Unmasking features of the auto-epitope essential for β(1)-adrenoceptor activation by autoantibodies in chronic heart failure

AIMS: Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β(1)-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β(1)-adrenoceptor (β(1) EC(II)) that is targeted by stimulating β(1)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. METHODS AND RESULTS: Non-conserved amino acids within the β(1) ECII loop (compared with the amino acids constituting the ECII loop of the β(2)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine-serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β(1) ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β(1)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β(1)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β(1) ECII loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK(211-214) motif and (ii) the intra-loop disulfide bond C(209)↔C(215). Of note, aberrant intra-loop disulfide bond C(209)↔C(216) almost fully disrupted the functional auto-epitope in cyclopeptides. CONCLUSIONS: The conformational auto-epitope targeted by cardio-pathogenic β(1)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β(1) EC(II) loop bearing the NDPK(211-214) motif and the C(209)↔C(215) bridge while lacking cysteine C(216). Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β(1)-autoantibody-positive CHF

Modulation of DNA topoisomerase II activity and expression in melanoma cells with acquired drug resistance

The role of DNA topoisomerases (Topo) IIα and IIβ was investigated in various drug-resistant melanoma cells. Melanoma cells resistant to etoposide, exhibited an up to tenfold reduced Topo II activity corresponding to an increasing degree of drug resistance indicating that modulation of Topo II activity contribute to the drug-resistant phenotype. The reduction of Topo II activity was reflected by decreased nuclear amounts of both Topo II isoforms. © 2000 Cancer Research Campaig

Massively Parallel Haplotyping on Microscopic Beads for the High-Throughput Phase Analysis of Single Molecules

In spite of the many advances in haplotyping methods, it is still very difficult to characterize rare haplotypes in tissues and different environmental samples or to accurately assess the haplotype diversity in large mixtures. This would require a haplotyping method capable of analyzing the phase of single molecules with an unprecedented throughput. Here we describe such a haplotyping method capable of analyzing in parallel hundreds of thousands single molecules in one experiment. In this method, multiple PCR reactions amplify different polymorphic regions of a single DNA molecule on a magnetic bead compartmentalized in an emulsion drop. The allelic states of the amplified polymorphisms are identified with fluorescently labeled probes that are then decoded from images taken of the arrayed beads by a microscope. This method can evaluate the phase of up to 3 polymorphisms separated by up to 5 kilobases in hundreds of thousands single molecules. We tested the sensitivity of the method by measuring the number of mutant haplotypes synthesized by four different commercially available enzymes: Phusion, Platinum Taq, Titanium Taq, and Phire. The digital nature of the method makes it highly sensitive to detecting haplotype ratios of less than 1∶10,000. We also accurately quantified chimera formation during the exponential phase of PCR by different DNA polymerases

Living long and well: prospects for a personalized approach to the medicine of ageing

Research into ageing and its underlying molecular basis enables us to develop and implement targeted interventions to ameliorate or cure its consequences. However, the efficacy of interventions often differs widely between individuals, suggesting that populations should be stratified or even individualized. Large-scale cohort studies in humans, similar systematic studies in model organisms as well as detailed investigations into the biology of ageing can provide individual validated biomarkers and mechanisms, leading to recommendations for targeted interventions. Human cohort studies are already ongoing, and they can be supplemented by in silico simulations. Systematic studies in animal models are made possible by the use of inbred strains or genetic reference populations of mice. Combining the two, a comprehensive picture of the various determinants of ageing and ‘health span' can be studied in detail, and an appreciation of the relevance of results from model organisms to humans is emerging. The interactions between genotype and environment, particularly the psychosocial environment, are poorly studied in both humans and model organisms, presenting serious challenges to any approach to a personalized medicine of ageing. To increase the success of preventive interventions, we argue that there is a pressing need for an individualized evaluation of interventions such as physical exercise, nutrition, nutraceuticals and calorie restriction mimetics as well as psychosocial and environmental factors, separately and in combination. The expected extension of the health span enables us to refocus health care spending on individual prevention, starting in late adulthood, and on the brief period of morbidity at very old ag

Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules

<p>Abstract</p> <p>Background</p> <p>Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from <it>Canavalia gladiata </it>seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, α-aminobutyric acid (Abu), is bound.</p> <p>Results</p> <p>The overall structure of native CGL and complexed with α-methyl-mannoside and Abu have been refined at 2.3 Å and 2.31 Å resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry.</p> <p>Conclusion</p> <p>The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.</p

Testing the optimal defence hypothesis for two indirect defences: extrafloral nectar and volatile organic compounds

