PubMed Health⌕ Search

Biomedical subjects

Thomas Meier

Publications and source records attributed to Thomas Meier.

13 recordsLinked to original sources
PubMed2003

Mitochondria-targeted antioxidants protect Friedreich Ataxia fibroblasts from endogenous oxidative stress more effectively than untargeted antioxidants.

Friedreich Ataxia (FRDA), the most common inherited ataxia, arises from defective expression of the mitochondrial protein frataxin, which leads to increased mitochondrial oxidative damage. Therefore, antioxidants targeted to mitochondria should be particularly effective at slowing disease progression. To test this hypothesis, we compared the efficacy of mitochondria-targeted and untargeted antioxidants derived from coenzyme Q10 and from vitamin E at preventing cell death due to endogenous oxidative stress in cultured fibroblasts from FRDA patients in which glutathione synthesis was blocked. The mitochondria-targeted antioxidant MitoQ was several hundredfold more potent than the untargeted analog idebenone. The mitochondria-targeted antioxidant MitoVit E was 350-fold more potent than the water soluble analog Trolox. This is the first demonstration that mitochondria-targeted antioxidants prevent cell death that arises in response to endogenous oxidative damage. Targeted antioxidants may have therapeutic potential in FRDA and in other disorders involving mitochondrial oxidative damage.

Antioxidants↗
PubMed2003

Observing membrane protein diffusion at subnanometer resolution.

Single sodium-driven rotors from a bacterial ATP synthase were embedded into a lipid membrane and observed in buffer solution at subnanometer resolution using atomic force microscopy (AFM). Time-lapse AFM topographs show the movement of single proteins within the membrane. Subsequent analysis of their individual trajectories, in consideration of the environment surrounding the moving protein, allow principal modes of the protein motion to be distinguished. Within one trajectory, individual proteins can undergo movements assigned to free as well as to obstacled diffusion. The diffusion constants of these two modes of motion are considerably different. Without the structural information about the membrane environment restricting the moving proteins, it would not be possible to reveal insight into these mechanisms. The high-resolution AFM topographs suggest that, in future studies, such data revealed under various physiological conditions will provide novel insights into molecular mechanisms that drive membrane protein assembly and supply excellent boundary conditions to model protein-protein arrangements.

Diffusion↗
PubMed2003

Complete DNA sequence of the atp operon of the sodium-dependent F1Fo ATP synthase from Ilyobacter tartaricus and identification of the encoded subunits.

The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F(1)F(o) ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that of atpB coding for the a subunit, were identified by the corresponding N-terminal amino acid sequence. The hydrophobic a subunit was identified by MALDI mass spectrometry. The atp genes of I. tartaricus are arranged in one operon with the sequence atpIBEFHAGDC comprising 6,992 base pairs with a GC content of 38.1%. The F(1)F(o) ATP synthase of I. tartaricus has a calculated molecular mass of 510 kDa and includes 4,810 amino acids. The gene sequences and products reveal significant identities to atp genes of other Na(+)-translocating F(1)F(o) ATP synthases, especially in the F(o) subunits a and c which are directly involved in ion translocation.

Adenosine Triphosphate↗
PubMed2003

Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases.

The Na(+)-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c(11) rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7A resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c(11) ring of I.tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F(1)F(o) holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na(+). From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5mM NaCl, respectively. These pK values are identical with those determined for the F(1)F(o) complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na(+) from the cytoplasm through access channels within the c-ring itself.

Bacterial Proteins↗
PubMed2003

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

Amino Acid Motifs↗
PubMed2003

Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes.

This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging with high spatial resolution and provides numerous possibilities in cellular and pharmaceutical research.

Animals↗
PubMed2002

A cellular model for Friedreich Ataxia reveals small-molecule glutathione peroxidase mimetics as novel treatment strategy.

Friedreich Ataxia (FRDA), the most prevalent of the inherited ataxias, is a multi-systemic disease with loss of sensory neurons and life-threatening hypertrophic cardiomyopathy as its most severe manifestations. Reduced levels of the mitochondrial protein frataxin lead to cell-damaging oxidative stress and consequently FRDA is considered as a model for more common neurodegenerative disorders in which reactive radicals and oxidative stress are involved. We have developed a cellular assay system that discriminates between fibroblasts from FRDA patients and unaffected donors on the basis of their sensitivity to pharmacological inhibition of de novo synthesis of glutathione. With this assay we observed that supplementation with selenium effectively improved the viability of FRDA fibroblasts, indicating that basal selenium concentrations are not sufficient to allow an adequate increase in the activity of certain detoxification enzymes (such as GPX). Furthermore, we characterized potential drug candidates and found that idebenone, a mitochondrially localized antioxidant that ameliorates cardiomyopathy in FRDA patients, as well as other lipophilic antioxidants protected FRDA cells from cell death. Our results also demonstrate for the first time that small-molecule GPX mimetics have potential as a novel treatment strategy for Friedreich Ataxia and presumably also for other neurodegenerative diseases with mitochondrial impairment.

