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1.
Sci Rep ; 5: 13521, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-26335545

RESUMO

Actin-based cellular protrusions are an ubiquitous feature of cells, performing a variety of critical functions ranging from cell-cell communication to cell motility. The formation and maintenance of these protrusions relies on the transport of proteins via myosin motors, to the protrusion tip. While tip-directed motion leads to accumulation of motors (and their molecular cargo) at the protrusion tip, it is observed that motors also form rearward moving, periodic and isolated aggregates. The origins and mechanisms of these aggregates, and whether they are important for the recycling of motors, remain open puzzles. Motivated by novel myosin-XV experiments, a mass conserving reaction-diffusion-advection model is proposed. The model incorporates a non-linear cooperative interaction between motors, which converts them between an active and an inactive state. Specifically, the type of aggregate formed (traveling waves or pulse-trains) is linked to the kinetics of motors at the protrusion tip which is introduced by a boundary condition. These pattern selection mechanisms are found not only to qualitatively agree with empirical observations but open new vistas to the transport phenomena by molecular motors in general.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Fluidez de Membrana/fisiologia , Modelos Biológicos , Proteínas Motores Moleculares/fisiologia , Animais , Tamanho Celular , Simulação por Computador , Humanos
2.
Hear Res ; 315: 49-60, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25016142

RESUMO

In the preservation of tissues in as 'close to life' state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 µm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues.


Assuntos
Criopreservação/métodos , Orelha Interna/patologia , Preservação de Órgãos/métodos , Fixação de Tecidos/métodos , Animais , Artefatos , Orelha Interna/ultraestrutura , Gerbillinae , Cobaias , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Modelos Animais , Notophthalmus viridescens , Estereocílios/patologia , Estereocílios/ultraestrutura , Fatores de Tempo
3.
Phys Rev E Stat Nonlin Soft Matter Phys ; 74(4 Pt 1): 041903, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17155092

RESUMO

Membrane ruffle fluctuations of amphibian epithelial cells A6 (CCL102) cultured in normal and upside down oriented plates have been analyzed through video microscopy. Our results reveal that their edge ruffle fluctuations exhibit a stochastic dynamics with 1/f(alpha) power spectrum over at least two decades at low frequencies and long range correlated, self-affine lateral border profiles. In a few and small areas of the membrane, probably nearby focal contacts, we found periodic oscillations which could be induced by myosin driven contraction of stress fibers. Furthermore, whereas the different gravitational orientations had none or little effect on the structure (power spectra and surface roughness) of these membrane ruffle fluctuations, their dynamic parameters were differentially affected. Indeed, the decay time of ruffles remained unchanged, but the period of lamellipodia oscillations near the focal adhesion points was significantly altered in A6 cells cultured upside down.


Assuntos
Relógios Biológicos/fisiologia , Membrana Celular/fisiologia , Células Epiteliais/fisiologia , Gravitação , Mecanotransdução Celular/fisiologia , Fluidez de Membrana/fisiologia , Modelos Biológicos , Anfíbios , Animais , Linhagem Celular , Simulação por Computador , Modelos Estatísticos , Movimento/fisiologia , Processos Estocásticos
4.
J Membr Biol ; 209(2-3): 71-88, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16773495

RESUMO

The gating-spring theory of hair cell mechanotransduction channel activation was first postulated over twenty years ago. The basic tenets of this hypothesis have been reaffirmed in hair cells from both auditory and vestibular systems and across species. In fact, the basic findings have been reproduced in every hair cell type tested. A great deal of information regarding the structural, mechanical, molecular and biophysical properties of the sensory hair bundle and the mechanotransducer channel has accumulated over the past twenty years. The goal of this review is to investigate new data, using the gating spring hypothesis as the framework for discussion. Mechanisms of channel gating are presented in reference to the need for a molecular gating spring or for tethering to the intra- or extracellular compartments. Dynamics of the sensory hair bundle and the presence of motor proteins are discussed in reference to passive contributions of the hair bundle to gating compliance. And finally, the molecular identity of the channel is discussed in reference to known intrinsic properties of the native transducer channel.


