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Richard Wagner

Publications and source records attributed to Richard Wagner.

18 recordsLinked to original sources
PubMed2006

Tim50 maintains the permeability barrier of the mitochondrial inner membrane.

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello. We found that the intermembrane space domain of Tim50 induced the Tim23 channel to close. Presequences overcame this effect and activated the channel for translocation. Thus, the hydrophilic cis domain of Tim50 maintains the permeability barrier of mitochondria by closing the translocation pore in a presequence-regulated manner.

Cell Membrane Permeability↗
PubMed2006

OEP37 is a new member of the chloroplast outer membrane ion channels.

The chloroplast outer envelope protein OEP37 is a member of the growing beta-barrel protein family of the outer chloroplast membrane. The reconstituted recombinant protein OEP37 from pea forms a rectifying high conductance channel with a main conductance (lambda) of Lambda= 500 picosiemens (symmetrical 250 mm KCl). The OEP37 channel is cation-selective (P(K+)/P(K-) = 14:1) with a voltage-dependent open probability maximal at V(mem) = 0 mV. The channel pore reveals an hourglass-shaped form with different diameters for the vestibule and restriction zone. The diameters of the vestibule at the high conductance side were estimated by d = 3.0 nm and the restriction zone by d = 1.5 nm. The OEP37 channel displayed a nanomolar affinity for the precursor of the chloroplast inner membrane protein Tic32, which is imported into the chloroplast through a yet unknown pathway. Pre-proteins imported through the usual Toc pathway and synthetic control peptides, however, did not show a comparable block of the OEP37 channel. In addition to the electrophysiological characterization, we studied the gene expression of OEP37 in the model plant Arabidopsis thaliana. Here, transcripts of AtOEP37 are ubiquitously expressed throughout plant development and accumulate in early germinating seedlings as well as in late embryogenesis. The plastid intrinsic protein could be detected in isolated chloroplasts of cotyledons and rosette leaves. However, the knock-out mutant oep37-1 shows that the proper function of this single copy gene is not essential for development of the mature plant. Moreover, import of Tic32 into chloroplasts of oep37-1 was not impaired when compared with wild type. Thus, OEP37 may constitute a novel peptide-sensitive ion channel in the outer envelope of plastids with function during embryogenesis and germination.

Arabidopsis↗
PubMed2006

Molecular properties of Oep21, an ATP-regulated anion-selective solute channel from the outer chloroplast membrane.

The flux of phosphorylated carbohydrates, the major export products of chloroplasts, is regulated at the level of the inner and presumably also at the level of the outer membrane. This is achieved through modulation of the outer membrane Oep21 channel currents and tuning of its ion selectivity. Refined analysis of the Oep21 channel properties by biochemical and electrophysiological methods revealed a channel formed by eight beta-strands with a wider pore vestibule of dvest approximately 2.4 nm at the intermembrane site and a narrower filter pore of drestr approximately 1 nm. The Oep21 pore contains two high affinity sites for ATP, one located at a relative transmembrane electrical distance delta = 0.56 and the second close to the vestibule at the intermembrane site. The ATP-dependent current block and reduction in anion selectivity of the Oep21 channel is relieved by the competitive binding of phosphorylated metabolic intermediates like 3-phosphoglycerate and glycerinaldehyde 3-phosphate. Deletion of a C-terminal putative FX4K binding motif in Oep21 decreased the capability of the channel to tune its ion selectivity by about 50%, whereas current block remained unchanged.

Adenosine Triphosphate↗
PubMed2006

How to successfully implement E-learning for both students and teachers.

