Biomedical subjects

Bechara Kachar

Publications and source records attributed to Bechara Kachar.

28 records · Page 2Linked to original sources

PubMed2003

Envelope ultrastructure of uncultured naturally occurring magnetotactic cocci.

Magnetotactic bacteria are microorganisms that respond to magnetic fields. We studied the surface ultrastructure of uncultured magnetotactic cocci collected from a marine environment by transmission electron microscopy using freeze-fracture and freeze-etching. All bacteria revealed a Gram-negative cell wall. Many bacteria possessed extensive capsular material and a S-layer formed by particles arranged with hexagonal symmetry. No indication of a metal precipitation on the surface of these microorganisms was observed. Numerous membrane vesicles were observed on the surface of the bacteria. Flagella were organized in bundles originated in a depression on the surface of the cells. Occasionally, a close association of the flagella with the magnetosomes that remained attached to the replica was observed. Capsules and S-layers are common structures in magnetotactic cocci from natural sediments and may be involved in inhibition of metal precipitation on the cell surface or indirectly influence magnetotaxis.

Cell Membrane↗

PubMed2003

Localization and functional studies of pendrin in the mouse inner ear provide insight about the etiology of deafness in pendred syndrome.

Immunolocalization studies of mouse cochlea and vestibular end-organ were performed to study the expression pattern of pendrin, the protein encoded by the Pendred syndrome gene (PDS), in the inner ear. The protein was restricted to the areas composed of specialized epithelial cells thought to play a key role in regulating the composition and resorption of endolymph. In the cochlea, pendrin was abundant in the apical membrane of cells in the spiral prominence and outer sulcus cells (along with their root processes). In the vestibular end-organ, pendrin was found in the transitional cells of the cristae ampullaris, utriculi, and sacculi as well as in the apical membrane of cells in the endolymphatic sac. Pds-knockout (Pds-/-) mice were found to lack pendrin immunoreactivity in all of these locations. Histological studies revealed that the stria vascularis in Pds-/- mice was only two-thirds the thickness seen in wild-type mice, with the strial marginal cells showing irregular shapes and sizes. Functional studies were also performed to examine the role of pendrin in endolymph homeostasis. Using double-barreled electrodes placed in both the cochlea and the utricle, the endocochlear potential and endolymph potassium concentration were measured in wild-type and Pds-/- mice. Consistent with the altered strial morphology, the endocochlear potential in Pds-/- mice was near zero and did not change during anoxia. On the other hand, the endolymphatic potassium concentration in Pds-/- mice was near normal in the cochlea and utricle. Together, these results suggest that pendrin serves a key role in the functioning of the basal and/or intermediate cells of the stria vascularis to maintain the endocochlear potential, but not in the potassium secretory function of the marginal cells.

Animals↗

PubMed2003

Regulation of outer hair cell cytoskeletal stiffness by intracellular Ca2+: underlying mechanism and implications for cochlear mechanics.

Two Ca(2+)-dependent mechanisms have been proposed to regulate the mechanical properties of outer hair cells (OHCs), the sensory-motor receptors of the mammalian cochlea. One involves the efferent neurotransmitter, acetylcholine, decreasing OHC axial stiffness. The other depends on elevation of intracellular free Ca(2+) concentration ([Ca(2+)](i)) resulting in OHC elongation, a process known as Ca(2+)-dependent slow motility. Here we provide evidence that both these phenomena share a common mechanism. In whole-cell patch-clamp conditions, a fast increase of [Ca(2+)](i) by UV-photolysis of caged Ca(2+) or by extracellular application of Ca(2+)-ionophore, ionomycin, produced relatively slow (time constant approximately 20s) cell elongation. When OHCs were partially collapsed by applying minimal negative pressure through the patch pipette, elevation of the [Ca(2+)](i) up to millimole levels (estimated by Fura-2) was unable to restore the cylindrical shape of the OHC. Stiffness measurements with vibrating elastic probes showed that the increase of [Ca(2+)](i) causes a decrease of OHC axial stiffness, with time course similar to that of the Ca(2+)-dependent elongation, without developing any measurable force. We concluded that, contrary to a previous proposal, Ca(2+)-induced OHC elongation is unlikely to be driven by circumferential contraction of the lateral wall, but is more likely a passive mechanical reaction of the turgid OHC to Ca(2+)-induced decrease of axial stiffness. This may be the key phenomenon for controlling gain and operating point of the cochlear amplifier.