Assuntos
Células Ciliadas Auditivas/fisiologia , Audição/fisiologia , Animais , Cílios/fisiologia , Células Ciliadas Auditivas/ultraestrutura , Ativação do Canal Iônico/fisiologia , Mecanorreceptores/fisiologia , Microscopia Eletrônica de Transmissão , Modelos Biológicos
5.
Mol Cell Neurosci ; 20(2): 343-53, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12093165

RESUMO

Sensory (hair) cells of the inner ear contain two specialized areas of membrane delivery. The first, located at the cell base, is the afferent synapse where rapid delivery of synaptic vesicles is required to convey information about auditory signals with exceedingly high temporal precision. The second area is at the apex. To accommodate the continuous movement of stereocilia and facilitate their repair, recycling of membrane components is required. Intense vesicular traffic is restricted to a narrow band of cytoplasm around the cuticular plate, which anchors stereocilia. Our previous analyses showed that SNARE proteins (syntaxin 1A/SNAP25/VAMP1) are concentrated at both poles of hair cells, consistent with their involvement in membrane delivery at both locations. To investigate further the molecules involved in membrane delivery at these two sites, we constructed a two-hybrid library of the organ of Corti and probed it with syntaxin 1A. Here we report the cloning of a novel syntaxin-binding protein that is concentrated in a previously uncharacterized organelle at the apex of inner hair cells.


Assuntos
Proteínas de Transporte/isolamento & purificação , Compartimento Celular/fisiologia , Endossomos/metabolismo , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/isolamento & purificação , Transporte Proteico/fisiologia , Membranas Sinápticas/metabolismo , Animais , Sequência de Bases/genética , Proteínas de Transporte/genética , Cílios/metabolismo , Cílios/ultraestrutura , Clonagem Molecular , DNA Complementar/análise , Endossomos/ultraestrutura , Proteínas de Fluorescência Verde , Cobaias , Células HeLa , Audição/fisiologia , Humanos , Imuno-Histoquímica , Indicadores e Reagentes/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Organelas/metabolismo , Organelas/ultraestrutura , Proteínas Qa-SNARE , Homologia de Sequência de Aminoácidos , Membranas Sinápticas/ultraestrutura , Sintaxina 1
6.
Ann N Y Acad Sci ; 942: 162-78, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11710459

RESUMO

The first part of this review deals with recent advances in the understanding of biochemical mechanisms of otoconial morphogenesis. Most important in this regard is the molecular characterization of otoconin 90, the principal matrix protein of mammalian calcitic otoconia, which was found to be a homologue of the phospholytic enzyme PLA2. The unique and unexpected expression pattern of this protein required radical rethinking of traditional concepts. The new data, when integrated with existing information, provide a rational basis for an explanation of the mechanisms leading to crystal nucleation and growth. Based on this information, a hypothetical model is presented that posits interaction of otoconin 90 with microvesicles derived from the supporting cells as a key event in the formation of otoconia. The second part of the review is directed at the controversial subject of maintenance of mature otoconia and systematically analyzes the available indirect information on this topic. A synthesis of these theoretical considerations is viewed in relation to the pathogenesis of the important otoneurologic entities of BPPN and senile otoconial degeneration. The last part of the review deals with several animal models that promise to help elucidate normal and abnormal mechanisms of otoconial morphogenesis, including mineral deficiencies, mutations with selective otoconial agenesis, as well as targeted disruption of essential genes.


