A Conus regularis Conotoxin with a Novel Eight-Cysteine Framework Inhibits CaV2.2 Channels and Displays an Anti-Nociceptive Activity

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits Ca_V2.2 channel current in a dose-dependent manner with an EC₅₀ of 2.8 μM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for Ca_V2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.     

A retrospective study of non-suppurative encephalitis in beef cattle from western Canada

Non-suppurative encephalitis occurs sporadically in beef cattle in western Canada, leading to loss of animals. This retrospective study investigated the presence of viral, bacterial, and protozoal antigens or DNA in 37 western Canadian feedlot cattle with non-suppurative encephalitis for which a cause had not been identified. Cases were selected based on the age of the animal (> 7 months), and clinical history of recumbency and depression. The identification of rabies in 1 case stresses the importance of including this viral disease in the list of differential diagnoses. Because there was variation in the severity, distribution, and type of lesions, it is possible that there may be more than 1 cause, but failure to identify an infectious agent might also suggest that non-infectious agents could play a role.     

Fatty Acid Profile,Total Carotenoids,and Free Radical-Scavenging from the Lipophilic Fractions of 12 Native Mexican Avocado Accessions

Plant Foods for Human Nutrition - Mexico is the world’s largest producer, exporter, and consumer of avocados. “Hass” avocado is the most commercialized cultivar, while the native...     

Potential factors controlling the population viability of newly introduced endangered marble trout populations

While several population viability analyses (PVAs) have been performed on anadromous salmonids, less attention has been given to stream-living salmonids. In this work, we explore the role of PVA as a tool in the recovery of threatened stream-living salmonid species. The analysis has been performed with reference to marble trout Salmo marmoratus, a salmonid with a limited geographic distribution and at risk of extinction due to hybridization with the non-native introduced brown trout. Demographic parameters, such as survival, fecundity and density-dependent patterns were estimated from an eight year on-going monitoring program of two translocated marble trout populations in pristine, previously fishless streams (Zakojska and Gorska) in the Soca and Idrijca river basins (Slovenia). To explore the importance of disturbance events such as floods on marble trout population dynamics, we performed a PVA under three scenarios: (1) occurrence of both severe and moderate floods; (2) occurrence of only moderate floods; (3) no flood events. Our analysis shows that population viability is threatened only by severe flood events, otherwise the two populations prove to be fairly stable with population abundance fluctuating around stream carrying capacity. A sensitivity analysis performed on model parameters highlighted that density-dependence in first-year survival and the magnitude of reduction in population size after a severe flood are the two most crucial parameters affecting population abundance and quasi-extinction probability, respectively. While only extreme floods can drive the population to extinction, the increase in juvenile survival when population abundance collapses after a major flood may allow the populations to quickly recover from few reproductive individuals back to stream carrying capacity.     

Synthesis and phytotoxic activity of ozonides

The [4 + 3] cycloaddition of the proper furans with the oxyallyl cation, generated in situ from 2,4-dibromopentan-3-one, produced a series of 8-oxabicyclo [3.2.1]oct-6-en-3-ones. Exposure of the oxabicycles to ozone afforded the corresponding 8,9,10,11-tetraoxatricyclo[5.2.1.1 (2,6)]undecan-4-ones in variable yields (7-100%). The phytotoxic properties of these ozonides (or 1,2,4-trioxolanes) and their oxabicycle precursors were evaluated as the ability to interfere with the growth of Sorghum bicolor and Cucumis sativus seedlings. Among oxabicycles, the highest inhibitory activity was shown by compounds possessing a alpha,beta-unsaturated carbonyl moiety. A differential sensitivity of the two crops was evident with ozonides. The most active compounds were also tested against the weed species Ipomoea grandifolia and Brachiaria decumbens. To the best of our knowledge, this is the first article describing ozonides as potential herbicides.     

