Biomedical subjects

Thomas Meier

Publications and source records attributed to Thomas Meier.

At least 19 recordsLinked to original sources

PubMed2006

Activation of muscle-specific receptor tyrosine kinase and binding to dystroglycan are regulated by alternative mRNA splicing of agrin.

Agrin induces the aggregation of postsynaptic proteins at the neuromuscular junction (NMJ). This activity requires the receptor-tyrosine kinase MuSK. Agrin isoforms differ in short amino acid stretches at two sites, called A and B, that are localized in the two most C-terminal laminin G (LG) domains. Importantly, agrin isoforms greatly differ in their activities of inducing MuSK phosphorylation and of binding to alpha-dystroglycan. By using site-directed mutagenesis, we characterized the amino acids important for these activities of agrin. We find that the conserved tripeptide asparagineglutamate-isoleucine in the eight-amino acid long insert at the B-site is necessary and sufficient for full MuSK phosphorylation activity. However, even if all eight amino acids were replaced by alanines, this agrin mutant still has significantly higher MuSK phosphorylation activity than the splice version lacking any insert. We also show that binding to alpha-dystroglycan requires at least two LG domains and that amino acid inserts at the A and the B splice sites negatively affect binding.

Agrin↗

PubMed2006

Structural investigations of the membrane-embedded rotor ring of the F-ATPase from Clostridium paradoxum.

The Na(+)-translocating F-ATPase of the thermoalkaliphilic bacterium Clostridium paradoxum harbors an oligomeric ring of c subunits that resists dissociation by sodium dodecyl sulfate. The c ring has been isolated and crystallized in two dimensions. From electron microscopy of these c-ring crystals, a projection map was calculated to 7 A resolution. In the projection map, each c ring consists of two concentric, slightly staggered, packed rings, each composed of 11 densities representing the alpha-helices. On the basis of these results, it was determined that the F-ATPase from C. paradoxum contains an undecameric c ring.

Amino Acid Sequence↗

PubMed2006

Low-molecular-weight heparin for prevention of restenosis after femoropopliteal percutaneous transluminal angioplasty: a randomized controlled trial.

BACKGROUND: Restenosis after angioplasty is essentially due to intimal hyperplasia. Low-molecular-weight heparins (LMWHs) have experimentally been shown to have antiproliferative effects in addition to their antithrombotic properties. Their potential in reducing restenosis remains to be established. Therefore, we wanted to test the hypothesis that LMWH plus aspirin is more effective than aspirin alone in reducing the incidence of restenosis/reocclusion in patients undergoing percutaneous transluminal angioplasty (PTA) of femoropopliteal arteries. Further, different effects of LMWH in patients treated for critical limb ischemia (CLI) or claudication only should be investigated. METHODS: After successful PTA, 275 patients with symptomatic peripheral arterial disease (claudication or critical limb ischemia) and femoropopliteal obstructions were randomized to receive either 2500 IU of dalteparin subcutaneously for 3 months plus 100 mg of aspirin daily (n = 137), or 100 mg aspirin daily alone (n = 138). The primary end point was restenosis or reocclusion documented by duplex ultrasonography imaging at 12 months. RESULTS: Restenosis/reocclusion occurred in 58 patients (44%) in the dalteparin group and in 62 patients (50%) in the control group (P = .30). In a subgroup analysis according to the severity of peripheral arterial disease, we found that in patients treated for claudication, restenosis/reocclusion developed in 43 (43%) in the dalteparin group, and in 35 (41%) in the control group (P = .70); in patients treated for CLI, restenosis/reocclusion was significantly lower in the dalteparin group (15, 45%) than in the control group (27, 72%; P = .01). No major bleeding events occurred in either group. CONCLUSIONS: Treatment with 2500 IU dalteparin subcutaneously given for 3 months after femoropopliteal PTA failed to reduce restenosis/reocclusion at 12 months. However, dalteparin may be beneficial in the subgroup of patients with CLI at 12 months follow-up.

Aged↗

PubMed2006

Catalytic and mechanical cycles in F-ATP synthases. Fourth in the Cycles Review Series.

Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle, in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive; a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F1F(o) ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded F(o) sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated.

Adenosine Triphosphate↗

PubMed2006

Social structures and social relations--an archaeological and anthropological examination of three early Medieval separate burial sites in Bavaria.

As part of an interdisciplinary cooperation, three early medieval separated burial sites from central Bavaria were subjected to archaeological and anthropological examinations. Separated burial sites are probably an expression of social stratification. This paper focuses on two characteristic aspects of these separated burial sites: (1) structure of the site, and (2) biological manifestation of a social upper class. The separated burial grounds Etting/Sandfeld (31 individuals), Grossmehring (44 individuals) and Kelheim (43 individuals), all located in southern Bavaria, were analyzed. Sex and age at death of all individuals were determined, and dietary behaviour was reconstructed by means of carbon and nitrogen isotope ratios in bone collagen. Local conditions such as climate, soil conditions or the intensity of agriculture can provoke significant variations in plant delta15N-values, which could lead to a shift of the baseline values of the corresponding trophic web. To facilitate the comparison of isotopic data from different sites, delta15N values of cattle bones were taken as a reference for the human data (presuming the diet to consumer chain). The results of dietary reconstruction indicate that the populations enjoyed very good living conditions with a primarily animal protein-based diet. Furthermore, the isotope analysis revealed more detailed indications for certain individuals regarding their social status. Some individuals can even be appointed as possible chiefs of the population, since anthropological and archaeological interpretations were in total agreement: It was possible to identify persons of a higher social status based on the structure of the burial site, grave goods and the isotopic analysis.

