Precursors to damascenone: synthesis and hydrolysis of isomeric 3,9-dihydroxymegastigma-4,6,7-trienes

A series of four isomeric 3,9-dihydroxymegastigma-4,6,7-trienes, 8, has been prepared. The (3S,6R,9S) isomer of 8 proved to be identical to an isomer of this compound tentatively identified as an intermediate in the formation of damascenone from an allene triol. Each of the four isomers, when hydrolyzed independently of each other at pH 3.0 and 25 degrees C, produced product mixtures in which the major product was damascenone (1). Contrary to expectation, 3-hydroxydamascone (5) was not observed in any of the hydrolyses. Consequently, the mechanism of formation of damascenone proposed earlier requires modification. In each hydrolysis, the product mixtures showed the presence of a second isomer of 8, produced by epimerization during hydrolysis. Chiral analysis on a Cyclosil B column revealed that this epimerization was occurring at C(3) in each of the hydrolyses.     

Determination of capsaicinoids in habanero peppers by chemometric analysis of UV spectral data

A novel spectrophotometric method for the determination of capsaicinoids in habanero pepper extracts is described that does not require prior analyte separation. The method uses partial-least-squares (PLS-1) multivariate regression modeling techniques in conjunction with ordinary UV absorption spectral data obtained on alcoholic extracts of habanero peppers (Capsicum chinese). The PLS-1 regression models were developed by correlating the known total concentration of the two major capsaicinoids (capsaicin and dihydrocapsaicin) in the extracts as determined by high-performance liquid chromatography with the spectral data. The regression models were subsequently validated with laboratory-prepared test sets. The validation studies revealed that the root-mean-square error of prediction varied from 4 to 8 ppm, based on the results obtained from models prepared from nine test sets. Once a regression model has been developed and validated, analyses of the extracts can be accomplished rapidly by ordinary spectrophotometric procedures without any prior separation steps.     

Phospholipid interactions protect the milk allergen alpha-lactalbumin from proteolysis during in vitro digestion

Interactions with food components may alter the resistance of food proteins to digestion, a property thought to play an important role in determining allergenic properties. The kinetics of breakdown of the bovine milk allergen alpha-lactalbumin during in vitro gastrointestinal digestion was found to be altered by interactions with physiologically relevant levels of phosphatidylcholine (PC), a surfactant that is abundant both in milk and is actively secreted by the stomach. Breakdown during gastric digestion was slowed in the presence of PC and accompanied by small alterations in the profile of resulting peptides, with little effect being observed during subsequent duodenal digestion. alpha-Lactalbumin was found to unfold at gastric (acid) pH, giving a CD spectrum similar to that obtained for the partially folded state it is known to adopt at pH values below its isoelectric point. Fluorescence polarization studies performed at low pH indicated that this partially unfolded form of the protein was able to penetrate into the PC vesicles. These interactions are probably responsible for the slowing of gastric digestion by reducing the accessibility of the protein to pepsin. These findings show that interactions with other food components, such as lipids, may alter the rate of breakdown of food proteins in the gastrointestinal tract. It underlines the importance of the food matrix in affecting patterns of food allergen digestion and hence presentation to the immune system and that in vitro digestion systems used for assessing digestibility of allergens must take account of surfactants.     

Parameters for specific detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers by multiplexed PCR-ELISA

A multiplex PCR-ELISA protocol for detection ofClavibacter michiganensis subsp.sepedonicus (Cms) was developed that is based on primers for amplification of three single-copy, unique DNA sequences, Cms50, Cms72, and Cms85. The three sequences were simultaneously amplified from the genomes of all 42 strains of Cms that were tested including variant mucoid forms, but not from strains representing five related subspecies, andRathayibacter rathayi andRhodococcus faciens. The lowest limit of detection by gel electrophoresis was estimated to be approximately 300 CFU per mL when cells were spiked into potato core fluid, but sensitivity increases approximately 10-fold using PCR-ELISA. Inclusion of a sea anemone DNA fragment engineered so it could be amplified from the Cms72 primer set provided the simultaneous signal that the system functioned properly when any sample was free of the pathogen. The addition of hydrolyzed casein to the reaction mix was demonstrated to markedly reduce or eliminate inhibition of PCR by plant cell components or contaminants. Multiplex PCR-ELISA detection of Cms was determined to be verifiable for analysis of both stems and tubers based on the amplification of multiple sites in its genome, it provides absolute specificity, and it was more sensitive than detection based on gel electrophoresis of PCR products and serological approaches.     

