Measurement of progesterone in sheep using a commercial ELISA kit for human plasma

Determination of serum or plasma progesterone (P4) concentrations is important to recognize pregnant and non-pregnant ewes, and also to predict the number of carried lambs. The 2 most common methodologies for the detection of plasma P4 are radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is very expensive, and not all laboratories are equipped to perform this test; EIA is commercially available for human use, but only a few companies produce species-specific kits, which are expensive. We verified for ovine plasma a less expensive and easily available ELISA kit (DiaMetra) designed to quantify P4 in humans. Pools of ovine and human plasma were used to compare repeatability, accuracy, sensitivity, and stability of P4 measured by the DiaMetra kit. Repeatability data were within 15%, and accuracy values were ~90% for both plasma matrices. Stability data showed a loss of <20% for freeze–thaw and <30% for 30-d storage. All parameters were acceptable under international guidelines for method validation. The human ELISA kit was used successfully to quantify plasma P4 in 26 ewes during pregnancy until delivery. P4 concentrations were also correlated with the number of carried lambs.     

Assessment of tissue culture-derived ‘Gala’ and ‘Royal Gala’ apples (Malus × domestica Borkh.) for somaclonal variation

To assess somaclonal variation, ‘Gala’ and ‘Royal Gala’ trees obtained via axillary and adventitious bud formation were compared ex vitro to conventionally grafted trees. In general, tissue culture-derived trees were relatively erect in comparison to grafted trees. Their branch angles were narrower than those of grafted trees. All trees that flowered had pink blossoms. There were no obvious differences in flowering time or in floral morphology. Most of the seven-year-old grafted control trees produced more fruits than either axillary or regenerated trees. Although there were differences in the range of fruit color between ‘Royal Gala’ and ‘Gala’ apples in both the control and tissue culture-derived plants (the fruits of ‘Royal Gala’ were darker red and more striped than those of ‘Gala’) and also in the degree of pigmentation from tree-to-tree, none of the variation exceeded that observed among apples harvested from an individual ‘Royal Gala’ or ‘Gala’ control tree for either the plants derived from axillary buds or adventitiously. Since both ‘Gala’ and ‘Royal Gala’ axillary buds showed very little somaclonal variation for the morphological and reproductive traits we studied, it appears that tissue culture may be a useful way to propagate these cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schr?t, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.     

Proximal Digital Annular Ligament Desmotomy in Horses: A Cadaveric Study

Proximal digital annular ligament (PDAL) desmitis occurs in horses for which PDAL desmotomy is the only reported treatment. The presented work aims to develop a technique for safely performing PDAL desmotomy in horses. Twenty hind limbs and 13 forelimbs, harvested from horses dead or euthanized for reasons unrelated to any structure in the pastern region, were used. All horses were free of disease at the level of their PDAL. Proximal digital annular ligament desmotomy was performed using a minimally invasive approach to the digital flexor tendon sheath between the palmar (plantar) annular ligament and the PDAL, using a medial or a lateral access. Limbs were immediately dissected after the procedure to assess the completeness of the PDAL desmotomy and any possible damage to surrounding structures. The PDAL was successfully transected in all limbs except one. Damage to the surrounding tissue included transection of the palmar digital nerve (n = 1), longitudinal tear of the superficial digital flexor tendon (n = 3), and transection of the ligament of the ergot (n = 21). A medial approach was found to be safer than a lateral approach. In conclusion, PDAL desmotomy can be performed in a safe, repeatable, and technically easy way. This is, to our knowledge, the first report of a standardized technique for performing a PDAL desmotomy in the horse.     

Perfrin,a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis

Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.     

Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS

Calorie restriction extends life span in organisms ranging from yeast to mammals. Here, we report that calorie restriction for either 3 or 12 months induced endothelial nitric oxide synthase (eNOS) expression and 3',5'-cyclic guanosine monophosphate formation in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and adenosine triphosphate production, and an enhanced expression of sirtuin 1. These effects were strongly attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.     

Stress-induced mutagenesis in bacteria

The evolutionary significance of stress-induced mutagenesis was evaluated by studying mutagenesis in aging colonies (MAC) of Escherichia coli natural isolates. A large fraction of isolates exhibited a strong MAC, and the high MAC variability reflected the diversity of selective pressures in ecological niches. MAC depends on starvation, oxygen, and RpoS and adenosine 3',5'-monophosphate regulons; thus it may be a by-product of genetic strategies for improving survival under stress. MAC could also be selected through beneficial mutations that it generates, as shown by computer modeling and the patterns of stress-inducible and constitutive mutagenesis. We suggest that irrespective of the causes of their emergence, stress-induced mutations participate in adaptive evolution.