Mutations in protocadherin 15 and cadherin 23 affect tip links and mechanotransduction in mammalian sensory hair cells

Immunocytochemical studies have shown that protocadherin-15 (PCDH15) and cadherin-23 (CDH23) are associated with tip links, structures thought to gate the mechanotransducer channels of hair cells in the sensory epithelia of the inner ear. The present report describes functional and structural analyses of hair cells from Pcdh15av3J (av3J), Pcdh15av6J (av6J) and Cdh23v2J (v2J) mice. The av3J and v2J mice carry point mutations that are predicted to introduce premature stop codons in the transcripts for Pcdh15 and Cdh23, respectively, and av6J mice have an in-frame deletion predicted to remove most of the 9th cadherin ectodomain from PCDH15. Severe disruption of hair-bundle morphology is observed throughout the early-postnatal cochlea in av3J/av3J and v2J/v2J mice. In contrast, only mild-to-moderate bundle disruption is evident in the av6J/av6J mice. Hair cells from av3J/av3J mice are unaffected by aminoglycosides and fail to load with [3H]-gentamicin or FM1-43, compounds that permeate the hair cell's mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [3H]-gentamicin, and are aminoglycoside sensitive. Transducer currents can be recorded from hair cells of all three mutants but are reduced in amplitude in all mutants and have abnormal directional sensitivity in the av3J/av3J and v2J/v2J mutants. Scanning electron microscopy of early postnatal cochlear hair cells reveals tip-link like links in av6J/av6J mice, substantially reduced numbers of links in the av3J/av3J mice and virtually none in the v2J/v2J mice. Analysis of mature vestibular hair bundles reveals an absence of tip links in the av3J/av3J and v2J/v2J mice and a reduction in av6J/av6J mice. These results therefore provide genetic evidence consistent with PCDH15 and CDH23 being part of the tip-link complex and necessary for normal mechanotransduction

Adjustable Ellipsoid Nanoparticles Assembled from Re-engineered Connectors of the Bacteriophage Phi29 DNA Packaging Motor

A 24 x 30 nm ellipsoid nanoparticle containing 84 subunits or 7 dodecamers of the re-engineered core protein of the bacteriophage phi29 DNA packaging motor was constructed. Homogeneous nanoparticles were obtained with simple one-step purification. Electron microscopy and analytical ultracentrifugation were employed to elucidate the structure, shape, size, and mechanism of assembly. The formation of this structure was mediated and stabilized by N-terminal peptide extensions. Reversal of the 84-subunit ellipsoid nanoparticle to its dodecamer subunit was controlled by the cleavage of the extended N-terminal peptide with a protease. The 84 outward-oriented C-termini were conjugated with a streptavidin binding peptide which can be used for the incorporation of markers. This further extends the application of this nanoparticle to pathogen detection and disease diagnosis by signal enhancement

Spiral ligament fibrocyte-derived MCP-1/CCL2 contributes to inner ear inflammation secondary to nontypeable H. influenzae-induced otitis media

<p>Abstract</p> <p>Background</p> <p>Otitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation.</p> <p>Methods</p> <p>Live NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2.</p> <p>Results</p> <p>Transtympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the <it>in vitro </it>data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation <it>in vivo</it>. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs.</p> <p>Conclusions</p> <p>Taken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.</p

A Chaperonin Subunit with Unique Structures Is Essential for Folding of a Specific Substrate

Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60β1–β3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4. Furthermore, the unique C-terminus of Cpn60β4 is required for the full activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates

Electronic Microscopy of Olygomerous Ferments

Available from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Cytochrome P-450: hexameric structure of the purified LM4 form

AbstractPurified cytochrome P-450LM4 was found to be monodisperse in 20% glycerol by analytical ultracentrifugation. Its S20,w value was quite similar to that of hexameric P-450LM2. At lower glycerol concentrations the P-450LM4 oligomers showed a tendency to aggregate. The P-450LM4 oligomers were immobilized on Ultrogel A4 under conditions allowing only one covalent link to the matrix per oligomer. In the presence of SDS, the oligomers dissociated leaving only 16th of the initial amount of bound protein on the matrix, suggesting that the purified P-450LM4 is a hexamer. This was confirmed by electron microscopy. The quaternary structure of the P-450LM4 was similar to that demonstrated earlier for P-450LM2

Helical Structure of Hair Cell Stereocilia Tip Links In the Chinchilla Cochlea

Outer-hair-cell stereocilia tip-link structure in the chinchilla cochlea was studied by transmission electron microscopy using tannic acid and Ruthenium red/Alcian blue histochemical procedures. Tannic acid and Ruthenium red/Alcian blue treatments showed the tip link as a compact strand of filaments 9–12 nm thick. Fourier analysis of tip-link images showed that the strand is a three-start helical bundle of fine, coiled filaments which had an axial period of 22.5 ± 1.5 nm. Each of three coiled filaments in the strand showed globular structures, 4.3 ± 0.3 nm in diameter. The globular structures may correspond to individual protein subunits or they may be repeating identical domains of one polypeptide. The three filaments of the helical array may provide a rigidity to the tip link during stereocilia deflections. Alternatively, changes in the subunit or domain structure of each filament may result in a lengthening or shortening of the tip-link strand

The bacteriophage phi 29 head-tail connector shows 13-fold symmetry in both hexagonally packed arrays and as single particles.

The symmetry of the phi 29 head-tail connector is controversial: several studies of two-dimensional arrays of the connector have found a 12-fold symmetry, while a recent study of isolated particles has found a 13-fold symmetry. To investigate whether a polymorphism of the structure might explain these different results, electron microscopy and image analysis were used to study both isolated connectors and particles in hexagonally packed arrays. The hexagonally packed arrays have a P1 symmetry, and the connectors displayed 13 subunits both in the arrays and as isolated single particles. While we do not observe a polymorphism between connectors in two-dimensional arrays and as isolated particles, data show that the connectors can exist with either 12 or 13 subunits. A three-dimensional reconstruction of our 13-fold connector was generated by combining an averaged side-view projection with the known symmetry. The structure of rosettes of the connectors formed in the presence of phi 29 prohead RNA (pRNA) was also examined. These rosettes contain five connectors arranged about a single connector in the center, and this arrangement may reflect an essential role of the pRNA in mediating a symmetry mismatch between either a 12- or 13-fold symmetric connector and a putative fivefold symmetric prohead portal vertex into which the connector fits