Leonardo Rodrigues Andrade

Cell Biology, Histology, Marine Biology

PhD
35.53

Publications

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    Leonardo R. Andrade · Felipe T. Salles · M’hamed Grati · Uri Manor · Bechara Kachar
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    ABSTRACT: All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and β tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α - and β –tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4 ± 1.5 nm, width), resembling another collagen type protein, or chains of globular elements (15 ± 3.2 nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α - and β -tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.
    Full-text · Article · Jan 2016 · Journal of Structural Biology
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    ABSTRACT: Background: Galectin-3 is known to be a lectin that plays an important role in inflammatory processes, acting as pro-inflammatory mediator in activation and migration of neutrophils and macrophages, as well as in the phagocytic function of these cells. The injection of mineral oils into the peritoneal cavity of mice, such as 2, 6, 10, 14-tetramethylpentadecane (pristane), induce a chronic granulomatous inflammatory reaction which is rich in macrophages, B cells and peritoneal plasma cells known as oil granuloma. In addition, this inflammatory microenvironment provided by oil granulomas is also an important site of plasmacytoma induction, which are dependent on cytokine production and cellular mobilization. Here, we have analyzed the role of galectin-3 in inflammatory cells mobilization and organization after pristane injection characterizing granulomatous reaction through the formation of oil granulomas. Results: In galectin-3 deficient mice (gal-3(-/-)), the mobilization of inflammatory cells, between peritoneal cavity and bone marrow, was responsible for the formation of disorganized oil granulomas, which presented scattered cells, large necrotic areas and low amounts of extracellular matrix. The production of inflammatory cytokines partially explained the distribution of cells through peritoneal cavity, since high levels of IL-6 in gal-3(-/-) mice led to drastically reduction of B1 cells. The previous pro-inflammatory status of these animals also explains the excess of cell death and disruption of oil granulomas architecture. Conclusions: Our data indicate, for the first time, that the disruption in the inflammatory cells migration in the absence of galectin-3 is a crucial event in the formation and organization of oil granulomas.
    Full-text · Article · Dec 2015 · BMC Immunology
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    ABSTRACT: The present study described with different microscopy approaches chloroplasts lobes in laurenciasensu latu (Rhodophyta) species and found inter-specific differences among them. Chloroplasts were investigated using confocal laser scanning microscopy (LSM), transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HRSEM). Using and TEM and HRSEM images we distinguished chloroplasts with lobes than chloroplasts without lobes in Yuzurua poiteauivar. gemmifera(Harvey) M. J. Wynne and laurencia dendroideaJ. Agardh cortical cells. The LSM images showed chloroplasts lobes (CLs) with different morphologies, varying from thicker and longer undulated projections in Y. poiteauivar. and I. dendroideato very small and thin tubules as in laurencia translucidaFujii & Cordeiro-Marino. The diameter and length of CLs from Y. poiteauivar. and I. dendroideawere significantly higher than I. translucidaCLs (p < 0.01). Based on LSM observations, we suggest that lobes morphology has a taxonomic validity only to characterize I. translucidaspecies.
    Full-text · Article · Nov 2015
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    ABSTRACT: Intrathymic lipid-laden multilocular cells (LLMC) are known to express pro-inflammatory factors that might regulate functional activity of the thymus. However, the phenotype of age-associated intrathymic LLMC is still controversial. In this study, we evaluated LLMC density in the aging thymus and better characterized their distribution, ultrastructure and phenotype. Our results show an increased density of LLMC in the thymus from 03 to 24 months of age. Morphologically, intrathymic LLMC exhibit fibroblastoid fusiform, globular or stellate shapes and can be found in the subcapsular region as well as deeper in the parenchyma, including the perivascular area. Some parenchymal LLMC were like telocytes accumulating lipids. We identified lipid droplets with different electrondensities, lipofuscin granules and autolipophagosome-like structures, indicating heterogeneous lipid content in these cells. Autophagosome formation in intrathymic LLMC was confirmed by positive staining for beclin-1 and perilipin (PLIN), marker for lipid droplet-associated proteins. We also found LLMC in close apposition to thymic stromal cells, endothelial cells, mast cells and lymphocytes. Phenotypically, we identified intrathymic LLMC as preadipocytes (PLIN+PPARγ2+), brown adipocytes (PLIN+UCP1+), macrophages (PLIN+Iba-1+) or pericytes (PLIN+NG2+) but not epithelial cells (PLIN- panCK+). These data indicate that intrathymic LLMC are already present in the young thymus and their density significantly increases with age. We also suggest that LLMC, which are morphologically distinct, establish direct contact with lymphocytes and interact with stromal cells. Finally, we evidence that intrathymic LLMC correspond to not only one but to distinct cell types accumulating lipids.
    Full-text · Article · Oct 2015 · PLoS ONE
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    Leonardo R. Andrade

