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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.
    Developmental Biology 01/2014; · 3.87 Impact Factor
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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.
    Molecular &amp Cellular Proteomics 12/2013; · 7.25 Impact Factor
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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.
    Cytoskeleton 11/2013; · 2.87 Impact Factor
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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.
    Plant Physiology and Biochemistry 03/2013; 67C:120-125. · 2.78 Impact Factor
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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.
    Journal of Structural Biology 12/2012; · 3.36 Impact Factor
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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.
    Hearing research 07/2012; 292(1-2):14-25. · 2.18 Impact Factor
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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.
    Developmental Biology 03/2012; 365(2):350-62. · 3.87 Impact Factor
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    ABSTRACT: The most common neurodegenerative disorder afflicting the aging human population is Alzheimer's disease (AD). A major hallmark of AD is dementia from a loss of neuronal function, attributed to the presence and accumulation of β-amyloid (Aβ) peptide into senile plaques. Preceding senile plaque formation, abnormalities in axons can be observed as changes in morphologies and intracellular trafficking. Recently, it has been recognized that Aβ also accumulates within neurons and this intraneuronal Aβ accumulation has been reported to be critical in the disruption of synapses and cognitive function. Here, we report on the internalization of a fluorescently labeled Aβ peptide into cultured chick retinal neurons. The pattern of Aβ distribution during the time course of incubation is reminiscent of the endocytic pathway. Furthermore, the distribution of the internalized Aβ peptide converges with that of myosin Vb and both relocalize from the axon to cell body. These observations are consistent with the hypothesis that AD proceeds as a result of an imbalance between Aβ production and Aβ clearance, suggesting a role for myosin Vb in this process.
    Cytoskeleton 01/2012; 69(3):166-78. · 2.87 Impact Factor
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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.
    PLoS ONE 01/2012; 7(7):e42162. · 3.53 Impact Factor
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    ABSTRACT: It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.
    Memórias do Instituto Oswaldo Cruz 12/2011; 106(8):1014-6. · 1.36 Impact Factor
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    ABSTRACT: Connective tissue growth factor (CTGF/CCN2) is a protein of the CCN family that modulates cell-ECM interactions in a variety of cell types. In this study, we investigated the chemotactic and adhesive properties of CCN2 protein in embryonic teratocarcinoma P19 cells. Initially, P19 cells were attracted to CCN2-coated agarose beads. In Boyden chamber experiments, CCN2-containing medium induced a threefold greater migration of P19 cells. CCN2 adhesion properties were studied by using optical tweezers. The specific adhesion times of P19 cells to polystyrene beads coated with laminin, fibronectin, CCN2 and bovine serum albumin were 1.8 ± 0.5s, 2.7 ± 0.4s, 10 ± 2s and 13 ± 2s, respectively, revealing an unexpectedly low adhesive capacity of CCN2 protein for P19 cells. In conclusion, our findings support the chemoattractive role of CCN2 for P19 cells, but not its adhesive role when compared to laminin or fibronectin.
    Biochemical and Biophysical Research Communications 09/2011; 413(4):582-7. · 2.28 Impact Factor
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    ABSTRACT: In this work, a simple method was developed to increase cell wall porosity and to achieve partial hydrolysis of β-glucan. First, the cells were permeabilized using ethanol under agitation. Then the cells were treated with glass beads to increase cell wall porosity and subjected to an enzymatic treatment to degrade glycogen at 65 °C for 30 minutes. Finally, the cells were incubated at 57 °C for 2 hours to partially degrade β-glucan. Whole cells, permeabilized cells and permeabilized cells with partially hydrolyzed β-glucan were evaluated by optical microscopy and transmission electron microscopy. From the images of permeabilized cells with partially hydrolyzed β-glucan it was possible to visualize structures named “ghosts”, which are alike along microcapsules containing β-glucan. These structures can be used to aggregate β-glucan in food industrialized products.
    Catal. Sci. Technol. 08/2011; 1(6):1068-1071.
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    ABSTRACT: Peritoneal dialysis (PD) is a renal substitutive therapy based on the infusion of a dialysate in the peritoneum, which induces through an osmotic gradient the ultrafiltration of water and the clearance of blood stream impurities by the peritoneal membrane. The colonization of Tenckhoff catheters (TCs) used in PD by pathogenic microorganisms can lead to peritonitis, and probably catheter removal. Here, optical microscopy and scanning electron microscopy were applied to study biofilm formation in 11 TCs. Biofilms varied in their morphology and thickness. Short-term catheters (6 months) presented thinner deposits (3 μm) with granular or flat morphologies, either on the intraluminal or external surfaces. Bacterial colonies were found on catheters from infected patients. A tendency was observed for long-term catheters (6-8 years) to present thicker biofilms (30-35 μm). Surprisingly, patients' cells colonized the deep layers of the thicker biofilms, forming a complex multicelullar community. It was concluded that the presence of a biofilm is not necessarily related with peritonitis, and biofilm features may correlate to the therapy time.
