Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle

Peroxynitrite generated in arteries fromsuperoxide and nitric oxide (NO) may damage their function. Here, wecompare the effects of peroxynitrite and peroxynitrite/NO-generatingagents SIN-1 (3-morpholinosydnonimine hydrochloride), SNAP(S-nitroso-N-acetyl-penicillamine), SNP (sodiumnitroprusside), and NONOate (spermine NONOate) on pig coronary artery.Deendothelialized artery rings were pretreated with these agents andthen washed before examining their contractility. Pretreatment with allagents (200 µM) results in a decrease in the force of contraction inresponse to the sarco(endo)plasmic Ca²⁺ (SERCA) pumpinhibitor cyclopiazonic acid (CPA): SNAP > NONOate  peroxynitrite  SIN-1 > SNP. Pretreatment with SNAP,NONOate, or SIN-1 also inhibits the force of contraction produced with 30 mM KCl, with SNAP being the most potent. Including catalase plussuperoxide dismutase (SOD) during the preincubation has no effect. Including an NO scavenger[2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or a guanylate cyclase inhibitor(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of cultured cells with peroxynitrite, but not with SNAP, inhibits the Ca²⁺transients produced in response to CPA. Pretreating isolated membranevesicles with peroxynitrite inhibits the Ca²⁺ uptake due tothe SERCA pump, with all the other agents being less effective. Thusperoxynitrite and NO both inhibit the CPA-induced contractions indeendothelialized artery rings, peroxynitrite by damage to the SERCApump and NO possibly by a step downstream from the increase incytosolic Ca²⁺.

     

Novel inhibitors of influenza sialidases related to GG167 structure-activity, crystallographic and Molecular dynamics studies with 4H-pyran-2-carboxylic acid 6-carboxamides

The structure-activity relationships of a series of 4-amino and guanidino-4H-pyran-2-carboxylic acid 6-carboxamides are described. These compounds represent a new class of inhibitor of influenza sialidases and are particularly active against influenza A sialidase. The binding of the N-phenethyl-N-propylamide 41 to influenza A and B sialidases has been investigated using X-ray crystallography and molecular dynamics simulations. Our results suggest that formation of a hitherto unobserved intramolecular salt bridge within the enzymes may account for the observed activity and selectivity of the series.     

King penguin demography since the last glaciation inferred from genome-wide data

How natural climate cycles, such as past glacial/interglacial patterns, have shaped species distributions at the high-latitude regions of the Southern Hemisphere is still largely unclear. Here, we show how the post-glacial warming following the Last Glacial Maximum (ca 18 000 years ago), allowed the (re)colonization of the fragmented sub-Antarctic habitat by an upper-level marine predator, the king penguin Aptenodytes patagonicus. Using restriction site-associated DNA sequencing and standard mitochondrial data, we tested the behaviour of subsets of anonymous nuclear loci in inferring past demography through coalescent-based and allele frequency spectrum analyses. Our results show that the king penguin population breeding on Crozet archipelago steeply increased in size, closely following the Holocene warming recorded in the Epica Dome C ice core. The following population growth can be explained by a threshold model in which the ecological requirements of this species (year-round ice-free habitat for breeding and access to a major source of food such as the Antarctic Polar Front) were met on Crozet soon after the Pleistocene/Holocene climatic transition.     

Correlating early evolution of parasitic platyhelminths to Gondwana breakup

Investigating patterns and processes of parasite diversification over ancient geological periods should involve comparisons of host and parasite phylogenies in a biogeographic context. It has been shown previously that the geographical distribution of host-specific parasites of sarcopterygians was guided, from Palaeozoic to Cainozoic times, mostly by evolution and diversification of their freshwater hosts. Here, we propose phylogenies of neobatrachian frogs and their specific parasites (Platyhelminthes, Monogenea) to investigate coevolutionary processes and historical biogeography of polystomes and further discuss all the possible assumptions that may account for the early evolution of these parasites. Phylogenetic analyses of concatenated rRNA nuclear genes (18S and partial 28S) supplemented by cophylogenetic and biogeographic vicariance analyses reveal four main parasite lineages that can be ascribed to centers of diversity, namely Australia, India, Africa, and South America. In addition, the relationships among these biogeographical monophyletic groups, substantiated by molecular dating, reflect sequential origins during the breakup of Gondwana. The Australian polystome lineage may have been isolated during the first stages of the breakup, whereas the Indian lineage would have arisen after the complete separation of western and eastern Gondwanan components. Next, polystomes would have codiverged with hyloid sensu stricto and ranoid frog lineages before the completion of South American and African plate separation. Ultimately, they would have undergone an extensive diversification in South America when their ancestral host families diversified. Therefore, the presence of polystome parasites in specific anuran host clades and in discrete geographic areas reveals the importance of biogeographic vicariance in diversification processes and supports the occurrence and radiation of amphibians over ancient and recent geological periods.     

