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Christian Soeller

Publications and source records attributed to Christian Soeller.

13 recordsLinked to original sources
PubMed2006

TRPC-like conductance mediates restoration of intracellular Ca2+ in cochlear outer hair cells in the guinea pig and rat.

Ca2+ signalling is central to cochlear sensory hair cell physiology through its influence on sound transduction, membrane filter properties and neurotransmission. However, the mechanism for establishing Ca2+ homeostasis in these cells remains unresolved. Canonical transient receptor potential (TRPC) Ca2+ entry channels provide an important pathway for maintaining intracellular Ca2+ levels. TRPC3 subunit expression was detected in guinea pig and rat organ of Corti by RT-PCR, and localized to the sensory and neural poles of the inner and outer hair cells (OHCs) by confocal immunofluorescence imaging. A cation entry current with a TRPC-like phenotype was identified in guinea pig and rat OHCs by whole-cell voltage clamp. This slowly activating current was induced by the lowering of cytosolic Ca2+ levels ([Ca2+]i) following a period in nominally Ca2+-free solution. Activation was dependent upon the [Ca2+]o and was sustained until [Ca(2+)]i was restored. Ca2+ entry was confirmed by confocal fluorescence imaging, and rapidly recruited secondary charybdotoxin- and apamin-sensitive K(Ca) currents. Dual activation by the G protein-coupled receptor (GPCR)-phospholipase C-diacylglycerol (DAG) second messenger pathway was confirmed using the analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Ion substitution experiments showed that the putative TRPC Ca2+ entry current was selective for Na+ > K+ with a ratio of 1: 0.6. The Ca2+ entry current was inhibited by the TRPC channel blocker 2-aminoethyl diphenylborate (2APB) and the tyrosine kinase inhibitor, erbstatin analogue. We conclude that TRPC Ca2+ entry channels, most likely incorporating TRPC3 subunits, support cochlear hair cell Ca2+ homeostasis and GPCR signalling.

Animals↗
PubMed2006

Label-free detection of DNA hybridization based on a novel functionalized conducting polymer.

A new functionalized pyrrole monomer, 3-pyrrolylacrylic acid (PAA) was synthesized. It was used to prepare a copolymer with pyrrole, poly(Py-co-PAA), which was investigated by reflective FT-IR, UV-vis spectroscopy and cyclic voltammetry. A label-free DNA sensor was prepared based on a poly(Py-co-PAA) film. Hybridization with complementary and non-complementary DNA targets was studied by electrochemical impedance spectroscopy. Results show a significant increase in the charge-transfer resistance upon addition of complementary target. The impedance spectra were analyzed by using a modified Randles and Ershler equivalent circuit model. The change in charge-transfer resistance that was used as an index of sensor response was found to be linear with logarithmic target concentration in the range of 2 x 10(-9) to 2 x 10(-7)M. The detection limit was 0.98 nM.

DNA↗
PubMed2006

Three-dimensional distribution of ryanodine receptor clusters in cardiac myocytes.

The clustering of ryanodine receptors (RyR2) into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling. Using immunolabeling and confocal microscopy, we have analyzed the distribution of RyR2 clusters in rat and ventricular atrial myocytes. The resolution of the three-dimensional structure was improved by a novel transverse sectioning method as well as digital deconvolution. In contrast to earlier reports, the mean RyR2 cluster transverse spacing was measured 1.05 microm in ventricular myocytes and estimated 0.97 microm in atrial myocytes. Intercalated RyR2 clusters were found interspersed between the Z-disks on the cell periphery but absent in the interior, forming double rows flanking the local Z-disks on the surface. The longitudinal spacing between the adjacent rows of RyR2 clusters on the Z-disks was measured to have a mean value of 1.87 microm in ventricular and 1.69 microm in atrial myocytes. The measured RyR2 cluster distribution is compatible with models of Ca2+ wave generation. The size of the typical RyR2 cluster was close to 250 nm, and this suggests that approximately 100 RyR2s might be present in a cluster. The importance of cluster size and three-dimensional spacing for current E-C coupling models is discussed.

Animals↗
PubMed2005

Electrochemical detection of DNA hybridization amplified by nanoparticles.

Detection of specific oligonucleotide (ODN) fragments has become an important field in many areas of biomedicine. We describe a novel ODN sensor based on electropolymerization of a conducting polymer (polypyrrole) in the presence of a sample containing ODN(s). The resulting trapped ODN(s) are then probed by addition of complimentary sequence ODN. By incorporating CdS nanoparticles with the probe, a significant improvement in sensor sensitivity was observed. Impedance spectroscopy suggested that optimal detection of hybridization occurred at frequencies>or=3000 Hz (for a 0.07 cm2 85 nm thick film). At these frequencies, the impedance signal was almost linear with the logarithm of ODN concentration in the range 3.7-370 nM with a detection limit of approximately 1 nM ODN (for the sensor fabricated). Importantly, the sensor could be regenerated by removing hybridized ODN with NaOH suggesting possibility of the sensor re-use.

