthyretin (TTR) or TTR variants has been linked to the etiology of systemic amyloidosis and familial amyloid polyneuropathy, respectively. Similarly, amyloid fibril formation by alpha-synuclein (alpha-syn) has been linked to neurodegeneration in Parkinson's disease, a movement disorder characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Here we show that consecutive cycles of compression-decompression under aggregating conditions lead to reversible dissociation of TTR and alpha-syn fibrils. The high sensitivity of amyloid fibrils toward high hydrostatic pressure (HHP) indicates the existence of packing defects in the fibril core. In addition, through the use of HHP we are able to detect differences in stability between fibrils formed from WT TTR and the familial amyloidotic polyneuropathy-associated variant V30M. The fibrils formed by WT alpha-syn were less susceptible to pressure denaturation than the Parkinson's disease-linked variants, A30P and A53T. This finding implies that fibrils of alpha-syn formed from the variants would be more easily dissolved into small oligomers by the cellular machinery. This result has physiological importance in light of the current view that the pathogenic species are the small aggregates rather the mature fibrils. Finally, the HHP-induced formation of fibrils from TTR is relatively fast (approximately 60 min), a quality that allows screening of antiamyloidogenic drugs.

Alzheimer Disease↗

PubMed2002

Elemental composition of acidocalcisomes of Trypanosoma cruzi bloodstream trypomastigote forms.

The elemental composition of acidocalcisomes present in Trypanosoma cruzi bloodstream trypomastigotes was analyzed by X-ray microanalysis. Acidocalcisomes appear as electron-dense vesicles dispersed throughout the cytoplasm. X-ray microanalysis of whole cells adhered to Formvar-coated grids showed that they contained C, O, Na, Mg, P, S, Ca and Zn, although in some acidocalcisomes iron was also detected. However, in ultra-thin sections of parasites processed according to routine procedures, most elements were lost. Living parasites were incubated with gold-labeled holo-transferrin to test if the iron was taken up by receptor-mediated endocytosis. After incubation at 4 degrees C, labeling was observed only in the flagellar pocket membrane, showing that receptors to this protein are already present in bloodstream trypomastigotes. In parasites incubated at 28 degrees C no intracellular tracer was observed, despite the labeling in the flagellar pocket membrane. Our data show that some acidocalcisomes of T. cruzi bloodstream trypomastigotes contain iron and that this element was not taken up by receptor-mediated endocytosis.

Animals↗