Many plants respond to herbivory with an increased production of extrafloral nectar (EFN) and/or volatile organic compounds (VOCs) to attract predatory arthropods as an indirect defensive strategy. In this study, we tested whether these two indirect defences fit the optimal defence hypothesis (ODH), which predicts the within-plant allocation of anti-herbivore defences according to trade-offs between growth and defence. Using jasmonic acid-induced plants of Phaseolus lunatus and Ricinus communis, we tested whether the within-plant distribution pattern of these two indirect defences reflects the fitness value of the respective plant parts. Furthermore, we quantified photosynthetic rates and followed the within-plant transport of assimilates with 13C labelling experiments. EFN secretion and VOC emission were highest in younger leaves. Moreover, the photosynthetic rate increased with leaf age, and pulse-labelling experiments suggested transport of carbon to younger leaves. Our results demonstrate that the ODH can explain the within-plant allocation pattern of both indirect defences studied

Genetic Crossovers Are Predicted Accurately by the Computed Human Recombination Map

Hotspots of meiotic recombination can change rapidly over time. This instability and the reported high level of inter-individual variation in meiotic recombination puts in question the accuracy of the calculated hotspot map, which is based on the summation of past genetic crossovers. To estimate the accuracy of the computed recombination rate map, we have mapped genetic crossovers to a median resolution of 70 Kb in 10 CEPH pedigrees. We then compared the positions of crossovers with the hotspots computed from HapMap data and performed extensive computer simulations to compare the observed distributions of crossovers with the distributions expected from the calculated recombination rate maps. Here we show that a population-averaged hotspot map computed from linkage disequilibrium data predicts well present-day genetic crossovers. We find that computed hotspot maps accurately estimate both the strength and the position of meiotic hotspots. An in-depth examination of not-predicted crossovers shows that they are preferentially located in regions where hotspots are found in other populations. In summary, we find that by combining several computed population-specific maps we can capture the variation in individual hotspots to generate a hotspot map that can predict almost all present-day genetic crossovers

Positive Selection for New Disease Mutations in the Human Germline: Evidence from the Heritable Cancer Syndrome Multiple Endocrine Neoplasia Type 2B

Multiple endocrine neoplasia type 2B (MEN2B) is a highly aggressive thyroid cancer syndrome. Since almost all sporadic cases are caused by the same nucleotide substitution in the RET proto-oncogene, the calculated disease incidence is 100–200 times greater than would be expected based on the genome average mutation frequency. In order to determine whether this increased incidence is due to an elevated mutation rate at this position (true mutation hot spot) or a selective advantage conferred on mutated spermatogonial stem cells, we studied the spatial distribution of the mutation in 14 human testes. In donors aged 36–68, mutations were clustered with small regions of each testis having mutation frequencies several orders of magnitude greater than the rest of the testis. In donors aged 19–23 mutations were almost non-existent, demonstrating that clusters in middle-aged donors grew during adulthood. Computational analysis showed that germline selection is the only plausible explanation. Testes of men aged 75–80 were heterogeneous with some like middle-aged and others like younger testes. Incorporating data on age-dependent death of spermatogonial stem cells explains the results from all age groups. Germline selection also explains MEN2B's male mutation bias and paternal age effect. Our discovery focuses attention on MEN2B as a model for understanding the genetic and biochemical basis of germline selection. Since RET function in mouse spermatogonial stem cells has been extensively studied, we are able to suggest that the MEN2B mutation provides a selective advantage by altering the PI3K/AKT and SFK signaling pathways. Mutations that are preferred in the germline but reduce the fitness of offspring increase the population's mutational load. Our approach is useful for studying other disease mutations with similar characteristics and could uncover additional germline selection pathways or identify true mutation hot spots

Linear Collider Physics Resource Book for Snowmass 2001, 3: Studies of Exotic and Standard Model Physics

This Resource Book reviews the physics opportunities of a next-generation e+e- linear collider and discusses options for the experimental program. Part 3 reviews the possible experiments on that can be done at a linear collider on strongly coupled electroweak symmetry breaking, exotic particles, and extra dimensions, and on the top quark, QCD, and two-photon physics. It also discusses the improved precision electroweak measurements that this collider will make available.This Resource Book reviews the physics opportunities of a next-generation e+e- linear collider and discusses options for the experimental program. Part 3 reviews the possible experiments on that can be done at a linear collider on strongly coupled electroweak symmetry breaking, exotic particles, and extra dimensions, and on the top quark, QCD, and two-photon physics. It also discusses the improved precision electroweak measurements that this collider will make available