Biosensing Techniques↗
PubMed2005

Label-free electrochemical DNA sensor based on functionalised conducting copolymer.

A simple and label-free electrochemical sensor for recognition of the DNA hybridization event was prepared based on a new functionalised conducting copolymer, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid]. This precursor copolymer can be easily electrodeposited on the electrode surface and shows high electroactivity in an aqueous medium. An amino-substituted oligonucleotide (ODN) probe was covalently grafted onto the surface of the copolymer in a one step procedure and tested on hybridization with complementary ODN segments. The cyclic voltammogram of ODN probe-modified copolymer showed very little change when incubated in presence of non-complementary ODN, while a significant, and reproducible, modification of the voltammogram was observed after addition of complementary ODN. The AC impedance spectrum showed an increased charge transfer resistance (Rct) and double layer capacitance of the sensor film after hybridisation. Sensors with thinner films showed higher sensitivity than thicker films, suggesting that hybridisation at or near the surface of the film produces a larger change in electrical properties than that within the body of the film.

Biosensing Techniques↗
PubMed2005

C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation.

The present study provides functional characterization of alternative splicing of the NTPDase2 (ecto-nucleoside triphosphate diphosphohydrolase-2) involved in the regulation of extracellular nucleotide concentrations in a range of organ systems. A novel NTPDase2beta isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2alpha isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2alpha or NTPDase2beta cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2alpha was predominantly membrane-bound, whereas NTPDase2beta had combined cell surface and intracellular localization. alpha and beta isoforms showed variations in divalent cation dependence and substrate specificity for nucleoside-5'-triphosphates and nucleoside-5'-diphosphates. NTPDase2beta exhibited reduced ATPase activity and no apparent ADPase activity. NTPDase2 isoforms demonstrated similar sensitivity to inhibitors such as suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid, and differential regulation by protein kinases. NTPDase2beta was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2alpha activity was down-regulated by protein kinase C phosphorylation. The results demonstrate that alternative coding of the intracellular C-terminal domain contributes distinctive phenotypic variation with respect to extracellular nucleotide specificity, hydrolysis kinetics, protein kinase-dependent intracellular regulation and protein trafficking. These findings advance the molecular physiology of this enzyme system by characterizing the contribution of the C-terminal domain to many of the enzyme's signature properties.

Adenosine Monophosphate↗
PubMed2004

Functional imaging: new views on lens structure and function.

1. We have developed an experimental imaging approach that allows the distribution of lens membrane proteins to be mapped with subcellular resolution over large distances as a function of fibre cell differentiation. 2. Using this approach in the rat lens, we have localized precisely histological sites of connexin 46 cleavage, quantitatively mapped changes in gap junction distribution and fibre cell morphology and correlated these changes to differences in intercellular dye transfer. 3. Profiling of glucose transporter isoform expression showed that lens epithelial cells express GLUT1, whereas deeper cortical fibre cells express the higher-affinity GLUT3 isoform. Near the lens periphery, GLUT3 was located in the cytoplasm of fibre cells, but it underwent a differentiation-dependent membrane insertion. 4. Similarly, the putative adhesion protein membrane protein 20 is inserted into fibre cell membranes at the stage when the cells lose their nuclei. This redistribution is strikingly rapid in terms of fibre cell differentiation and correlates with a barrier to extracellular diffusion. 5. Our imaging-orientated approach has facilitated new insights into the relationships between fibre cell differentiation and lens function. Taken together, our results indicate that a number of strategies are used by the lens during the course of normal differentiation to change the subcellular distribution, gross spatial location and functional properties of key membrane transport proteins.

Animals↗
PubMed2004

Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens.