Acetylcholine↗

PubMed2003

Expression of prestin, a membrane motor protein, in the mammalian auditory and vestibular periphery.

Hair cells are specialized mechanoreceptors common to auditory and vestibular sensory organs of mammalian and non-mammalian species. Different hair cells are believed to share common features related to their mechanosensory function. It has been shown that hair cells possess various forms of motile properties that enhance their receptor function. Membrane-based electromotility is a form of hair cell motility observed in isolated outer hair cells (OHCs) of the cochlea. A novel membrane protein, prestin, recently cloned from gerbil and rat tissues, is presumably responsible for electromotility. We cloned prestin from mouse organ of Corti and confirmed strong homology of this protein among different rodent species. We explored whether or not prestin is present in hair cells of the vestibular system. Using reverse transcription-polymerase chain reaction, we demonstrated that prestin is expressed in mouse and rat auditory and vestibular organs, but not in chicken auditory periphery. In situ hybridization and immunolocalization studies confirmed the presence of prestin in OHCs as well as in vestibular hair cells (VHCs) of rodent saccule, utricle and crista ampullaris. However, in the VHCs, staining of varying intensity with anti-prestin antibodies was observed in the cytoplasm, but not in the lateral plasma membrane or in the stereociliary membrane. Whole-cell patch-clamp recordings showed that VHCs do not possess the voltage-dependent capacitance associated with membrane-based electromotility. We conclude that although prestin is expressed in VHCs, it is unlikely that it supports the form of somatic motility observed in OHCs.

Amino Acid Sequence↗

PubMed2003

Membrane vesicles in magnetotactic bacteria.

Magnetotactic bacteria are microorganisms that respond to magnetic fields. We have studied the surface ultrastructure of Magnetospirillum magnetotacticum and uncultured magnetotactic bacteria from a marine environment using transmission electron microscopy and freeze-etching. Numerous membrane vesicles were observed on the surface of Magnetospirillum magnetotacticum bacteria. All uncultured magnetotactic bacteria presented membrane vesicles on their surface in addition to an extensive capsular material and an S-layer formed by particles arranged in a hexagonal symmetry. We did not observe any indication of electron-dense precipitation on the surface of these microorganisms. Our results indicate that membrane vesicles are a common characteristic of magnetotactic bacteria in natural sediments.

Bacteria↗

PubMed2002

Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice.

Deafness in spontaneously occurring mouse mutants is often associated with defects in cochlea sensory hair cells, opening an avenue to systematically identify genes critical for hair cell structure and function. The classical semidominant mouse mutant varitint-waddler (Va) exhibits early-onset hearing loss, vestibular defects, pigmentation abnormalities, and perinatal lethality. A second allele, Va(J), which arose in a cross segregating for Va, shows a less severe phenotype. By using a positional cloning strategy, we identify two additional members of the mucolipin gene family (Mcoln2 and Mcoln3) in the 350-kb Va(J) minimal interval and provide evidence for Mcoln3 as the gene mutated in varitint-waddler. Mcoln3 encodes a putative six-transmembrane-domain protein with sequence and motif similarities to the family of nonselective transient-receptor-potential (TRP) ion channels. In the Va allele an Ala419Pro substitution occurs in the fifth transmembrane domain of Mcoln3, and in Va(J), a second sequence alteration (Ile362Thr) occurring in cis partially rescues the Va allele. Mcoln3 localizes to cytoplasmic compartments of hair cells and plasma membrane of stereocilia. Hair cell defects are apparent by embryonic day 17.5, assigning Mcoln3 an essential role during early hair cell maturation. Our data suggest that Mcoln3 is involved in ion homeostasis and acts cell-autonomously. Hence, we identify a molecular link between hair cell physiology and melanocyte function. Last, MCOLN2 and MCOLN3 are candidate genes for hereditary and/or sporadic forms of neurosensory disorders in humans.