RATIONALE AND OBJECTIVES: Electronic learning (e-learning) may provide a means to enhance learning efficacy. However, introduction of e-learning often fails. We describe a strategy of how an e-learning curriculum was successfully implemented. MATERIALS AND METHODS: The curriculum was designed based on published evidence. It consists of self-directed learning, an online discussion forum, and discussion rounds. The e-content in nuclear medicine and radiotherapy was produced by the k-MED team of medical authors, web designers, and psychologists. The online courses were delivered via a dedicated learning management system. The e-content for diagnostic radiology and physics was provided as PDF/HTML script by the respective teachers who objected to participate in the k-MED project. The exam was taken online. Online evaluation of the curriculum by the students was taken at the end of the course. RESULTS: The new curriculum proved very effective. The time for the preparation for the clinical part of the radiology course could be reduced from 4 to 2 weeks. The students particularly enjoyed the self-directed learning. Although the material provided by k-MED received 90%-99% positive scores, the HTML and PDF scripts scored worse (13%-67% positive ratings). The positive results of the evaluation convinced the teachers responsible for physics and diagnostic radiology to participate in k-MED. CONCLUSIONS: As our example shows, new e-learning curricula can successfully be introduced. The strategy of implementation should be based on the existing evidence from the literature. The new curriculum helped to increase the efficacy of teaching and save time as the duration of the respective part of the course could be reduced by half.

Computer-Assisted Instruction↗
PubMed2005

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore.

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.

Binding Sites↗
PubMed2005

Nanopore unitary permeability measured by electrochemical and optical single transporter recording.

For the analysis of membrane transport processes two single molecule methods are available that differ profoundly in data acquisition principle, achievable information, and application range: the widely employed electrical single channel recording and the more recently established optical single transporter recording. In this study dense arrays of microscopic horizontal bilayer membranes between 0.8 microm and 50 microm in diameter were created in transparent foils containing either microholes or microcavities. Prototypic protein nanopores were formed in bilayer membranes by addition of Staphylococcus aureus alpha-hemolysin (alpha-HL). Microhole arrays were used to monitor the formation of bilayer membranes and single alpha-HL pores by confocal microscopy and electrical recording. Microcavity arrays were used to characterize the formation of bilayer membranes and the flux of fluorescent substrates and inorganic ions through single transporters by confocal microscopy. Thus, the unitary permeability of the alpha-HL pore was determined for calcein and Ca(2+) ions. The study paves the way for an amalgamation of electrical and optical single transporter recording. Electro-optical single transporter recording could provide so far unresolved kinetic data of a large number of cellular transporters, leading to an extension of the nanopore sensor approach to the single molecule analysis of peptide transport by translocases.

Bacterial Toxins↗
PubMed2004

Thyroxine-thyroid hormone receptor interactions.

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3), 3,5,3'-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T(4), 3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless, T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH, our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design.

Animals↗
PubMed2004

IFNalpha induces Fas expression and apoptosis in hedgehog pathway activated BCC cells through inhibiting Ras-Erk signaling.

Basal cell carcinoma (BCC), the most common form of human cancer, is understood to be associated with activation of the sonic hedgehog pathway, through loss-of-function mutations of tumor suppressor PTCH1 or gain-of-function mutations of smoothened. Interferon (IFN)-based therapy is quite effective in BCC treatment, but the molecular basis is not well understood. Here we report a novel mechanism by which IFNalpha mediates apoptosis in BCCs. In the presence of IFNalpha, we observed increased apoptosis in a BCC cell line ASZ001, in which PTC is null, and therefore with constitutive activation of the sonic hedgehog pathway. We demonstrate that SMO agonist Ag-1.4 mediates activation of extracellular signal-regulated kinase (Erk) phosphorylation, which is abrogated by IFNalpha in sonic hedgehog responsive C3H10T1/2 cells. In transient transfection experiments, we demonstrate that IFNalpha inhibits Erk phosphorylation and serum response element activation induced by expression of SMO, Gli1, PDGFRalpha and activated Raf, but not activated mitogen-activated Erk-regulating kinase (Mek), suggesting that IFNalpha targets mainly on Mek function. We further show that IFNalpha induces expression of Fas in BCC cells through interfering with Mek function. The role of the Fas-L/Fas signaling axis in IFNalpha-mediated apoptosis is demonstrated by the fact that addition of Fas-L neutralizing antibodies, just as caspase-8 inhibitor Z-IETD-FMK, effectively prevents IFNalpha-mediated apoptosis. Thus, our data indicate that IFNalpha-based BCC therapy induces Fas expression and apoptosis through interfering with Mek function.