Comparison of release mechanisms for botanical oils to control Varroa destructor (Acari: Varroidae) and Acarapis woodi (acari: Tarsonemidae) in colonies of honey bees (Hymenoptera: Apidae)

Two major parasitic pests threaten honey bee populations, the external mite Varroa destructor and the internal mite Acarapis woodi (Rennie). Varroa are beginning to develop resistance to the main chemical defense fluvalinate, and alternative control methods are being pursued. Previous studies have shown that botanical oils, especially thymol, can be effective. Six release devices for either thymol or a blend of botanical oils known as Magic 3 were tested in beehives. The release devices were as follows: (1) low density polyethylene (LDPE) sleeves filled with Magic 3, (2) Magic 3-infused florist blocks, (3) thymol infused florist blocks, (4) a canola oil and thymol mixture wick release, (5) a plastic strip coated with calcium carbonate and Magic 3, and (6) an untreated control. There were significant decreases in varroa levels with the use of Magic 3 sleeves, but brood levels also decreased. Tracheal mite levels significantly decreased with the Magic 3 sleeve treatment, the Magic 3 florist block treatment, and the thymol canola wick treatment. A second experiment showed that changing the location of Magic 3 sleeves in the colony did not detrimentally effect brood levels, but also did not effectively control varroa mites.     

Survival of Mycobacterium avium subsp. paratuberculosis in dam water and sediment

In a previous longitudinal study, Mycobacterium avium subsp. paratuberculosis survived for 55 weeks in fecal material in the shade, but for much shorter periods in exposed locations. In this experiment, the survival of the organism was studied in 250 liters of dam water and sediment in large water troughs that were placed in either a semiexposed location or in a shaded location and compared to survival in fecal material and soil in the shaded location. Survival in water and/or sediment in the shade was for up to 48 weeks compared to 36 weeks in the semiexposed location. Survival in sediment was 12 to 26 weeks longer than survival in the water column. Survival in soil and fecal material in the terrestrial environment in the shaded location was only 12 weeks. Although disturbance to sediment could not be ruled out as a factor, there was evidence of dormancy in both the water column and the sediment, since the organism could not be recovered for several months before again becoming detectable. The results suggest that water may be a significant reservoir of M. avium subsp. paratuberculosis infection. Further research on the biology of the organism in aquatic environments is warranted. Animal health authorities will need to provide appropriate advice to farmers to minimize exposure of livestock to potentially infected water sources. Survival of the organism in water destined for human consumption will need to be addressed if the organism is found to be involved in the etiology of Crohn's disease.     

On-line screening of conformationally constrained nicotines and anabasines for agonist activity at the alpha3beta4- and alpha4beta2-nicotinic acetylcholine receptors using immobilized receptor-based liquid chromatographic stationary phases

Liquid chromatography columns containing stationary phases based upon immobilized nicotinic acetylcholine receptors (nAChRs) were used to screen a series of conformationally constrained nicotine and anabasine derivatives for agonist activity. The alpha3beta4 nAChR and alpha4beta2 nAChR subtypes were used to prepare the chromatographic columns and [(3)H] epibatidine dihydrochloride ([(3)H] EB) was used as the marker ligand. Single displacement experiments were conducted with the test ligands and with nicotine and carbachol. Nicotine was used as an internal control for compounds with agonist activity and carbachol was used as an internal control for compounds with very weak agonistic activity (K(d) > 4700 nM for alpha3beta4). The displacement of [(3)H] EB by each of the test compounds and internal controls was calculated and expressed as Deltaml. Functional studies were then conducted using a stably transfected cell line that expresses the alpha3beta4 nAChR and EC(50) values were determined for the test compounds and the internal controls. A comparison of the Deltaml and EC(50) values indicated that 9/11 compounds had been correctly identified as agonists or non-agonists of the alpha3beta4 nAChR. A similar comparison could not be made for the alpha4beta2 nAChR, since the intact cell line was not available for testing. The results of the study suggest that the immobilized nAChR columns can be used for the rapid on-line screening of compounds for their relative affinities for the immobilized receptor and as an initial determination of qualitative functional activities.     

Cytochrome P450IIE1 (CYP2E1) is induced by skatole and this induction is blocked by androstenone in isolated pig hepatocytes

Skatole, a derivative of tryptophan, is produced in the hind-gut of pigs and is metabolised via hepatic cytochrome P4502E1 (CYP2E1). Excessive accumulation of skatole together with androstenone, a metabolite of testosterone, in adipose tissue in some pigs is a major cause of 'boar taint' and is associated with defective expression of CYP2E1. This phenomenon is not understood because factors regulating CYP2E1 expression in pig liver have not yet been characterised. Therefore effects of skatole and androstenone on CYP2E1 expression were studied using isolated pig hepatocytes as a model system. Skatole induced CYP2E1 protein expression to the same degree as did acetone, a known CYP2E1 inducer. Induction by skatole was maximum between 20 and 28 h and a half-maximum effect was obtained at a skatole concentration of 0.2 mM. Induction of CYP2E1 by skatole was protein-synthesis dependent. Androstenone antagonised the effect of skatole on CYP2E1 expression but did not affect the CYP2E1 protein level when added alone. These results suggest that defective expression of CYP2E1 in some pigs is due to excessive concentrations of androstenone which prevent CYP2E1 induction by its substrate skatole. As a result, skatole metabolism is reduced and skatole is accumulated in adipose tissue.