[Impact of CI use and CI fitting on speech production in very early cochlear-implanted infants]. / Bedeutung von CI-Nutzungsverhalten und CI-Anpassung für sprachproduktive Leistungen sehr früh cochleaimplantierter Kinder.

BACKGROUND: Children's age at implantation is an important factor for their outcome in auditory and language skills with a cochlear implant (CI). CI use and frequency of CI fitting may also influence speech performance. Purpose of this study was to evaluate CI use and CI fitting of very early implanted infants and its potential influence on age-related speech production performance. METHODS: Data of 34 bilaterally cochlea-implanted infants (age at CI in months: M = 8,8; SD = 1,7) were included. During the third year of life speech production performance was evaluated and related to datalogging-based CI use and number of CI fitting sessions. RESULTS: About half of the cohort achieved speech production level within the normal range of hearing peers. Daily time of CI use was approximately 8 h. Analysis of listening environment showed that infants were exposed most of the time to quiet environment and least amount of time to speech in noise. Daily time of CI use seems to be a significant predictor of speech production, speech-exposition particularly predicts word production. Number of daily disconnection between CI-processor and implant as well as the monthly number of CI fitting sessions were not correlated with speech production. CONCLUSION: Very early cochlear implanted infants may achieve age-appropriate speech production performance in the third year of life. Time of daily CI use in the study cohort is comparable to results of other studies. Time of daily CI use and exposure to speech seem to be important factors for early speech production. These findings should be integrated in pre- and postoperative parent counselling.

[Auditory group therapy in adult cochlear implant rehabilitation]. / Hörtherapeutische Gruppenkonzepte in der Cochleaimplantat-Rehabilitation bei Erwachsenen.

BACKGROUND: Auditory training is an established intervention in adult cochlear implant (CI) aural rehabilitation. In most cases, training is implemented in an individual therapy setting. Increasing patient numbers and the associated time-economic and cost-related demands as well as psychosocial and communicative aspects support the use of aural group interventions. OBJECTIVES: This study aimed 1) to describe concepts and contents of group interventions for adult CI users and 2) to present results of a questionnaire-based evaluation. METHODS: Group interventions have been offered at the CI Centre Erlangen CICERO for several years. In Auditory Training Groups, exercises have priority, while Thematic Group Workshops focus on psychosocial aspects and provide information for the participants. The Auditory Training Groups were evaluated based on a patient questionnaire. Additionally, the reliability of the questionnaire was analyzed. RESULTS: The median overall satisfaction of CI users participating in Auditory Training Groups was rated as good. Training of speech perception in noise as well as communicative exchange are of great importance for CI users. They rated the therapeutic design as very good to good. Reliability analysis showed significant positive intercorrelations of the questionnaire items. CONCLUSION: Group interventions are well accepted by CI users and represent a useful complement to individual therapy during the rehabilitation process. As group interventions place special demands on patients and therapists, structured and well-proven concepts should be used in practice.

NMR and fluorescence spectroscopy approaches to secondary and primary active multidrug efflux pumps.

Multidrug efflux pumps are found in all major transporter families. Along with a lack of three-dimensional structure information, the mechanism of drug recognition, energy coupling with drug translocation and the catalytic cycle are so far not understood. In the present study, we present first data of a fluorescence-based assay to study the pH-gradient-mediated activity of the multidrug antiporter EmrE, by co-reconstitution with the light-driven proton pump bacteriorhodopsin. In addition to biochemical approaches, the emerging technique, solid-state NMR, can be used for the investigation of these transporters. A number of experiments based on MAS (magic angle sample spinning) NMR are available to provide data on protein structure and dynamics, drug binding and protein-lipid interactions. However, these experiments dictate a number of constraints with respect to sample preparation that will be discussed for proteins from the SMR (small multidrug resistance transporter) family. In addition, 2H-NMR is used to probe protein mobility of Lactococcus lactis ABC transporter, LmrA.

REDOR NMR on a hydrophobic peptide in oriented membranes.