Assuntos
Membrana dos Otólitos/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Proteínas da Matriz Extracelular , Glicoproteínas/metabolismo , Gravitação , Camundongos , Modelos Animais , Morfogênese , Membrana dos Otólitos/enzimologia , Membrana dos Otólitos/crescimento & desenvolvimento , Fosfolipases A/metabolismo , Fosfolipases A2
7.
J Neurosci ; 21(14): 5066-78, 2001 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11438582

RESUMO

Mechanoelectrical transduction channels of hair cells allow for the entry of appreciable amounts of Ca(2+), which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca(2+) that enters transduction channels is extruded by the plasma membrane Ca(2+)-ATPase (PMCA), a Ca(2+) pump that is highly concentrated in hair bundles and may be essential for normal hair cell function. Because PMCA isozymes and splice forms are regulated differentially and have distinct biochemical properties, we determined the identity of hair bundle PMCA in frog and rat hair cells. By screening a bullfrog saccular cDNA library, we identified abundant PMCA1b and PMCA2a clones as well as rare PMCA2b and PMCA2c clones. Using immunocytochemistry and immunoprecipitation experiments, we showed in bullfrog sacculus that PMCA1b is the major isozyme of hair cell and supporting cell basolateral membranes and that PMCA2a is the only PMCA present in hair bundles. This complete segregation of PMCA1 and PMCA2 isozymes holds for rat auditory and vestibular hair cells; PMCA2a is the only PMCA isoform in hair bundles of outer hair cells and vestibular hair cells and is the predominant PMCA of hair bundles of inner hair cells. Our data suggest that hair cells control plasma membrane Ca(2+)-pumping activity by targeting specific PMCA isozymes to distinct subcellular locations. Because PMCA2a is the only Ca(2+) pump present at appreciable levels in hair bundles, the biochemical properties of this pump must account fully for the physiological features of transmembrane Ca(2+) pumping in bundles.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Células Ciliadas Auditivas/metabolismo , Processamento Alternativo/genética , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Clonagem Molecular , DNA Complementar/isolamento & purificação , Células Ciliadas Auditivas/citologia , Células Ciliadas Vestibulares/citologia , Células Ciliadas Vestibulares/metabolismo , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Órgão Espiral/citologia , Órgão Espiral/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática , Testes de Precipitina , Rana catesbeiana , Ratos , Sáculo e Utrículo/citologia , Sáculo e Utrículo/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
9.
Synapse ; 40(4): 258-68, 2001 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-11309841

RESUMO

PDZ-domain containing proteins of the MAGUK (membrane-associated guanylate kinase) family target, anchor, and cluster receptors and channels to subcellular sites. Among the MAGUK proteins, the members of the PSD-95 family (MAGUKs: PSD-95, PSD-93, SAP-97, and SAP-102) target and anchor glutamate receptors to the synaptic terminals. Associations of glutamate receptors with MAGUKs have been described in the brain but not in the cochlea. In this study, RT-PCR, immunofluorescence microscopy, and immunoelectron microscopy were used to investigate the presence and distribution of MAGUK proteins in the organ of Corti. The presence of the mRNA for PSD-95, PSD-93, SAP-97, and SAP-102 in the organ of Corti was confirmed by RT-PCR. Immunocytochemistry using a "pan-MAGUK" antibody, which recognizes all four MAGUK proteins, and selective antibodies against these proteins revealed that all four MAGUKs are present within the base of inner hair cells while all except SAP-97 are found within the base of the outer hair cells. In addition, PSD-93 and PSD-95 are found in postsynaptic afferent terminals on inner hair cells, while postsynaptic afferent terminals on outer hair cells have PSD-93.


Assuntos
Células Ciliadas Auditivas Internas/química , Células Ciliadas Auditivas Externas/química , Proteínas do Tecido Nervoso/análise , Núcleosídeo-Fosfato Quinase/análise , Membranas Sinápticas/química , Animais , Guanilato Quinases , Cobaias , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/ultraestrutura , Imuno-Histoquímica , Microscopia Imunoeletrônica , Neuropeptídeos/análise , Canais de Potássio/metabolismo , Receptores de Glutamato/metabolismo , Gânglio Espiral da Cóclea/química , Gânglio Espiral da Cóclea/ultraestrutura , Membranas Sinápticas/ultraestrutura
10.
J Physiol ; 531(Pt 3): 667-76, 2001 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11251049