Amino Acid Sequence↗

PubMed2002

Rapid renewal of auditory hair bundles.

Stereocilia, also known as hair bundles, are mechanosensitive organelles of the sensory hair cells of the inner ear that can detect displacements on a nanometre scale and are supported by a rigid, dense core of actin filaments. Here we show that these actin-filament arrays are continuously remodelled by the addition of actin monomers to the stereocilium tips, and that the entire core of the stereocilium is renewed every 48 hours. This unexpected dynamic feature of stereocilia will help our understanding of how auditory sensory function develops and is maintained.

Acoustic Stimulation↗

PubMed2002

Role of membrane proteins in permeability barrier function: uroplakin ablation elevates urothelial permeability.

Although water, small nonelectrolytes, and gases are freely permeable through most biological membranes, apical membranes of certain barrier epithelia exhibit extremely low permeabilities to these substances. The role of integral membrane proteins in this barrier function has been unclear. To study this problem, we have ablated the mouse gene encoding uroplakin III (UPIII), one of the major protein subunits in urothelial apical membranes, and measured the permeabilities of these membranes. Ablation of the UPIII gene greatly diminishes the amounts of uroplakins on the apical urothelial membrane (Hu P, Deng FM, Liang FX, Hu CM, Auerbach AB, Shapiro E, Wu XR, Kachar B, and Sun TT. J Cell Biol 151: 961-972, 2000). Our results indicate that normal mouse urothelium exhibits high transepithelial resistance and low urea and water permeabilities. The UPIII-deficient urothelium exhibits a normal transepithelial resistance (normal 2,024 +/- 122, knockout 2,322 +/- 114 Omega. cm(2); P > 0.5). However, the UPIII-deficient apical membrane has a significantly elevated water permeability (normal 0.91 +/- 0.06, knockout 1.83 +/- 0.14 cm/s x 10(-5); P < 0.05). The urea permeability of the UPIII-deficient membrane also increased, although to a lesser extent (normal 2.22 +/- 0.24, knockout 2.93 +/- 0.31 cm/s x 10(-6); P = 0.12). These results indicate that reduced targeting of uroplakins to the apical membrane does not significantly alter the tight junctional barrier but does double the water permeability. We provide the first demonstration that integral membrane proteins contribute to the apical membrane permeability barrier function of urothelium.

Animals↗

PubMed2002

Determination of elastic moduli of thin layers of soft material using the atomic force microscope.

We address three problems that limit the use of the atomic force microscope when measuring elastic moduli of soft materials at microscopic scales. The first concerns the use of sharp cantilever tips, which typically induce local strains that far exceed the linear material regime. We show that this problem can be alleviated by using microspheres as probes, and we establish the criteria for their use. The second relates to the common use of the Hertz contact mechanics model, which leads to significant errors when applied to thin samples. We develop novel, simple to use corrections to apply for such cases. Samples that are either bonded or not bonded to a rigid substrate are considered. The third problem concerns the difficulty in establishing when contact occurs on a soft material. We obtain error estimates for the elastic modulus resulting from such uncertainty and discuss the sensitivity of the estimation methods to error in contact point. The theoretical and experimental results are compared to macroscopic measurements on poly(vinyl-alcohol) gels.

Calibration↗

PubMedDate not supplied

Vesicle targeting in hair cells.

The mammalian hair cell has two distinct plasma membrane domains separated by tight junctions, the apical domain which contains the stereocilia and the basolateral domain which contains the presynaptic region. Little is known concerning the mechanisms that regulate vesicle trafficking to these two domains. Using SNAP 25 and syntaxin as baits, we carried out a yeast two-hybrid screen of the organ of Corti. We identified a novel syntaxin interacting protein, ocsyn, that is enriched in inner hair cells and concentrated at the apical pole. Our results are consistent with ocsyn playing a role in vesicle trafficking to the apical membrane of the hair cell.

Animals↗