Synthetic muO-conotoxin MrVIB blocks TTX-resistant sodium channel NaV1.8 and has a long-lasting analgesic activity

MuO-conotoxin MrVIB is a blocker of voltage-gated sodium channels, including TTX-sensitive and -resistant subtypes. A comprehensive characterization of this peptide has been hampered by the lack of sufficient synthetic material. Here, we describe the successful chemical synthesis and oxidative folding of MrVIB that has made an investigation of the pharmacological properties and therapeutic potential of the peptide feasible. We show for the first time that synthetic MrVIB blocks rat NaV1.8 sodium channels and has potent and long-lasting local anesthetic effects when tested in two pain assays in rats. Furthermore, MrVIB can block propagation of action potentials in A- and C-fibers in sciatic nerve as well as skeletal muscle in isolated preparations from rat. Our work provides the first example of analgesia produced by a conotoxin that blocks sodium channels. The emerging diversity of antinociceptive mechanisms targeted by different classes of conotoxins is discussed.     

An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.     

Equine cloning: in vitro and in vivo development of aggregated embryos

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.     

Conantokins derived from the Asprella clade impart conRl-B, an N-methyl d-aspartate receptor antagonist with a unique selectivity profile for NR2B subunits

Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-methyl d-aspartate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity with the most studied NR2B selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogues for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings for Tyr5 substitutions in other conantokins, conRl-B[L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis, and pharmacology for discovering new bioactive natural products.     

Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice

As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92Da; calculated monoisotopic mass 2884.20Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63Da; calculated monoisotopic mass 3308.34Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.     

The Socioeconomic Impact of Truffle Cultivation in Rural Spain<Superscript>1</Superscript>

The Socioeconomic Impact of Truffle Cultivation in Rural Spain. Commercial black truffle (Tuber melanosporum) plantations have been promoted in Europe with the intention of benefiting rural economies while conserving biodiversity through the expansion of oak woodlands. In this context, a socioeconomic study was conducted around the town of Sarrión in eastern Spain, where government subsidies have supported oak reforestation and truffle cultivation in unproductive hilly areas since 1987. Currently there are about 4,500 ha of truffle orchards in the surrounding county and 530 members in the local truffle association, which has provided a key forum for truffle cultivators to share technical, financial and administrative experiences. Structured interviews were carried out in 2002 with a number of orchard owners, as well as representatives of financial and governmental institutions. Truffles, which are harvested using trained dogs, typically fetch local cultivators average prices of 220–670 EUR/kg, although retail prices of high-quality specimens may reach twice this amount. In addition to the direct economic impact, an increase in local land prices was also documented, as well as a tendency for continued expansion of truffle orchards, and thus oak reforestation. In conclusion, the promotion of truffle cultivation through autonomous community and provincial government subsidies, in conjunction with support by local banks, a dedicated local truffle association, and growing interest on behalf of local farmers, seems to have achieved the mutual goals of biodiversity conservation and improving the rural economy in this region of Spain.     

Changes in activities of antioxidant enzymes during <Emphasis Type="Italic">Chenopodium murale</Emphasis> seed germination

The activities and isoenzyme pattern of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) have been studied during germination of Chenopodium murale seeds. CAT and SOD activities were similar in dry seeds and during first 2 d of imbibition. CAT activity increased during radicle protrusion and early seedling development. The maximum SOD activity was found at final stages of germination and early seedling development. POD activity was not detected until the 6^th day of germination, indicating POD involvement not until early seedling development. Gibberellic acid (GA₃, 160 μM) delayed and synchronized C. murale germination.     

Partial purification and characterization of a non-specific acid phosphatase in leaves and root nodules of Phaseolus vulgaris.

Acid phosphatase (ACP) activity in common bean grown with or without 1.5 mM of phosphate has been examined. Leaves and root nodules responded to the absence of an exogenous phosphate source with an increase in ACP activity. Increases in enzyme activity were not associated with the synthesis of new isoforms of the enzyme. We partially purified and characterized the ACPs, which consisted of three proteins, one of leaf and two of nodule. Proteins of leaf migrated at 72 and 51 kDa in SDS-PAGE, whereas that of nodule migrated at 72, 49, 41 and 34 kDa. Enzymes of both organs had a pH optimum of 5.6, and were relatively heat stable. The enzymes exhibit a broad substrate selectivity, with maximal activity obtained with alpha-naphthyl-phosphate, ribulose 1,5-bisphosphate and p-nitrophenyl-phosphate (p-NPP). Potent inhibition by Zn2+, Hg2+, Cu2+, Pb2+, Al3+ and (MoO4)2- was observed.