    Full-text · Dataset · Aug 2015
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    ABSTRACT: The peritoneum is a thin membrane that covers most of the abdominal organs, composed of a monolayer of mesothelial cells and subjacent submesothelial loose connective tissue. Cells from the peritoneal wall are correlated with peritoneal fibrosis and epithelial-to-mesenchymal transition. However, the distinct involvement of mesothelial or submesothelial cells in such phenomena is still not clear. Here, we propose a new strategy to obtain stromal cells from anterior peritoneal wall explant cultures. These cells migrated from peritoneal tissues and proliferated in vitro for 4 weeks as adherent fibroblast-like cells. Optical and electronic microscopy analyses of the fragments revealed a significant submesothelial disorganization. The obtained cells were characterized as cytokeratin- vimentin+ laminin+ α-smooth muscle actin+, suggesting a connective tissue origin. Moreover, at the third passage, these stromal cells were CD90+CD73+CD29+Flk-1+CD45-, a phenotype normally attributed to cells of mesenchymal origin. These cells were able to support hematopoiesis, expressing genes involved in myelopoiesis (SCF, G-CSF, GM-CSF, IL-7 and CXCL-12), and differentiated into osteogenic and adipogenic cell lineages. The methodology demonstrated in this work can be considered an excellent experimental model to understand the physiology of the peritoneal wall in healthy and pathological processes. Moreover, this work shows for the first time that submesothelial stromal cells have properties similar to those of mesenchymal cells from other origins.
    Full-text · Article · May 2015 · Cells Tissues Organs
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    Leonardo R. Andrade
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    ABSTRACT: Cytoplasmic actin isoforms beta (β-) and gamma (γ-) perform crucial physiological roles in inner ear hair cells (HC). The stereocilium, which is structured by parallel actin filaments composed of both isoforms, is the responsive organelle to mechanical stimuli such as sound, gravity and head movements. Modifications in isoform proportions affect the function of the stereocilia as previously shown in genetic studies of mutant mice. Here, immunogold labeling TEM studies in mice showed that both β- and γ-actin isoforms colocalize throughout stereocilia actin filaments, adherens junctions and cuticular plates as early as embryonic stage 16.5. Gold-particle quantification indicated that there was 40% more γ- actin than β-actin at E16.5. In contrast, β- and γ-actin were equally concentrated in adult stereocilia of cochlear and vestibular HC. Interestingly, all actin-based structures presented almost five-fold more β-actin than γ-actin in 22 month- old mice, suggesting that γ-actin is probably under-expressed during the aging process. These data provide evidence of dynamic modifications of the actin isoforms in stereocilia, cuticular plates and cell junctions during the whole HC life. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Full-text · Article · May 2015 · Cytoskeleton
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    Full-text · Article · Jan 2015 · Journal of Phycology
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    ABSTRACT: Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB-Efnb2 signaling-deficient mice.
    Full-text · Article · Jun 2014 · Developmental Biology
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    Full-text · Dataset · Feb 2014
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    Full-text · Dataset · Feb 2014
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    Full-text · Dataset · Feb 2014
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    Full-text · Dataset · Feb 2014
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    ABSTRACT: Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. PTPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Jan 2014 · Cytoskeleton
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    ABSTRACT: During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e., fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein (CAPZ), a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. CAPZ levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development.
    Full-text · Article · Dec 2013 · Molecular & Cellular Proteomics
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    ABSTRACT: The TCP class of genes is found only in plants and is represented by the first three identified genes: teosinte branched1, cycloidea and pcf. Members belonging to this class are important regulators of plant growth, development and control multiple traits in diverse plant species, including flower and petal asymmetry, plant architecture, leaf morphogenesis and senescence, embryo growth and circadian rhythm. Here we described a member of the TCP-P subfamily called AtTCP23. Using qRT-PCR we present evidence that AtTCP23 is ubiquitously express in all organs examined. To ascertain AtTCP23 localization, we fused GFP at the C-terminal position and analyzed stable expression by confocal microscopy. Transgenic lines harboring the full-length protein (OxTCP23:GFP) seems to accumulate GFP in the nucleus. In order to analyze AtTCP23 function, we obtained a T-DNA insertional line and developed AtTCP23 over-expression (OxTCP23) lines. Phenotypic analysis indicates that tcp23-1 knockout line has an early-flowering phenotype while overexpression lines (OxTCP23 and OxTCP23:eGFP) presents opposite phenotype. Besides that those lines have leaf morphology alteration, pale leaf borders and smaller roots. Thus we propose in this study that AtTCP23 may be involved in flowering time control and plant development.
    Full-text · Article · Mar 2013 · Plant Physiology and Biochemistry