Genetic potential for residual feed intake and diet fed during early- to mid-gestation influences post-natal DNA methylation of imprinted genes in muscle and liver tissues in beef cattle

Discovery of epigenetic modifications associated with feed efficiency or other economically important traits would increase our understanding of the molecular mechanisms underlying these traits. In combination with known genetic markers, this would provide opportunity to improve genomic selection accuracy in cattle breeding programs. It would also allow cattle to be managed to improve favorable gene expression. The objective of this study was to identify variation in DNA methylation between beef cattle of differential pre-natal nutrition and divergent genetic potential for residual feed intake (RFI). Purebred Angus offspring with the genetic potential for either high (HRFI) or low (LRFI) RFI were prenatally exposed to either a restricted maternal diet of 0.5 kg/d average daily gain (ADG) or a moderate maternal diet of 0.7 kg/d ADG from 30 to 150 d of gestation. We performed DNA methylation analysis of differentially methylated regions (DMR) of imprinted genes (Insulin-like growth factor 2 (IGF2) DMR2, IGF2/H19 imprinting control region (ICR) and IGF2 receptor (IGF2R) DMR2) using post-natal samples of longissimus dorsi (LD) muscle taken from male and female calves at birth and weaning, and of LD muscle, semimembranosus (SM) muscle, and liver samples collected from steers at slaughter (17 months of age). Interestingly, for all three DMR investigated in liver, LRFI steers had higher levels of methylation than HRFI steers. In LD muscle, IGF2/H19 ICR methylation differences for heifers at birth were due to pre-natal diet, while for steers at birth they were mostly the result of genetic potential for RFI with LRFI steers again having higher levels of methylation than HRFI steers. While results from repeated measures analysis of DNA methylation in steers grouped by RFI revealed few differences, in steers grouped by diet, we found higher methylation levels of IGF2 DMR2 and IGF2R DMR2 in LD muscle of restricted diet steers at weaning and slaughter than at birth, as well as increased methylation in LD muscle of restricted diet steers compared with moderate diet steers at weaning and/or slaughter. Our results suggest that differential pre-natal nutrition, and divergent genetic potential for RFI, induces tissue- and sex-specific alterations in post-natal IGF2 and IGF2R methylation patterns and that these patterns can vary with age in Angus beef cattle.     

The importance of manual white blood cell differential counts and platelet estimates in elephant hematology: blood film review is essential

Unique features of elephant hematology are known challenges in analytical methodology like two types of monocytes typical for members of the Order Afrotheria and platelet counts of the comparatively small elephant platelet. To investigate WBC differential and platelet data generated by an impedance-based hematology analyzer without availability of validated species-specific software for recognition of elephant WBCs and platelets, compared to manual blood film review. Blood samples preserved in ethylenediaminetetraacetic acid (EDTA) of 50 elephants (n = 35 Elephas maximus and n = 15 Loxodonta africana) were used. A Mann-Whitney test for independent samples was used to compare parameters between methods and agreement was tested using Bland-Altman bias plots. All hematological variables, including absolute numbers of heterophils, lymphocytes, monocytes, eosinophils, basophils, and platelets, were significantly different (p < 0.0001) between both methods of analysis, and there was no agreement using Bland-Altman bias plots. Manual review consistently produced higher heterophil and monocyte counts as well as platelet estimates, while the automated analyzer produced higher lymphocyte, eosinophil, and basophil counts. The hematology analyzer did not properly differentiate elephant lymphocytes and monocytes, and did not accurately count elephant platelets. These findings emphasize the importance of manual blood film review as part of elephant complete blood counts in both clinical and research settings and as a basis for the development of hematological reference intervals.     

Pre-laying behaviour in battery cages

The pre-laying behaviour of hens from two strains of domestic fowls housed in battery cages was studied. Hens from one strain exhibited stereotyped pacing before laying, whereas hens from the other tended to sit during the pre-laying period. Hens from both strains performed vacuum nest-building behaviour before laying. Implications of these findings in relation to the improvement of animal welfare are discussed.