PURPOSE: To map changes in the structure and function of fiber cell gap junctions that occur with lens differentiation. METHODS: Equatorial lens sections were fluorescently labeled with antibodies to the gap junction protein connexin (Cx)46, the membrane marker wheat germ agglutinin, and the nuclear stain propidium iodide. Two-photon microscopy and digital image analysis were used to quantify label and cell morphology as a function of radial distance (r/a) across the lens. Loop- and tail-specific Cx46 antibodies were used to identify regions of posttranslational modification. Local fiber cell coupling was imaged in situ using two-photon flash photolysis of caged fluorescein. RESULTS: Antibody labeling showed that the cytoplasmic tail of Cx46 was removed in two zones (r/a approximately 0.9 and r/a approximately 0.7). In addition, with increasing depth, the large radially aligned plaques of peripheral fiber cells became fragmented and dispersed around the cell membrane, and cells became more circular in cross section. Fluorescein transfer between peripheral fiber cells was highly anisotropic and occurred predominantly within a column of fiber cells, resulting in radially directed transport. In regions beyond the zone of nuclear loss, transport was more isotropic and occurred across columns of fiber cells. CONCLUSIONS: The cleavage of Cx46 is associated with a spatial redistribution of gap junction plaques. The distribution of gap junction plaques around the cell membrane can explain the observed directionality of intercellular solute transfer. The findings suggest that the processing and redistribution of gap junction proteins is central to controlling radial and circumferential solute gradients in different regions within the lens.

Animals↗
PubMed2003

Resolving morphology and antibody labeling over large distances in tissue sections.

Protein expression patterns are a primary determinant of tissue function and in this study we developed methods to study protein expression over macroscopic distances at subcellular levels of detail. Using the mammalian lens as a model tissue system, we show that by combining two-photon microscopy with novel image montage methods (fast beam blanking coupled with mathematical alignment tools) we have extended the limited field of view of laser scanning microscopes. To illustrate the utility of our approach, the distribution of connexin-46 was visualized across equatorial sections of the rat mammalian lens. By optimizing fixation protocols, good morphological preservation could be achieved over the thickness of the lens (approximately 4 mm) while preserving antigenicity of lens proteins. Using the same image data, changes in lens fiber cell morphology were mapped quantitatively by automatic image analysis routines. The methods presented should be generally applicable to any tissue system where changes in antibody labeling and tissue structure occur over large and small distances.

Animals↗
PubMed2002

Estimation of the sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks.

Using a combination of experimental and numerical approaches, we have tested two different approaches to calculating the sarcoplasmic reticulum (SR) Ca2+ release flux, which gives rise to cardiac muscle Ca2+ sparks. By using two-photon excited spot photolysis of DM-Nitrophen, known Ca2+ release flux time courses were generated to provide the first experimental validation of spark flux reconstruction algorithms. These artificial Ca2+ sparks show that it is possible to calculate the SR Ca2+ release waveform with reasonable accuracy, provided the flux equations reasonably reflect the properties of the experimental system. Within cardiac muscle cells, we show that Ca2+ flux reconstruction is complicated by the substantial dye binding to proteins, a factor that has not been adequately addressed in previous flux reconstruction algorithms. Furthermore, our numerical experiments suggest that the calculated time course of release flux inactivation based on conventional flux reconstruction algorithms is likely to be in error. We therefore developed novel algorithms based on an explicit dye binding scheme. When these algorithm were applied to evoked Ca2+ sparks in rat cardiac ventricular myocytes, the reconstructed Ca2+ release waveform peaked in ~5 ms and decayed with a halftime of approximately 5 ms. The peak flux magnitude was 7-12 pA, suggesting that sparks must arise from clusters of >15 ryanodine receptors.

Algorithms↗
PubMedDate not supplied

Analysing cardiac excitation-contraction coupling with mathematical models of local control.

Cardiac excitation-contraction (E-C) coupling describes the process that links sarcolemmal Ca2+ influx via L-type Ca2+ channels to Ca2+ release from the sarcoplasmic reticulum via ryanodine receptors (RyRs). This process has proven difficult to study experimentally, and complete descriptions of how the cell couples surface membrane and intracellular signal transduction proteins to achieve both stable and sensitive intracellular calcium release are still lacking. Mathematical models provide a framework to test our understanding of how this is achieved. While no single model is yet capable of describing all features of cardiac E-C coupling, models of increasing complexity are revealing unexpected subtlety in the process. In particular, modelling has established a general failure of 'common-pool' models and has emphasized the requirement for 'local control' so that microscopic sub-cellular domains can separate local behaviour from the whole-cell average (common-pool) behaviour. The micro-architecture of the narrow diadic cleft in which the local control takes place is a key factor in determining local Ca2+ dynamics. There is still considerable uncertainty about the number of Ca2+ ions required to open RyRs within the cleft and various gating models have been proposed, many of which are in reasonable agreement with available experimental data. However, not all models exhibit a realistic voltage dependence of E-C coupling gain. Furthermore, it is unclear which model features are essential to producing reasonable gain properties. Thus, despite the success of local-control models in explaining many features of cardiac E-C coupling, more work will be needed to provide a sound theoretical basis of cardiac E-C coupling.

Adaptation, Physiological↗