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    ABSTRACT: We examined the structure and biomineralization of prismatic magnetosomes in the magnetotactic marine vibrio Magnetovibrio blakemorei strain MV-1 and a non-magnetotactic mutant derived from it, using a combination of cryo-electron tomography and freeze-fracture. The vesicles enveloping the Magnetovibrio magnetosomes were elongated and detached from the cell membrane. Magnetosome crystal formation appeared to be initiated at a nucleation site on the membrane inner surface. Interestingly, while scattered filaments were observed in the surrounding cytoplasm, their association with the magnetosome chains could not be unequivocally established. Our data suggests fundamental differences between prismatic and octahedral magnetosomes in their mechanisms of nucleation and crystal growth as well as in their structural relationships with the cytoplasm and plasma membrane.
    Full-text · Article · Dec 2012 · Journal of Structural Biology
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    ABSTRACT: Top marine predators present high mercury concentrations in their tissues as consequence of biomagnification of the most toxic form of this metal, methylmercury (MeHg). The present study concerns mercury accumulation by Guiana dolphins (Sotalia guianensis), highlighting the selenium-mediated methylmercury detoxification process. Liver samples from 19 dolphins incidentally captured within Guanabara Bay (Rio de Janeiro State, Brazil) from 1994 to 2006 were analyzed for total mercury (THg), methylmercury (MeHg), total organic mercury (TOrgHg) and selenium (Se). X-ray microanalyses were also performed. The specimens, including from fetuses to 30-year-old dolphins, comprising 8 females and 11 males, presented high THg (0.53-132 µg/g wet wt.) and Se concentrations (0.17-74.8 µg/g wet wt.). Correlations between THg, MeHg, TOrgHg and Se were verified with age (p<0.05), as well as a high and positive correlation was observed between molar concentrations of Hg and Se (p<0.05). Negative correlations were observed between THg and the percentage of MeHg contribution to THg (p<0.05), which represents a consequence of the selenium-mediated methylmercury detoxification process. Accumulation of Se-Hg amorphous crystals in Kupffer Cells was demonstrated through ultra-structural analysis, which shows that Guiana dolphin is capable of carrying out the demethylation process via mercury selenide formation.
    Full-text · Article · Jul 2012 · PLoS ONE
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    ABSTRACT: Implementation of the deep-etch technique enabled unprecedented definition of substructural elements of otoconia, including the fibrillar meshwork of the inner core with its globular attachments. Subsequently the effects of the principal soluble otoconial protein, otoconin 90, upon calcite crystal growth in vitro were determined, including an increased rate of nucleation, inhibition of growth kinetics and significant morphologic changes. The logical next step, ultrastructural localization of otoconin 90, by means of immunogold TEM in young mature mice, demonstrated a high density of gold particles in the inner core in spite of a relatively low level of mineralization. Here gold particles are typically arranged in oval patterns implying that otoconin 90 is attached to a scaffold consisting of the hexagonal fibrillar meshwork, characteristic of otolin. The level of mineralization is much higher in the outer cortex where mineralized fiber bundles are arranged parallel to the surface. Following decalcification, gold particles, as well as matrix fibrils, presumed to consist of a linear structural phenotype of otolin, are aligned in identical direction, suggesting that they serve as scaffold to guide mineralization mediated by otoconin 90. In the faceted tips, the level of mineralization is highest, even though the density of gold particles is relatively low, conceivably due to the displacement by the dense mineral phase. TEM shows that individual crystallites assemble into iso-oriented columns. Columns are arranged in parallel lamellae which convert into mineralized blocks for hierarchical assembly into the complex otoconial mosaic. Another set of experiments based on immunogold TEM in young mice demonstrates that the fibrils interconnecting otoconia consist of the short chain collagen otolin. By two years of age the superficial layer of mouse otoconia (corresponding to mid-life human) has become demineralized resulting in weakening or loss of anchoring of the fibrils interconnecting otoconia. Consequently, otoconia detached from each other may be released into the endolymphatic space by minor mechanical disturbances. In humans, benign positional vertigo (BPV) is believed to result from translocation of otoconia from the endolymphatic space into the semi-circular canals rendering their receptors susceptible to stimulation by gravity causing severe attacks of vertigo. The combinations of these observations in humans, together with the presented animal experiments, provide a tentative pathogenetic basis of the early stage of BPV.
    Full-text · Article · Jul 2012 · Hearing research
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    ABSTRACT: Cholesterol-rich membrane microdomains (CRMMs) are specialized structures that have recently gained much attention in cell biology because of their involvement in cell signaling and trafficking. However, few investigations, particularly those addressing embryonic development, have succeeded in manipulating and observing CRMMs in living cells. In this study, we performed a detailed characterization of the CRMMs lipid composition during early frog development. Our data showed that disruption of CRMMs through methyl-β-cyclodextrin (MβCD) cholesterol depletion at the blastula stage did not affect Spemann's organizer gene expression and inductive properties, but impaired correct head development in frog and chick embryos by affecting the prechordal plate gene expression and cellular morphology. The MβCD anterior defect phenotype was recapitulated in head anlagen (HA) explant cultures. Culture of animal cap expressing Dkk1 combined with MβCD-HA generated a head containing eyes and cement gland. Together, these data show that during Xenopus blastula and gastrula stages, CRMMs have a very dynamic lipid composition and provide evidence that the secreted Wnt antagonist Dkk1 can partially rescue anterior structures in cholesterol-depleted head anlagen.
    Full-text · Article · Mar 2012 · Developmental Biology

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