Antimicrobial activity of the ornamental species Salvia corrugata, a potential new crop for extractive purposes

As a part of our search for biologically active compounds from cultivated Salvia spp. we investigated Salvia corrugata Vahl. The activity of two isolated icetaxane diterpene quinones, fruticuline A and demethylfruticuline A, was assessed against 46 bacterial pathogens, mostly resistant to several primary antibiotics. The MIC for all the inhibited Gram-positive pathogens tested showed a very narrow distribution and ranged from 32 to 64 mg/L, regardless of their resistance patterns to other antibiotics. Demethylfruticuline A was shown to be highly bactericidal (>3 log(10) CFU decrease within 24 h) against Staphylococcus aureus and S. epidermidis and bacteriostatic against Enterococcus faecalis and E. faecium. Fruticuline A manifested bacteriostatic activity against all tested strains. S. corrugata can be viewed as an interesting source for these diterpenes, which, if well tolerated in vivo, may represent new medical agents useful for the treatment of serious infections caused by resistant Gram-positive pathogens.     

Identification of the production chain of Asiago d'Allevo cheese by nuclear magnetic resonance spectroscopy and principal component analysis

In the present work, a rapid and simple NMR method to discriminate Asiago d'Allevo cheese samples from different production chains is described. A fast and reproducible extraction of the organic fraction was employed. By applying chemometric analysis to NMR data, it is possible to differentiate PDO Asiago cheese produced in alpine farms from that produced in lowland and mountain industrialized factories. PCA of both (1)H and (13)C NMR spectra showed a good separation of alpine farm products from the other ones, whereas the lowland and mountain industrialized cheeses are undistinguishable. The samples were differentiated on the basis of a higher content of unsaturated fatty acids, principally oleic, linoleic, linolenic, and conjugated linoleic acids for the alpine farm cheeses and a higher content of saturated fatty acids for the industrialized products. Conjugated linoleic acid and 1-pentene are also discriminating components.     

Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes

Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.     

Simulation of over-exploitation of ornamental fish and its consequences for the functional structure of assemblages of Amazonian streams

The consequences of over-exploitation may seriously impair the integrity and functioning of ecosystems. When loss of species is accompanied by the loss of ecological processes, the effects are no longer just taxonomic and may affect the stability of the environments. Ornamental fishing is one of the main economic activities of the middle Negro River. Such activity is directed to a small number of species and has the Cardinal Tetra (Paracheirodon axelrodi) as the main target species. Given the potential effects this pressure and the lack of information on its consequences, the present study analysed, through simulations, the possible effects of depletion of ornamental fish populations on the functional structure of fish assemblages, represented by functional diversity (FD) and functional redundancy (FR) indexes. We sampled 13 streams exploited by the ornamental fishery, where we collected 4,286 specimens of 110 species, of which 22 were targeted as ornamental fishing. We found that the ornamental species corresponded, on average, to 27.8% of the species and 33.3% of the FD of the assemblages. In a scenario of complete exclusion of ornamental species, local communities would lose 24.6% of the FD and reduce up to 12% of the FR. With these results, it is possible to infer that the local extinction of exploited species would cause negative impacts on the multifunctionality and the resilience of the streams ecosystems. In this way, we emphasise the need for proper management of the ornamental fisheries aiming to the sustainability of the activity and to the conservation of ecosystem functionality.     

rBmTI-6, a Kunitz-BPTI domain protease inhibitor from the tick Boophilus microplus, its cloning, expression and biochemical characterization

Boophilus microplus is a rich source of trypsin inhibitors, numerous Kunitz-BPTI (bovine pancreatic trypsin inhibitor) inhibitors have been described from larvae and eggs, named BmTIs. Among them, were characterized inhibitors for trypsin, human neutrophil elastase, human plasma kallikrein and plasmin. BmTIs elicited a protective immunological response against B. microplus infestation in cattle. However, only a small amount of purified natural BmTIs can be obtained from larvae and eggs by chromatographic methods, thus if BmTIs are to be used as vaccine antigens (immunogens) the production of recombinant BmTIs (rBmTIs) is essential. In this work we describe the cloning, expression, purification and characterization of rBmTI-6. rBmTI-6 is a three-headed Kunitz-BPTI inhibitor, expressed in the Pichia pastoris system. Although rBmTI-6 was processed by proteases and glycosylated during the expression process, these post-translational modifications did not alter the ability of rBmTI-6 to inhibit protease activity. Purified rBmTI-6 inhibited trypsin and plasmin.