Animals↗
PubMed2004

Talar dislocations.

From 1987-2003, 36 patients were treated for talar dislocation, 27 patients for subtalar, six for total talar, and three patients for peritalar dislocation. Luxatio pedis sub talo: We found 19 medial closed, seven lateral closed and one third degree open subtalar dislocations. Our therapeutic concept provides for immediate reduction, which is possible by closed procedure for the majority of medial dislocations. If there is a tendency to redislocation, we perform talonavicular K-wire transfixation. In the case of irreducibility, open reduction via lateral approach is the rule. The lateral dislocation type is often accompanied by additional fractures of the hindfoot and tarsus, frequently requiring primary open procedures via medial approach. 32 patients were followed-up in whom we found 17 excellent results, ten good, three mediocre and two poor results. With two thirds of the patients, low grade arthrosis at least was observed and two thirds showed a reduced amplitude of motion in one or more talar joints. A definite correlation between arthrosis and reduced function was not established. We did not find talar necroses, persisting instabilities, or redislocations. Luxatio tali totalis: We found three lateral and three medial complete dislocations. The therapeutic concept consists of immediate reduction-only possible by open procedure. A tendency to redislocation requires K-wire transfixation. All patients were followed-up. We found two good and four poor results, with two total and three partial necroses. As a secondary treatment, two lower ankle joint(LAJ) and two upper ankle joint (UAJ) arthrodeses were performed. There were no talectomies, amputations, or infections. Luxatio pedis cum talo: We found three anterolateral UAJ dislocations. Our therapeutic concept provides for immediate reduction. The whole capsular ligament apparatus was reconstructed by primary or secondary treatment, depending on the degree of soft tissue damage. Follow-up showed two excellent results

Adolescent↗
PubMed2003

Using advanced intercross lines for high-resolution mapping of HDL cholesterol quantitative trait loci.

Mapping quantitative trait loci (QTLs) with high resolution facilitates identification and positional cloning of the underlying genes. The novel approach of advanced intercross lines (AILs) generates many more recombination events and thus can potentially narrow QTLs significantly more than do conventional backcrosses and F2 intercrosses. In this study, we carried out QTL analyses in (C57BL/6J x NZB/BlNJ) x C57BL/6J backcross progeny fed either chow or an atherogenic diet to detect QTLs that regulate high-density lipoprotein cholesterol (HDL)concentrations, and in (C57BL/6J x NZB/BlNJ) F11 AIL progeny to confirm and narrow those QTLs. QTLs for HDL concentrations were found on chromosomes 1, 5, and 16. AIL not only narrowed the QTLs significantly more than did a conventional backcross but also resolved a chromosome 5 QTL identified in the backcross into two QTLs, the peaks of both being outside the backcross QTL region. We tested 27 candidate genes and found significant mRNA expression differences for 12 (Nr1i3, Apoa2, Sap, Tgfb2, Fgfbp1, Prom, Ppargc1, Tcf1, Ncor2, Srb1, App, and Ifnar). Some of these underlay the same QTL, indicating that expression differences are common and not sufficient to identify QTL genes. All the major HDL QTLs in our study had homologous counterparts in humans, implying that their underlying genes regulate HDL in humans.

Animals↗
PubMed2003

Protein insertion into the mitochondrial inner membrane by a twin-pore translocase.

The mitochondrial inner membrane imports numerous proteins that span it multiple times using the membrane potential Deltapsi as the only external energy source. We purified the protein insertion complex (TIM22 complex), a twin-pore translocase that mediated the insertion of precursor proteins in a three-step process. After the precursor is tethered to the translocase without losing energy from the Deltapsi, two energy-requiring steps were needed. First, Deltapsi acted on the precursor protein and promoted its docking in the translocase complex. Then, Deltapsi and an internal signal peptide together induced rapid gating transitions in one pore and closing of the other pore and drove membrane insertion to completion. Thus, protein insertion was driven by the coordinated action of a twin-pore complex in two voltage-dependent steps.