A method is presented for the calculation of REDOR dephasing for specifically labeled membrane-spanning peptides in uniformly aligned lipid bilayers under magic angle oriented sample spinning (MAOSS) conditions. Numerical simulations are performed for dephasing of (13)C signal by (15)N when the labels are placed in an alpha-helical peptide at the carbonyl of residue (i) and amide nitrogen of residue (i + 2) to show the dependency of REDOR echo intensity on the peptide tilt angle relative to the membrane normal. The approach was applied to the labeled transmembrane domain of phospholamban ([(15)N-Leu(37), (13)C-Leu(39)]PLBTM) incorporated into dimyristoylphosphatidylcholine bilayers. The dephasing observed for a random membrane dispersion showed that the peptide was alpha-helical in the region including the two labels, and dephasing in oriented membranes showed that the peptide helix was tilted by 25 degrees +/- 7 degrees relative to the bilayer normal. These results agree with those obtained by other spectroscopic methods.

31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE.

The binding of tetraphenylphosphonium (TPP+) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross-polarisation-magic-angle spinning nuclear magnetic resonance spectroscopy (CP-MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP-MAS could selectively distinguish binding of TPP+ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.

Observations of light-induced structural changes of retinal within rhodopsin.

Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 A) electron microscopy studies confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method. Significant structural changes are observed in the retinal following activation by light to the photo-activated M(I) state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.

Structural and orientational information of the membrane embedded M13 coat protein by (13)C-MAS NMR spectroscopy.

Oriented and unoriented M13 coat protein, incorporated into dimyristoyl phosphatidylcholine bilayers, has been studied by (13)C-magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Rotational resonance experiments provided two distance constraints between Calpha and C&z.dbnd6;O positions of the labelled residues Val-29/Val-30 (0.4+/-0.5nm) and Val-29/Val-31 (0.45+/-0. 5nm) in its hydrophobic domain. The derived dihedral angles (Phi, Psi) for Val-30 revealed a local alpha-helical conformation. (13)C-CP-MAS experiments on uniformly aligned samples (MAOSS experiments) using the (13)C&z.dbnd6;O labelled site of Val-30 allowed the determination of the helix tilt (20 degrees +/-10 degrees ) in the membrane. It is shown that one uniform MAS high-resolution solid state NMR approach can be used to obtain structural and orientational data.

Probing membrane surfaces and the location of membrane-embedded peptides by (13)C MAS NMR using lanthanide ions.

A simple but efficient (13)C MAS NMR method is presented for the determination of the location of embedded molecules such as peptides relative to biological membrane surfaces by exploiting the interaction with paramagnetic lanthanide ions. Using various aqueous Dy(3+) concentrations a distance-dependent differential paramagnetic quenching of NMR lipid resonance intensities for specific carbon sites was observed, with residues at the bilayer surface quenched effectively and hydrophobic sites unaffected by Dy(3+). Tested on the membrane-embedded 50 residue long M13 coat protein, (13)C labeled at its Val-29 and Val-31 residues, no paramagnetic quenching was observed for the peptide resonances by Dy(3+), suggesting that Val-29 and Val-31 are not in close proximity to the bilayer interface, but buried deeply inside the hydrophobic region of the lipid bilayer.

Magic angle-oriented sample spinning (MAOSS): A new approach toward biomembrane studies.

The application of magic angle sample spinning (MAS) NMR to uniformly aligned biomembrane samples is demonstrated as a new general approach toward structural studies of membrane proteins, peptides, and lipids. The spectral linewidth from a multilamellar lipid dispersion is dominated, in the case of protons, by the dipolar coupling. For low-gamma or dilute spins, however, the chemical shift anisotropy dominates the spectral linewidth, which is reduced by the two-dimensional order in a uniformly aligned lipid membrane. The remaining line broadening, which is due to orientational defects ("mosaic spread") can be easily removed at low spinning speeds. This orientational order in the sample also allows the anisotropic intermolecular motions of membrane components (such as rotational diffusion, tauc = 10(-10) s) for averaging dipolar interactions to be utilized, e.g., by placing the membrane normal parallel to the rotor axis. The dramatic resolution improvement for protons which are achieved in a lipid sample at only 220 Hz spinning speed in a 9.4 T field is slightly better than any data published to date using ultra-high fields (up to 17.6 T) and high-speed spinning (14 kHz). Additionally, the analysis of spinning sidebands provides valuable orientational information. We present the first 1H, 31P, and 13C MAS spectra of uniformly aligned dimyristoylphosphatidylcholine (DMPC) bilayers. Also, 1H resolution enhancement for the aromatic region of the M13 coat protein reconstituted into DMPC bilayers is presented. This new method combines the high resolution usually achieved by MAS with the advantages of orientational constraints obtained by working with macroscopically oriented samples. We describe the general potential and possible perspectives of this technique.

