Sequences 5'' to translation start regulate expression of petunia rbcS genes.

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes.     

A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator.

c[Arg-aB-(CH2+SCH3 phi)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (u-PA and t-PA) and plasmin. This compound inhibited u-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor: first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-1 and 9 microM, respectively at pH 7.5 and 25 degrees C. The activation of plasminogen by u-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than u-PA, respectively.     

Structure and genetic diversity of Ixora margaretae an endangered species

The dry forests of New Caledonia are an exceptional ecosystem because of their numerous endemic botanical species and their highly diversified fauna of insects, mollusks, reptiles and birds. Unfortunately, the area of the dry forests has been significantly reduced, mainly by human activities. Ecological, phenological and genetic analysis of Ixora margaretae, a symbolic species of the sclerophyll forest, has revealed contrasting traits among natural stands. The division of the natural range and then the separation of forest islands has greatly reduced the existing genetic variability of this species. The genetic diversity is strongly structured in genetic clusters which correspond well to specific ecotypes according to the environmental conditions and the forest types. Furthermore, genetic analysis of the reproductive and non-reproductive trees as well the half-sib families obtained by complete protection of mother trees has revealed substantial genetic drift which has resulted in increased loss of allelic variability. The total consumption of seeds by mainly rats confirms the observed absence of natural regeneration. All these results show that measures taken to protect the stands of dry forests will not be enough to maintain sufficient genetic variability of I. margaretae populations in the long term. Assisted regeneration with control of the increase in variability will be necessary to maintain the biodiversity of the species. The results obtained for I. margaretae must be confirmed with other symbolic species in order to take the necessary measures for the effective preservation of the dry forests in New Caledonia.     

CD44 Receptor Unfolding Enhances Binding by Freeing Basic Amino Acids to Contact Carbohydrate Ligand

The extracellular carbohydrate-binding domain of the Type I transmembrane receptor CD44 is known to undergo affinity switching, where change in conformation leads to enhanced binding of its carbohydrate ligand hyaluronan. Separate x-ray crystallographic and NMR experiments have led to competing explanations, with the former supporting minor conformational changes at the binding site and the latter a major order-to-disorder unfolding transition distant from the binding site. Here, all-atom explicit-solvent molecular dynamics studies employing adaptive biasing force sampling revealed a substantial favorable free-energy change associated with contact formation between the Arg⁴¹ side chain and hyaluronan at the binding site, independent of whether the distant site was ordered or disordered. Analogous computational experiments on Arg⁴¹Ala mutants showed loss of this favorable free-energy change, consistent with existing experimental data. More provocatively, the simulation data revealed the molecular mechanism by which the order-to-disorder transition enhances hyaluronan binding: in the disordered state, a number of basic residues gain sufficient conformational freedom—lacking in the ordered state—to spontaneously form side-chain contacts with hyaluronan. Mutation of these residues to Ala had been known to decrease binding affinity, but there had previously been no structural explanation, given their lack of proximity to the carbohydrate-binding site in existing structures of the complex.     

Conus venoms: a source of toxins which interact with membrane- potential-dependent sodium channels

Marine snails of the genus Conus, as they are carnivorous predators, have a venom apparatus used to capture their prey. The toxins contained in the venoms of Conidae, called conotoxins, are of a particular high degree of diversity and represent powerful tools in the neuroscience field. Indeed, these toxins specifically bind with a high affinity to receptors and ionic channels. Therefore, they provide original pharmacological tools which receive increasing investigation both to identify and study some functions of the nervous systems and to characterize new types and closely related subtypes of receptors or ionic channels. The voltage-gated sodium channel, because of its fundamental role in cell membrane excitability, is the specific target of a large number of animal and vegetal toxins. Actually, at least seven toxin receptor sites have been identified on this channel-protein. These toxins, and in particular conotoxins, are used to precise the role of different types and/or closely related subtypes of sodium channels in the peripheral and central nervous systems. The focus of the present review is to summarize our current knowledge of the consequences of physiological interactions between different conotoxin families and sodium channels.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Piperazine sulfonamide BACE1 inhibitors: Design,synthesis, and in vivo characterization

With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2′ sub-pocket of the enzyme culminated in identification of an analog that potently lowers peripheral Aβ₄₀ in transgenic mice with a single subcutaneous dose.     

Feeding behaviour of sheep fed lucerne v. grass hays with controlled post-ingestive consequences

Understanding what determines feeding behaviour in herbivores is essential to optimise the use of forages in breeding systems. Herbivores can evaluate foods by associative learning of their pre-ingestive characteristics (taste, odour, etc.) and their post-ingestive consequences. Post-ingestive consequences are acknowledged as influencing intake and food choices, but the role of pre-ingestive characteristics is still being debated. Our experiment was designed to test their separate effects on daily dry matter intake (DMI), intake patterns and short-term choices in sheep by crossing the nature of the hay orally consumed (o) ad libitum, lucerne (L) or grass (G), with the nature of the hay introduced into the rumen (r), L or G, at a rate of half the total amount of hay received the day before. We applied four treatments, Go/Gr, Go/Lr, Lo/Gr and Lo/Lr, to test the effects of (i) post-ingestive consequences with similar pre-ingestive characteristics (Go/Gr v. Go/Lr; Lo/Gr v. Lo/Lr) and (ii) pre-ingestive characteristics with similar post-ingestive consequences at the end of the feeding period (Go/Lr v. Lo/Gr). Six rumen-fistulated sheep underwent all the treatments over 11-day periods in a latin square design. Eating time was restricted to 6 h/day, intraruminal introductions were performed just before food offer and choice tests were conducted after food removal. For similar pre-ingestive characteristics, DMI increased when L hay was introduced into the rumen rather than G (P < 0.05), possibly owing to a lower fill effect of L due to its lower NDF content and higher rumen degradability. The increased DMI resulted from longer eating time when G was orally consumed (149 v. 192 min, P < 0.05), whereas it resulted from higher intake rate with L (4.8 v. 6.1 g/min, P < 0.05). For similar post-ingestive consequences at the end of the feeding period (Go/Lr and Lo/Gr), DMI were similar (P > 0.05). Pre-ingestive characteristics or palatability per se did not therefore influence daily intake, although they influenced eating patterns. Pre-ingestive characteristics also greatly influenced short-term choices in favour of the hay that was not previously consumed, independently of any post-ingestive influence. This study confirms the effects of post-ingestive consequences on daily intake, but demonstrates that these variations are obtained by different behavioural adjustments under the influence of pre-ingestive characteristics. Preference for novelty, regardless of post-ingestive consequences, thus suggests that sheep may seek a diverse diet more for pleasure than for functional purposes, with implications for animal welfare.     

Small conformationally restricted piperidine N-arylsulfonamides as orally active gamma-secretase inhibitors

The design and development of a new class of small 2,6-disubstituted piperidine N-arylsulfonamide gamma-secretase inhibitors is reported. Lowering molecular weight including the use of conformational constraint led to compounds with less CYP 3A4 liability compared to early leads. Compounds active orally in lowering Abeta levels in Tg CRND8 mice were identified as potential treatments for Alzheimer's disease.     

A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.