Carrier Proteins↗
PubMed2003

The Sec61p complex is a dynamic precursor activated channel.

Previous studies have shown that the rough endoplasmic reticulum (ER) contains nascent precursor polypeptide gated channels. Circumstantial evidence suggests that these channels are formed by the Sec61p complex. We reconstituted the purified Sec61p complex in a lipid bilayer and characterized its dynamics and regulation. The Sec61p complex is sufficient to form the precursor polypeptide activated channel under co- and posttranslational transport conditions. Activity of the Sec61p channel in both transport modes is induced by direct interaction with precursor protein. The Sec61p complex comprises a highly dynamic pore covering conductances corresponding to channel openings from approximately 6 to 60 A. Its properties are indistinguishable from those we observed with native ER channels, directly demonstrating that these channels are formed by the Sec61p complex.

ADP Ribose Transferases↗
PubMed2003

Combinatorial compound libraries for drug discovery: an ongoing challenge.

Almost 20 years of combinatorial chemistry have emphasized the power of numbers, a key issue for drug discovery in the current genomic era, in which it has been estimated that there might be more than 10,000 potential targets for which it would be desirable to have small-molecule modulators. Combinatorial chemistry is best described as the industrialization of chemistry; the chemistry has not changed, just the way in which it is now carried out, which is principally by exploiting instrumentation and robotics coupled to the extensive use of computers to efficiently control the process and analyse the vast amounts of resulting data. Many researchers have contributed to the general concepts as well as to the technologies in present use. However, some interesting challenges still remain to be solved, and these are discussed here in the context of the application of combinatorial chemistry to drug discovery.

Animals↗
PubMed2002

The preprotein conducting channel at the inner envelope membrane of plastids.

The preprotein translocation at the inner envelope membrane of chloroplasts so far involves five proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein specifically interacts with isolated inner envelope vesicles as well as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of beta-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Purified transit peptide causes strong flickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane.

Chloroplasts↗
PubMed2002

The chloroplast protein import channel Toc75: pore properties and interaction with transit peptides.

The channel properties of Toc75 (the protein import pore of the outer chloroplastic membrane) were further characterized by electrophysiological measurements in planar lipid bilayers. After improvement of the Toc75 reconstitution procedure the voltage dependence of the channel open probability resembled those observed for other beta-barrel pores. Studies concerning the pore size of the reconstituted Toc75 indicate the presence of a narrow restriction zone corresponding to the selectivity filter and a wider pore vestibule with diameters of approximately 14 A and 26 A, respectively. Interactions between Toc75 and different peptides (a genuine chloroplastic transit peptide, a synthetic peptide resembling a transit peptide, and a mitochondrial presequence) show that Toc75 itself is able to differentiate between these peptides and the recognition is based on both conformational and electrostatic interactions.

Amino Acid Sequence↗
PubMed2002

Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel.

The protein insertion complex of the mitochondrial inner membrane is crucial for import of the numerous multitopic membrane proteins with internal targeting signals. Little is known about the molecular mechanism of this complex, including whether it forms a real channel or merely acts as scaffold for protein insertion. We report the unexpected observation that Tim22 is the only essential membrane-integrated subunit of the complex. Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters. The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences. Thus, a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component.

Carrier Proteins↗
PubMedDate not supplied

Kaiser Permanente's evaluation and management of biotech drugs: assessing, measuring, and affecting use.

Two decades into the age of high-cost biotechnology medications, health care organizations are challenged to provide safe, effective treatment while managing costs. In a market lacking generic biotech drugs, health care organizations have few opportunities to negotiate reduced pricing; other strategies must be devised to manage use. Kaiser Permanente leverages its integrated health care delivery system to deploy management tools for costly therapies: evidence analysis, usage measurements, and multidisciplinary planning. Evidence-based medicine and realization of value provided by new therapies will depend on data capture and outcomes measurement, tools that are crucial to Kaiser Permanente's management of biotechnologies and effective use of members' financial resources.

Biotechnology↗