Structural analysis of a plant sucrose carrier using monoclonal antibodies and bacteriophage lambda surface display.

Monoclonal antibodies were raised and selected against recombinant Plantago major PmSUC2 sucrose carrier protein. Epitopes of two monoclonal antibodies (PS2-1A2 and PS2-4D4) were mapped using N-terminally truncated PmSUC2 proteins and a lambda library displaying random PmSUC2 peptides. PS2-1A2 recognizes an octapeptide close to the N-terminus of PmSUC2, PS2-4D4 binds to a decapeptide at the very C-terminus. Analyses of antibody binding to yeast protoplasts with functionally active, tagged PmSUC2 protein revealed that both epitopes are located in cytoplasmic domains of PmSUC2. These results support a model for plant sucrose transporters containing 12 transmembrane helices with the N-terminus and the C-terminus on the cytoplasmic side of the plasma membrane.     

Action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in Neurospora.

In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (repeat-induced point mutation). Affected sequences are subjected to multiple GC-to-AT mutations. To explore the pattern in which base changes are laid down by RIP we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event at the mtr locus. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication flanking the zeta-eta (zeta-eta) locus. The mtr sequence data were also used to further examine the basis for the differential severity of C-to-T mutations on the coding and noncoding strands in genes. The known bias of RIP toward CpA/TpG sites in conjunction with the sequence bias of Neurospora accounts for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) within the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications is consistent with the idea that reversion of long tandem duplications and RIP share a common step.     

Random field models for fitness landscapes

 In many cases fitness landscapes are obtained as particular instances of random fields by randomly assigning a large number of parameters. Models of this type are often characterized reasonably well by their covariance matrices. We characterize isotropic random fields on finite graphs in terms of their Fourier series expansions and investigate the relation between the covariance matrix of the random field model and the correlation structure of the individual landscapes constructed from this random field. Correlation measures are a good characteristic of “rugged landscapes” models as they are closely related to quantities like the number of local optima or the length of adaptive walks. Our formalism suggests to approximate landscape with known autocorrelation function by a random field model that has the same correlation structure. Received: 10 November 1995 / Revised version: 19 February 1996     

Ant attendance in aphids: why different degrees of myrmecophily?

1. Aphids show a range of associations with ants from nonattendance to obligate myrmecophily. Aphis fabae cirsiiacanthoides is facultatively associated with ants, while Symydobius oblongus is an obligate myrmecophile. The selection pressures that have shaped these associations are unknown. 2. The consequences for these aphids of their different degrees of associations with ants were determined, in terms of costs and benefits to individuals and colonies in laboratory and field experiments. In the laboratory, individuals of A. f. cirsiiacanthoides performed worse and those of Symydobius oblongus performed better when attended by the ant Lasius niger than when unattended. For example, when ant-attended, A. f. cirsiiacanthoides developed more slowly, were smaller, and invested less in gonads, whereas S. oblongus developed more quickly, were larger, and invested more in gonads. In addition, the ant regulated the population size of S. oblongus to an average of 50–70 individuals per birch sapling by removing aphids, but did not regulate the population size of A. f. cirsiiacanthoides. 3. Under field conditions, ant-attended colonies of both A. f. cirsiiacanthoides and S. oblongus achieved higher peak numbers and lasted longer, and ant-attended colonies of A. f. cirsiiacanthoides produced more alate dispersers than unattended colonies. 4. The implications of divergent selection pressures for the development of myrmecophily in aphids are discussed.     

SynBlast: Assisting the analysis of conserved synteny information

Motivation  

In the last years more than 20 vertebrate genomes have been sequenced, and the rate at which genomic DNA information becomes available is rapidly accelerating. Gene duplication and gene loss events inherently limit the accuracy of orthology detection based on sequence similarity alone. Fully automated methods for orthology annotation do exist but often fail to identify individual members in cases of large gene families, or to distinguish missing data from traceable gene losses. This situation can be improved in many cases by including conserved synteny information.     

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.     

Discovery of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine scaffold as a novel,potent and selective GABAA α5 inverse agonist series

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine was deemed the most promising GABA_A α5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.