RESUMO

1. Whole-cell patch-clamp recordings were obtained from isolated cochlear outer hair cells (OHCs) while applying 2,3-butanedione monoxime (BDM) by pressure. BDM (5 mM) shifted the range of voltage sensitivity of membrane capacitance and cell length in the hyperpolarised direction by -49.6 +/- 4.0 mV (n = 12; mean +/- S.E.M.), without appreciable effects on membrane conductance. The shift was completely reversible and dose dependent, with a Hill coefficient of 1.8 /- 0.4 and a half-maximal dose of 3.0 +/- 0.8 mM (values +/- S.D). 2. The shift of the capacitance curve was also reproducible in cells whose natural turgor had been removed. BDM had no detectable effect on the capacitance of Deiters' cells, a non-sensory cell type of the organ of Corti. 3. The effect of BDM on membrane capacitance was faster than that of salicylate. At similar saturating concentrations (20 mM), the time constant of the capacitance changes was 1.8 +/- 0.3 s (n = 3) for salicylate and 0.75 +/- 0.06 s (n = 3) for BDM. The recovery periods were 13 +/- 1 s and 1.7 +/- 0.4 s, respectively (means +/- S.E.M.). 4. The effect of BDM, a known inorganic phosphatase, was compared to the effects of okadaic acid, trifluoperazine and W-7, which are commonly used in studies of protein phosphorylation. Incubation of OHCs with okadaic acid (1 microM, 30-60 min) shifted the voltage sensitivity of the membrane capacitance in the hyperpolarised direction. Incubation with trifluoperazine (30 microM) and W-7 (150 microM) shifted it in the opposite, depolarised direction. BDM induced hyperpolarising shifts even in the presence of W-7. 5. Simultaneous measurement of membrane capacitance and intracellular free Ca2+ concentration ([Ca2+]i) showed that BDM action on OHC voltage-dependent capacitance and electromotility is not mediated by changes of [Ca2+]i. 6. Our results suggest that: (a) the effects of BDM are unrelated to its inorganic phosphatase properties, cell turgor conditions or Ca2+ release from intracellular stores; and (b) BDM may target directly the voltage sensor of the OHC membrane motor protein.


Assuntos
Diacetil/análogos & derivados , Diacetil/farmacologia , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/fisiologia , Animais , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Condutividade Elétrica , Eletrofisiologia , Cobaias , Células Ciliadas Auditivas Externas/citologia , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiologia , Concentração Osmolar , Fosforilação/efeitos dos fármacos , Pressão , Proteínas/metabolismo , Salicilatos/farmacologia
11.
J Physiol ; 531(Pt 3): 693-706, 2001 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11251051

RESUMO

1. Hensen's cells in the isolated cochlea were stimulated by extracellular adenosine 5'-triphosphate (ATP) applied to their endolymphatic surface while changes in membrane current and intracellular calcium concentration ([Ca2+]i) were measured simultaneously. The response consisted of (i) an initial rapid inward current accompanied by elevation of the [Ca2+]i, (ii) a more slowly rising inward current accompanied by a rise of the [Ca2+]i and (iii) a slowly developing reduction of input conductance. 2. The slower responses were maintained in the absence of extracellular Ca2+. Similar responses were produced by increasing the [Ca2+]i via UV flash photolysis of intracellular D-myo-inositol 1,4,5-trisphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester (caged InsP3) loaded at pipette concentrations of 8-16 microM. 3. The slow inward current, reversing around 0 mV, was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). 4. Bath application of U-73122 (1 microM), a phospholipase C inhibitor, eliminated the slow Ca2+-release component of the response to ATP. It is proposed that the effects of ATP are mediated by the co-activation of ionotropic P2X and metabotropic P2Y receptors. 5. Immunohistochemistry using light and electron microscopy revealed that inositol 1,4,5-trisphosphate (InsP3) receptors delineate a network within the cells. 6. The coupling ratio (CR) between cell pairs measured in dual patch-clamp recordings was 0.356 +/- 0.024. The coupling reversibly decreased to 51 % of the control within 2 min of applying 100 microM ATP. Flash photolysis of 32 microM intracellular caged InsP3 and 1 mM caged Ca2+ reduced CR to 42 and 62 % of the control, respectively. 7. We propose that endolymphatic ATP via P2X and P2Y receptors can control intercellular communication amongst Hensen's cells by reducing gap junction conductance in a Ca2+- and InsP3-dependent manner.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Comunicação Celular/fisiologia , Cóclea/citologia , Cóclea/fisiologia , Purinas/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Cóclea/efeitos dos fármacos , Cóclea/efeitos da radiação , Condutividade Elétrica , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/metabolismo , Cobaias , Imuno-Histoquímica , Inositol 1,4,5-Trifosfato/farmacologia , Membranas Intracelulares/metabolismo , Fotólise , Purinas/agonistas , Purinas/antagonistas & inibidores , Fatores de Tempo , Fosfolipases Tipo C/antagonistas & inibidores , Raios Ultravioleta
12.
Cell ; 104(1): 165-72, 2001 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-11163249

