Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.     

Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission

Herbivore feeding elicits defense responses in infested plants, including the emission of volatile organic compounds that can serve as indirect defense signals. Until now, the contribution of plant tissue wounding during the feeding process in the elicitation of defense responses has not been clear. For example, in lima bean (Phaseolus lunatus), the composition of the volatiles induced by both the insect caterpillar Spodoptera littoralis and the snail Cepaea hortensis is very similar. Thus, a mechanical caterpillar, MecWorm, has been designed and used in this study, which very closely resembles the herbivore-caused tissue damage in terms of similar physical appearance and long-lasting wounding period on defined leaf areas. This mode of treatment was sufficient to induce the emission of a volatile organic compound blend qualitatively similar to that as known from real herbivore feeding, although there were significant quantitative differences for a number of compounds. Moreover, both the duration and the area that has been mechanically damaged contribute to the induction of the whole volatile response. Based on those two parameters, time and area, which can replace each other to some extent, a damage level can be defined. That damage level exhibits a close linear relationship with the accumulation of fatty acid-derived volatiles and monoterpenes, while other terpenoid volatiles and methyl salicylate respond in a nonlinear manner. The results strongly suggest that the impact of mechanical wounding on the induction of defense responses during herbivore feeding was until now underestimated. Controlled and reproducible mechanical damage that strongly resembles the insect's feeding process represents a valuable tool for analyzing the role of the various signals involved in the induction of plant defense reactions against herbivory.     

Anionic Phospholipid Interactions of the Prion Protein N Terminus Are Minimally Perturbing and Not Driven Solely by the Octapeptide Repeat Domain

Although the N terminus of the prion protein (PrP^C) has been shown to directly associate with lipid membranes, the precise determinants, biophysical basis, and functional implications of such binding, particularly in relation to endogenously occurring fragments, are unresolved. To better understand these issues, we studied a range of synthetic peptides: specifically those equating to the N1 (residues 23–110) and N2 (23–89) fragments derived from constitutive processing of PrP^C and including those representing arbitrarily defined component domains of the N terminus of mouse prion protein. Utilizing more physiologically relevant large unilamellar vesicles, fluorescence studies at synaptosomal pH (7.4) showed absent binding of all peptides to lipids containing the zwitterionic headgroup phosphatidylcholine and mixtures containing the anionic headgroups phosphatidylglycerol or phosphatidylserine. At pH 5, typical of early endosomes, quartz crystal microbalance with dissipation showed the highest affinity binding occurred with N1 and N2, selective for anionic lipid species. Of particular note, the absence of binding by individual peptides representing component domains underscored the importance of the combination of the octapeptide repeat and the N-terminal polybasic regions for effective membrane interaction. In addition, using quartz crystal microbalance with dissipation and solid-state NMR, we characterized for the first time that both N1 and N2 deeply insert into the lipid bilayer with minimal disruption. Potential functional implications related to cellular stress responses are discussed.     

Volatiles modulate the development of plant pathogenic rust fungi

Rust fungi are obligate biotrophic pathogens that differentiate a series of specialized cells to establish infection. One of these cells, the haustorium, which serves to absorb nutrients from living host cells, normally develops only in planta. Here, we show that the rust fungus Uromyces fabae (Pers.) Schroet. stimulates volatile emission of its host, broad bean (Vicia faba L.). Volatiles were identified and shown to be perceived by the fungus in in vitro assays that excluded the host. Three of them, nonanal, decanal, and hexenyl acetate promoted the development of haustoria on artificial membranes. In contrast, the terpenoid farnesyl acetate suppressed this differentiation. In assays using whole plants, farnesyl acetate reduced rust disease not only on broad bean but also on several cereals and legumes including soybean. This natural substance was effective against all rusts tested when directly applied to the host. This demonstrated that farnesyl acetate may serve as a powerful novel tool to combat rust fungi including Phakopsora pachyrhizi that currently threatens the production of soybeans world-wide.     

Membrane interactions of antimicrobial peptides from Australian tree frogs

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic alpha-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.     