    Biofouling 07/2011; 27(6):603-8. · 3.40 Impact Factor
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    ABSTRACT: The translocator protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is important for many cellular functions in mammals and bacteria, such as steroid biosynthesis, cellular respiration, cell proliferation, apoptosis, immunomodulation, transport of porphyrins and anions. Arabidopsis thaliana contains a single TSPO/PBR-related gene with a 40 amino acid N-terminal extension compared to its homologs in bacteria or mammals suggesting it might be chloroplast or mitochondrial localized. To test if the TSPO N-terminal extension targets it to organelles, we fused three potential translational start sites in the TSPO cDNA to the N-terminus of GFP (AtTSPO:eGFP). The location of the AtTSPO:eGFP fusion protein was found to depend on the translational start position and the conditions under which plants were grown. Full-length AtTSPO:eGFP fusion protein was found in the endoplasmic reticulum and in vesicles of unknown identity when plants were grown in standard conditions. However, full length AtTSPO:eGFP localized to chloroplasts when grown in the presence of 150 mM NaCl, conditions of salt stress. In contrast, when AtTSPO:eGFP was truncated to the second or third start codon at amino acid position 21 or 42, the fusion protein co-localized with a mitochondrial marker in standard conditions. Using promoter GUS fusions, qRT-PCR, fluorescent protein tagging, and chloroplast fractionation approaches, we demonstrate that AtTSPO levels are regulated at the transcriptional, post-transcriptional and post-translational levels in response to abiotic stress conditions. Salt-responsive genes are increased in a tspo-1 knock-down mutant compared to wild type under conditions of salt stress, while they are decreased when AtTSPO is overexpressed. Mutations in tetrapyrrole biosynthesis genes and the application of chlorophyll or carotenoid biosynthesis inhibitors also affect AtTSPO expression. Our data suggest that AtTSPO plays a role in the response of Arabidopsis to high salt stress. Salt stress leads to re-localization of the AtTSPO from the ER to chloroplasts through its N-terminal extension. In addition, our results show that AtTSPO is regulated at the transcriptional level in tetrapyrrole biosynthetic mutants. Thus, we propose that AtTSPO may play a role in transporting tetrapyrrole intermediates during salt stress and other conditions in which tetrapyrrole metabolism is compromised.
    BMC Plant Biology 06/2011; 11:108. · 4.35 Impact Factor
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    ABSTRACT: Protocadherin 15 (PCDH15) is expressed in hair cells of the inner ear and in photoreceptors of the retina. Mutations in PCDH15 cause Usher Syndrome (deaf-blindness) and recessive deafness. In developing hair cells, PCDH15 localizes to extracellular linkages that connect the stereocilia and kinocilium into a bundle and regulate its morphogenesis. In mature hair cells, PCDH15 is a component of tip links, which gate mechanotransduction channels. PCDH15 is expressed in several isoforms differing in their cytoplasmic domains, suggesting that alternative splicing regulates PCDH15 function in hair cells. To test this model, we generated three mouse lines, each of which lacks one out of three prominent PCDH15 isoforms (CD1, CD2 and CD3). Surprisingly, mice lacking PCDH15-CD1 and PCDH15-CD3 form normal hair bundles and tip links and maintain hearing function. Tip links are also present in mice lacking PCDH15-CD2. However, PCDH15-CD2-deficient mice are deaf, lack kinociliary links and have abnormally polarized hair bundles. Planar cell polarity (PCP) proteins are distributed normally in the sensory epithelia of the mutants, suggesting that PCDH15-CD2 acts downstream of PCP components to control polarity. Despite the absence of kinociliary links, vestibular function is surprisingly intact in the PCDH15-CD2 mutants. Our findings reveal an essential role for PCDH15-CD2 in the formation of kinociliary links and hair bundle polarization, and show that several PCDH15 isoforms can function redundantly at tip links.
    Development 04/2011; 138(8):1607-17. · 6.60 Impact Factor
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    ABSTRACT: The aims of this paper were to quantify the heavy metals (HM) in the air of different sites in Rio de Janeiro (RJ) and Salvador (SA) using Tillandsia usneoides (Bromeliaceae) as a biomonitor, and to study the morphology and elemental composition of the air particulate matter (PM) retained on the Tillandsia surface. Tillandsia samples were collected in a noncontaminated area and exposed to the air of five sites in RJ State and seven in SA for 45 days, in two seasons. Samples were prepared to HM quantification by flame atomic absorption spectrophotometry, while morphological and elemental characterizations were studied by using scanning electron microscopy. HM concentrations were significantly higher when compared to control sites. We found an increasing metal concentration as follows: Cd < Cr < Pb < Cu < Zn. PM exhibited a morphology varying from amorphous- to polygonal-shaped particles. Size measurements indicated that more than 80% of particles were less than 10 μm. PM contained aluminosilicates iron-rich particles, but Zn, Cu, Cr, and Ba were also detected. HM input in the atmosphere was mainly associated with anthropogenic sources such as vehicle exhaust. Elemental analysis detected HM in the inhalable particles, indicating that those HMs may intensify the toxic effects of PM on human health. Our results indicated T. usneoides as an adequate biomonitor of HM in the PM belonging to the inhalable fraction.
    Environmental Science and Pollution Research 03/2011; 18(3):416-27. · 2.76 Impact Factor

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