RESUMO

Tight junctions in the cochlear duct are thought to compartmentalize endolymph and provide structural support for the auditory neuroepithelium. The claudin family of genes is known to express protein components of tight junctions in other tissues. The essential function of one of these claudins in the inner ear was established by identifying mutations in CLDN14 that cause nonsyndromic recessive deafness DFNB29 in two large consanguineous Pakistani families. In situ hybridization and immunofluorescence studies demonstrated mouse claudin-14 expression in the sensory epithelium of the organ of Corti.


Assuntos
Surdez/genética , Saúde da Família , Proteínas de Membrana/genética , Órgão Espiral/química , Mutação Puntual , Junções Íntimas/química , Northern Blotting , Claudinas , Consanguinidade , Genes Recessivos , Ligação Genética , Humanos , Proteínas de Membrana/análise , Dados de Sequência Molecular , Linhagem , RNA Mensageiro/análise , Homologia de Sequência de Aminoácidos
13.
Nat Genet ; 27(1): 103-7, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11138008

RESUMO

Mouse chromosome 10 harbors several loci associated with hearing loss, including waltzer (v), modifier-of deaf waddler (mdfw) and Age-related hearing loss (Ahl). The human region that is orthologous to the mouse 'waltzer' region is located at 10q21-q22 and contains the human deafness loci DFNB12 and USH1D). Numerous mutations at the waltzer locus have been documented causing erratic circling and hearing loss. Here we report the identification of a new gene mutated in v. The 10.5-kb Cdh23 cDNA encodes a very large, single-pass transmembrane protein, that we have called otocadherin. It has an extracellular domain that contains 27 repeats; these show significant homology to the cadherin ectodomain. In v(6J), a GT transversion creates a premature stop codon. In v(Alb), a CT exchange generates an ectopic donor splice site, effecting deletion of 119 nucleotides of exonic sequence. In v(2J), a GA transition abolishes the donor splice site, leading to aberrant splice forms. All three alleles are predicted to cause loss of function. We demonstrate Cdh23 expression in the neurosensory epithelium and show that during early hair-cell differentiation, stereocilia organization is disrupted in v(2J) homozygotes. Our data indicate that otocadherin is a critical component of hair bundle formation. Mutations in human CDH23 cause Usher syndrome type 1D and thus, establish waltzer as the mouse model for USH1D.