Profiling of structurally labile oxylipins in plants by in situ derivatization with pentafluorobenzyl hydroxylamine

A GC-MS-based method for the simultaneous quantification of common oxylipins along with labile and highly reactive compounds based on in situ derivatization with pentafluorobenzyl hydroxylamine to the corresponding O-2,3,4,5,6-pentafluorobenzyl oximes (PFB oximes) is presented. The approach covers oxo derivatives such as jasmonic acid (JA), 12-oxophytodienoic acid (OPDA), certain phytoprostanes, unsaturated oxo-acids, oxo-hydroxy acids, and aldehyde fragments from the polar head of fatty acids. In the positive electron impact-MS mode, the PFB oximes display characteristic fragment ions that greatly facilitate the identification of oxylipins in complex matrices. In addition, the fluorinated derivatives allow a highly selective and low-background analysis by negative chemical ionization. Besides showing the general value of the method for the identification of a broad range of oxylipins (18 examples), we also demonstrate sensitivity, linearity, and reproducibility for the quantification of JA, OPDA, 11-oxo-9-undecenoic acid, and 13-oxo-9,11-tridecadienoic acid. The efficiency of the method is demonstrated by differential profiling of these four oxylipins in lima bean leaves after mechanical wounding and feeding by the herbivore Spodoptera littoralis. Caterpillar feeding induced several oxylipins, whereas after wounding only the level of JA increased. The rapid in situ derivatization prevents the isomerization of cis-JA to trans-JA. The resting level of JA in lima beans showed an isomer ratio of 80:20 for trans/cis-JA. After wounding, de novo synthesis of JA alters the ratio to 20:80 in favor of the cis isomer.     

NFκB pathway is down-regulated by 1α,25(OH)(2)-vitamin D(3) in endothelial cells transformed by Kaposi sarcoma-associated herpes virus G protein coupled receptor

We have previously demonstrated that 1α,25 dihydroxy-vitamin D(3) (1α,25(OH)(2)D(3)) has antiproliferative effects on the growth of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR). In this work, we have investigated whether 1α,25(OH)(2)D(3) exerts its growth inhibitory effects by inhibiting the Nuclear Factor κ B (NFκB) pathway which is highly activated by vGPCR. Cell proliferation studies demonstrated that 1α,25(OH)(2)D(3), similarly to bortezomib, a proteosome inhibitor that suppresses the activation of NFκB, reduced the proliferation of endothelial cells transformed by vGPCR (SVEC-vGPCR). The activity of NFκB in these cells decreased by 70% upon 1α,25(OH)(2)D(3) treatment. Furthermore, time and dose response studies showed that the hormone significantly decreased NFκB and increased IκBα mRNA and protein levels in SVEC-vGPCR cells, whereas in SVEC only IκBα increased significantly. Moreover, NFκB translocation to the nucleus was inhibited and occurred by a mechanism independent of NFκB association with vitamin D(3) receptor (VDR). 1α,25(OH)(2)D(3)-induced increase in IκBα required de novo protein synthesis, and was independent of MAPK and PI3K/Akt pathways. Altogether, these results suggest that down-regulation of the NFκB pathway is part of the mechanism involved in the antiproliferative effects of 1α,25(OH)(2)D(3) on endothelial cells transformed by vGPCR.     

Population structure of the butternut canker fungus,Ophiognomonia clavigignenti‐juglandacearum,in North American forests

The occurrence of multiple introduction events, or sudden emergence from a host jump, of forest pathogens may be an important factor in successful establishment in a novel environment or on a new host; however, few studies have focused on the introduction and emergence of fungal pathogens in forest ecosystems. While Ophiognomonia clavigignenti‐juglandacearum (Oc‐j), the butternut canker fungus, has caused range‐wide mortality of butternut trees in North America since its first observation in 1967, the history of its emergence and spread across the United States and Canada remains unresolved. Using 17 single nucleotide polymorphic loci, we investigated the genetic population structure of 101 isolates of Oc‐j from across North America. Clustering analysis revealed that the Oc‐j population in North America is made up of three differentiated genetic clusters of isolates, and these genetic clusters were found to have a strong clonal structure. These results, in combination with the geographic distribution of the populations, suggest that Oc‐j was introduced or has emerged in North America on more than one occasion, and these clonal lineages have since proliferated across much of the range of butternut. No evidence of genetic recombination was observed in the linkage analysis, and conservation of the distinct genetic clusters in regions where isolates from two or more genetic clusters are present, would indicate a very minimal or non‐existent role of sexual recombination in populations of Oc‐j in North America.