Benzoquinones↗
PubMed2002

Intersubunit bridging by Na+ ions as a rationale for the unusual stability of the c-rings of Na+-translocating F1F0 ATP synthases.

The oligomeric c-rings of Na+-translocating F1F0 ATP synthases exhibit unusual stability, resisting even boiling in SDS. Here, we show that the molecular basis for this remarkable property is intersubunit crossbridging by Na+ or Li+ ions. The heat stability of c11 was dependent on the presence of Na+ or Li+ ions. For equal stability, 10 times higher Li+ than Na+ concentrations were required, reflecting the 10 times lower binding affinity for Li+ than for Na+. In a recent structural model of c11, the Na+ or Li+ binding ligands are located on neighboring c-subunits, which thus become crossbridged by the binding of either alkali ion with a concomitant increase in the stability of the ring. Site-directed mutagenesis strengthens the essential role of glutamate 65 in the crossbridging of the subunits and also corroborates the proposed stabilizing effect of an ion bridge including aspartate 2.

Bacterial Proteins↗
PubMed2002

Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase.

The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an alpha-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms.

Amino Acid Sequence↗
PubMed2002

Glucocorticoid-mediated regulation of utrophin levels in human muscle fibers.

Previous studies on transgenic mice indicate that upregulation of utrophin protein may offer a potential treatment strategy for Duchenne muscular dystrophy. We have analyzed the effect of the glucocorticoid 6alpha-methylprednisolone-21 sodium succinate on utrophin protein levels, using a cell-based assay with differentiated human myotubes, derived from biopsies of healthy individuals or Duchenne muscular dystrophy patients. We found that within 5-7 days 6alpha-methylprednisolone-21 sodium succinate increases utrophin protein up to approximately 40% in both normal and dystrophin-deficient myotubes compared to untreated control cultures. When analyzed in promoter-reporter assays 6alpha-methylprednisolone-21 sodium succinate activated a utrophin promoter A-fragment but did not activate a utrophin promoter B-fragment. Surprisingly, endogenous levels of utrophin mRNA in 6alpha-methylprednisolone-21 sodium succinate-treated muscle cells were unaltered indicating that the utrophin-inducing effect of glucocorticoids may be a result of post-transcriptional mechanisms. We have also analyzed 66 glucocorticoids for their effect on utrophin protein levels and found that glucocorticoids in general are able to induce utrophin protein in human myotubes.

Cytoskeletal Proteins↗
PubMed2002

Membrane embedded location of Na+ or H+ binding sites on the rotor ring of F1F0 ATP synthases.

Recent crosslinking studies indicated the localization of the coupling ion binding site in the Na+-translocating F1F0 ATP synthase of Ilyobacter tartaricus within the hydrophobic part of the bilayer. Similarly, a membrane embedded H+-binding site is accepted for the H+-translocating F1F0 ATP synthase of Escherichia coli. For a more definite analysis, we performed parallax analysis of fluorescence quenching with ATP synthases from both I. tartaricus and E. coli. Both ATP synthases were specifically labelled at their c subunit sites with N-cyclohexyl-N'-(1-pyrenyl)carbodiimide, a fluorescent analogue of dicyclohexylcarbodiimide and the enzymes were reconstituted into proteoliposomes. Using either soluble quenchers or spinlabelled phospholipids, we observed a deeply membrane embedded binding site, which was quantitatively determined for I. tartaricus and E. coli to be 1.3 +/- 2.4 A and 1.8 +/- 2.8 A from the bilayer center apart, respectively. These data show a conserved topology among enzymes of different species. We further demonstrated the direct accessibility for Na+ ions to the binding sites in the reconstituted I. tartaricus c11 oligomer in the absence of any other subunits, pointing to intrinsic rotor channels. The common membrane embedded location of the binding site of ATP synthases suggest a common mechanism for ion transfer across the membrane.

Adenosine Triphosphate↗
PubMed2000

The Role of Dystroglycan and Its Ligands in Physiology and Disease.

Dystroglycan contributes to the formation of basement membrane during embryonic development and enforces cell membrane integrity by bridging cytoskeleton and components of the extracellular matrix. In several forms of muscle disease, dystroglycan is reduced in abundance. Moreover, human viral and bacterial pathogens use dystroglycan as their cellular entry point.

Journal Article↗