Assuntos
Caderinas/genética , Células Ciliadas Auditivas Internas/patologia , Perda Auditiva Neurossensorial/genética , Mutação/genética , Sequência de Aminoácidos , Animais , Percepção Auditiva/fisiologia , Sequência de Bases , Caderinas/química , Caderinas/metabolismo , Clonagem Molecular , Cóclea/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/fisiopatologia , Células Ciliadas Auditivas Internas/ultraestrutura , Audição/fisiologia , Perda Auditiva Neurossensorial/patologia , Testes Auditivos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome
14.
Hum Mol Genet ; 10(2): 153-61, 2001 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11152663

RESUMO

Following the positional cloning of PDS, the gene mutated in the deafness/goitre disorder Pendred syndrome (PS), numerous studies have focused on defining the role of PDS in deafness and PS as well as elucidating the function of the PDS-encoded protein (pendrin). To facilitate these efforts and to provide a system for more detailed study of the inner-ear defects that occur in the absence of pendrin, we have generated a Pds-knockout mouse. Pds(-/-) mice are completely deaf and also display signs of vestibular dysfunction. The inner ears of these mice appear to develop normally until embryonic day 15, after which time severe endolymphatic dilatation occurs, reminiscent of that seen radiologically in deaf individuals with PDS mutations. Additionally, in the second postnatal week, severe degeneration of sensory cells and malformation of otoconia and otoconial membranes occur, as revealed by scanning electron and fluorescence confocal microscopy. The ultrastructural defects seen in the Pds(-/-) mice provide important clues about the mechanisms responsible for the inner-ear pathology associated with PDS mutations.


Assuntos
Proteínas de Transporte/genética , Orelha Interna/anormalidades , Bócio/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana Transportadoras , Animais , Bócio/patologia , Bócio/fisiopatologia , Células Ciliadas Auditivas/anormalidades , Células Ciliadas Auditivas/ultraestrutura , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/fisiopatologia , Camundongos , Camundongos Knockout , Camundongos Mutantes Neurológicos , Microscopia Eletrônica de Varredura , Transportadores de Sulfato , Síndrome , Glândula Tireoide/patologia , Glândula Tireoide/fisiopatologia , Doenças Vestibulares/genética , Doenças Vestibulares/patologia , Doenças Vestibulares/fisiopatologia , Vestíbulo do Labirinto/anormalidades , Vestíbulo do Labirinto/ultraestrutura
15.
Curr Protoc Neurosci ; Chapter 2: Unit 2.1, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18428456

RESUMO

The growing importance in biology and especially in neurobiology of fluorescence microscopy is due to (1) the extraordinary development of new fluorescent molecular probes and (2) the development of improved low light level imaging systems and confocal microscopy techniques. This overview covers fluorescent molecular probes, filters and filter sets, multiband filters and multidye fluorescence, light sources, microscope objectives, image resolution and the point-spread function, and general steps for immunolabeling.


Assuntos
Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Animais , Células Cultivadas , Corantes Fluorescentes , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Sondas Moleculares , Técnicas de Cultura de Tecidos/instrumentação , Técnicas de Cultura de Tecidos/métodos
16.
Curr Protoc Mol Biol ; Chapter 14: Unit 14.10, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18265106

RESUMO

This overview discusses the principle of fluorescence along with practical discussions of fluorescent molecular probes, filters and filter sets, multiband filters and multi-dye fluorescence, light sources, objective lenses, image resolution and the point-spread function, fluorescence microscopy of living cells, and immunolabeling.


Assuntos
Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Animais , Anticorpos/imunologia , Sobrevivência Celular , Corantes Fluorescentes , Imuno-Histoquímica
17.
Cell Commun Adhes ; 8(4-6): 393-9, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-12064625

RESUMO

Immunolabeling with antibodies against connexins 26 and 30 showed that, in the guinea pig cochlea, supporting Deiters' cells are massively interconnected and form an orderly network within the organ of Corti. In paired patch-clamp recordings the coupling ratio (CR) of adjacent Deiters' cells at the apex of the cochlea (approximately 0.31) was 3-fold smaller than in isolated cell pairs due to shunting afforded by multicellular connectivity. With sinusoidal current stimuli the delay in signal propagation between adjacent cells increased with increasing frequency whereas the amplitude did not change significantly up to 200 Hz (corner frequency Fc approximately 220 Hz). Depolarizing voltage commands applied to an outer hair cell (OHC) elicited outward potassium currents in the OHC and inward currents in the abutting Deiters' cells, supplying direct evidence for potassium buffering in the organ of Corti. Computational analysis indicates that electrical signals injected into a Deiters' cell are transmitted across a network segment spanning 8 cell diameters. Thus electrical coupling in the organ of Corti is unlikely to influence the selectivity of frequency filtering performed mechanically by the mammalian cochlea.