Adult↗

PubMed2005

Novel cell-penetrating alpha-keto-amide calpain inhibitors as potential treatment for muscular dystrophy.

Dipeptide-derived alpha-keto-amide compounds with potent calpain inhibitory activity have been identified. These reversible covalent inhibitors have IC(50) values down to 25nM and exhibit greatly improved activity in muscle cells compared to the reference compound MDL28170. Several novel calpain inhibitors have shown positive effects on histological parameters in an animal model of Duchenne muscular dystrophy demonstrating their potential as a treatment option for this fatal disease.

Amides↗

PubMed2005

Structure of the rotor ring of F-Type Na+-ATPase from Ilyobacter tartaricus.

In the crystal structure of the membrane-embedded rotor ring of the sodium ion-translocating adenosine 5'-triphosphate (ATP) synthase of Ilyobacter tartaricus at 2.4 angstrom resolution, 11 c subunits are assembled into an hourglass-shaped cylinder with 11-fold symmetry. Sodium ions are bound in a locked conformation close to the outer surface of the cylinder near the middle of the membrane. The structure supports an ion-translocation mechanism in the intact ATP synthase in which the binding site converts from the locked conformation into one that opens toward subunit a as the rotor ring moves through the subunit a/c interface.

Adenosine Triphosphatases↗

PubMed2005

The c15 ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory.

The oligomeric c ring of the F-ATP synthase from the alkaliphilic cyanobacterium Spirulina platensis was isolated and characterized. Mass spectroscopy analysis indicated a mass of 8,210 Da, reflecting that of a c monomer. The mass increased by 206 Da after treatment with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD), which indicated modification of the ion-binding carboxylate by DCCD. Atomic force microscopy topographs of c rings from S. platensis showed 15 symmetrically assembled subunits. The c15-mer reported here is the largest c ring that is isolated and does not show the classical c-ring mismatch to the three-fold symmetry of the F1 domain.

Crystallization↗

PubMed2005

Structural evidence for a constant c11 ring stoichiometry in the sodium F-ATP synthase.

The Na+-dependent F-ATP synthases of Ilyobacter tartaricus and Propionigenium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene, extracted from the membrane using detergent and then purified. Subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers. The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggregates of homogeneous c11 rings and/or assemblies of c11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crystalline or densely packed assemblies. Most of these rings represented completely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c11 and c1, as observed by SDS/PAGE. Our results are congruent with a stable c11 ring stoichiometry that seems to not be influenced by the expression level of subunit c in the bacteria.

Electrophoresis, Polyacrylamide Gel↗

PubMed2004

Histological parameters for the quantitative assessment of muscular dystrophy in the mdx-mouse.

Duchenne muscular dystrophy is a severe X-linked hereditary disease caused by the absence of functional dystrophin. The dystrophin-deficient mdx-mouse strain is a widely used animal model for dystrophin-deficiency. Several therapeutic approaches for muscular dystrophy have been proposed by different laboratories. In order to compare the efficacy of these therapies in the mdx-mouse, it is essential to implement standardized protocols for the assessment of functional and histological parameters in this mouse model. Here, we determine that the minimal 'Feret's diameter' is a geometrical parameter that allows for reliable measure of muscle fiber cross-sectional size. Using this geometrical parameter we calculate variance coefficients of the muscle fiber size and provide reference values for the quantitative assessment of dystrophic symptoms in frequently investigated muscles of wild-type and mdx-mouse. In addition, we compare the variance coefficients of the muscle fiber size with the percentage of muscle fibers with centralized nuclei; another histological hallmark of muscular dystrophy.

Age Factors↗

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

Electrical power fuels rotary ATP synthase.

ATP synthesis by F-type ATP synthases consumes energy stored in a transmembrane electrochemical gradient of protons or sodium ions. The electric component of the ion motive force is crucial for ATP synthesis. Here, we incorporate recent results on structure and function of the F(0) domain and present a mechanism for torque generation with the fundamental nature of the membrane potential as driving force in the core.

Bacterial Proton-Translocating ATPases↗

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

Detection of glucocorticoid-like activity in traditional Chinese medicine used for the treatment of Duchenne muscular dystrophy.

Anecdotal reports of positive influence of certain traditional Chinese medicines on the progression of neuromuscular diseases in general and Duchenne muscular dystrophy (DMD) in particular has raised interest in patient support groups and clinical experts alike. However, clinical signs of steroid-specific side effects in patients treated with a particular form of Chinese medicine raised the concern that they may contain glucocorticoids, which in turn could also explain the mild beneficial effects seen in some of the patients. We have extracted and fractionated capsules containing pulverized Chinese medicine that had been used for the treatment of DMD patients and analyzed their content for glucocorticoid-like activity using promoter-reporter assays. We demonstrate that extracts from this Chinese medicine activate a prototype glucocorticoid-response element, increase the level of utrophin protein in human muscle cells and activate the utrophin promoter A. Based on our bioassays we conclude that this particular Chinese medicine used for the treatment of muscular dystrophy patients contains glucocorticoids as one of its active ingredients.

Animals↗

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↗