Interaction of a type II myosin with biological membranes studied by 2H solid state NMR.

Deuterium nuclear magnetic resonance spectroscopy (2H NMR) has been employed to investigate the interaction of lung type II myosin protein with neutral bilayers containing dimyristoylphosphatidylcholine (DMPC) as the only constituent and mixed bilayers containing the negatively charged lipid dimyristoylphosphatidylglycerol (DMPG). DMPC was deuterated at its headgroup by substituting the four protons at the alpha- and beta-positions (DMPC-d4) and the nine protons at the gamma-position (DMPC-d9). DMPG was perdeuterated at its headgroup (DMPG-d5). No changes were observed in the quadrupole splittings or spin-lattice relaxation times for the deuterated DMPC headgroup segments when increasing amounts of myosin were added to liposomes, made exclusively of DMPC-d9 or of DMPC-d4. However, upon the insertion of the negatively charged lipid DMPG at 1:1 molar ratio into the DMPC bilayers, myosin was found to interact electrostatically with the liposomes, thereby affecting significantly both the quadrupole splittings and spin-lattice relaxation rates of the alpha-, beta-, and gamma-deuterons in labeled DMPC. Monitoring DMPG-d5 in mixed DMPC/DMPG bilayers revealed a direct electrostatic interaction of DMPG with the protein, where positively charged lysine residues located at the tail domain of myosin provide the necessary sites for the interaction to occur. When ATP and Mg2+ were complexed to the head domain of myosin, a reduced interaction with the negatively charged bilayers was observed. The results clearly indicate that a type II myosin can interact with membranes without the need for a specific hydrophobic domain or an anchor in the protein molecule, provided that negatively charged lipids are present in the bilayer.

Photoreceptor rhodopsin: structural and conformational study of its chromophore 11-cis retinal in oriented membranes by deuterium solid state NMR.

Rhodopsin is the retinal photoreceptor responsible for visual signal transduction. To determine the orientation and conformation of retinal within the binding pocket of this membrane bound receptor, an ab initio solid state 2H NMR approach was used. Bovine rhodopsin containing 11-cis retinal, specifically deuterated at its methyl groups at the C19 or C20 position, was uniaxially oriented in DMPC bilayers. Integrity of the membranes and quality of alignment were monitored by 31P NMR. Analysis of the obtained 2H NMR spectra provided angles for the individual labelled chemical bond vectors leading to an overall picture for the three dimensional structure of the polyene chain of the chromophore in the protein binding pocket around the Schiff base attachment site.

Macroscopic orientation of natural and model membranes for structural studies.

One approach for obtaining high-resolution structural and functional information for biomembranes and their proteins is by static solid-state NMR of oriented systems. Here, a general procedure to align fully functional biological membranes containing large membrane proteins (Mr >30,000) is described. The method, based on the isopotential spin-dry ultracentrifugation technique, relies on the centrifugation of membrane fragments onto a support with simultaneous, or subsequent, partial evaporation of the solvent which aids alignment. The quality of orientation, as shown by the mosaic spread of the samples, was monitored by static solid-state 31P NMR for the phospholipids and by 2H NMR for a deuterated retinal in bovine rhodopsin. The generality of this method is demonstrated with three different membranes containing bovine rhodopsin in reconstituted bilayers, natural membranes with the red cell anion exchange transport protein in erythrocytes, band 3, and the nicotinic acetylcholine receptor.