Assuntos
Comunicação Celular/fisiologia , Cóclea/metabolismo , Conexinas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Animais , Cálcio/metabolismo , Cóclea/ultraestrutura , Simulação por Computador , Conexina 26 , Conexina 30 , Cobaias , Células Ciliadas Auditivas Externas/ultraestrutura , Inositol 1,4,5-Trifosfato/metabolismo , Técnicas de Patch-Clamp
18.
J Cell Biol ; 151(5): 961-72, 2000 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-11085999

RESUMO

Urothelium synthesizes a group of integral membrane proteins called uroplakins, which form two-dimensional crystals (urothelial plaques) covering >90% of the apical urothelial surface. We show that the ablation of the mouse uroplakin III (UPIII) gene leads to overexpression, defective glycosylation, and abnormal targeting of uroplakin Ib, the presumed partner of UPIII. The UPIII-depleted urothelium features small plaques, becomes leaky, and has enlarged ureteral orifices resulting in the back flow of urine, hydronephrosis, and altered renal function indicators. Thus, UPIII is an integral subunit of the urothelial plaque and contributes to the permeability barrier function of the urothelium, and UPIII deficiency can lead to global anomalies in the urinary tract. The ablation of a single urothelial-specific gene can therefore cause primary vesicoureteral reflux (VUR), a hereditary disease affecting approximately 1% of pregnancies and representing a leading cause of renal failure in infants. The fact that VUR caused by UPIII deletion seems distinct from that caused by the deletion of angiotensin receptor II gene suggests the existence of VUR subtypes. Mutations in multiple gene, including some that are urothelial specific, may therefore cause different subtypes of primary reflux. Studies of VUR in animal models caused by well-defined genetic defects should lead to improved molecular classification, prenatal diagnosis, and therapy of this important hereditary problem.


Assuntos
Glicoproteínas de Membrana/genética , Urotélio/metabolismo , Urotélio/patologia , Refluxo Vesicoureteral/metabolismo , Refluxo Vesicoureteral/patologia , Animais , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Modelos Animais de Doenças , Deleção de Genes , Expressão Gênica/fisiologia , Hidronefrose/metabolismo , Hidronefrose/patologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutagênese/fisiologia , Tetraspaninas , Urina , Uroplaquina III , Uroplaquina Ib
19.
Proc Natl Acad Sci U S A ; 97(24): 13336-41, 2000 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-11087873

RESUMO

Transduction-channel gating by hair cells apparently requires a gating spring, an elastic element that transmits force to the channels. To determine whether the gating spring is the tip link, a filament interconnecting two stereocilia along the axis of mechanical sensitivity, we examined the tip link's structure at high resolution by using rapid-freeze, deep-etch electron microscopy. We found that the tip link is a right-handed, coiled double filament that usually forks into two branches before contacting a taller stereocilium; at the other end, several short filaments extend to the tip link from the shorter stereocilium. The structure of the tip link suggests that it is either a helical polymer or a braided pair of filamentous macromolecules and is thus likely to be relatively stiff and inextensible. Such behavior is incompatible with the measured elasticity of the gating spring, suggesting that the gating spring instead lies in series with the helical segment of the tip link.


Assuntos
Células Ciliadas Auditivas/ultraestrutura , Animais , Galinhas , Cóclea/ultraestrutura , Técnica de Congelamento e Réplica , Técnica de Fratura por Congelamento , Cobaias , Células Ciliadas Auditivas/fisiologia , Microscopia Eletrônica , Rana catesbeiana , Sáculo e Utrículo/ultraestrutura , Vestíbulo do Labirinto/ultraestrutura
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