Structure and quantitative determination of the major urinary metabolite of thromboxane B2 in the guinea pig.

2,3-Dinor-thromboxane B2 was the major urinary metabolite of thromboxane B2 in the guinea pig. The structure was assessed mainly by mass spectrometric analysis of a number of derivatives of the metabolite and by chemical degradation by oxidative ozonolysis. A method for quantitative determination of 2,3-dinor-thromboxane B2 in guinea pig urine based on multiple ion analysis and octadeuterated 2,3-dinor-thromboxane B2 as internal standard was developed. The basal excretion of the metabolite was 65 +/- 36 (S.D.) ng/kg x 24 h (n = 19; range 19--140 ng). This level corresponded to an endogenous synthesis of 543 +/- 300 ng of TXB2. No increase in the excretion was seen after anaphylaxis, in contrast to what has earlier been reported for PGF2 alpha.     

Quantitation of mast cell heparin by flow cytofluorometry.

Mast cells can be automatically identified in a mixed cell population by flow cytofluorometry after Berberine sulphate staining. Volume specific counts of the total number of cells and number of mast cells, as well as frequency distributions of fluorescence intensities of mast cells, based on a large number of cells, can be rapidly obtained. Results obtained by microscope fluorometry of cells identified by phase contrast microscopy showviously published results it may be inferred that the fluorescence intensity of individual mast cells is proportional to mast cell heparin content. The automated cell counts correlated very well with manual hemocytometer counts. Both cell counts and the determination of mean mast cell fluorescence showed excellent reproducibility.     

How do macrophytes growing in or close to water reduce their consumption by aquatic herbivores?

Larvae of Potamophylax cingulatus (Steph.) (Trichoptera) which are polyphagous herbi- and detritivorous, were used as test organism to investigate the palatability of aquatic and terrestrial macrophytes. In 7 out of 9 plant pairs the consumption rate was low on the species growing in or close to water. The consumption rate was negatively correlated with the nitrogen content of the plant. It is argued that the majority of the aquatic plants produce secondary plant substances, which reduce the attacks by aquatic herbivores. A restricted distribution, the long time of exposure to herbivores, as well as the relatively large probability of being discovered at the border of an inhabitable area, are factors suggested to have influenced the development of chemical defence mechanisms in aquatic macrophytes.     

Factors Controlling the Stable Nitrogen Isotopic Composition (δ15N) of Lipids in Marine Animals

Lipid extraction of biomass prior to stable isotope analysis is known to cause variable changes in the stable nitrogen isotopic composition (δ¹⁵N) of residual biomass. However, the underlying factors causing these changes are not yet clear. Here we address this issue by comparing the δ¹⁵N of bulk and residual biomass of several marine animal tissues (fish, crab, cockle, oyster, and polychaete), as well as the δ¹⁵N of the extracted lipids. As observed previously, lipid extraction led to a variable offset in δ¹⁵N of biomass (differences ranging from -2.3 to +1.8 ‰). Importantly, the total lipid extract (TLE) was highly depleted in ¹⁵N compared to bulk biomass, and also highly variable (differences ranging from -14 to +0.7 ‰). The TLE consisted mainly of phosphatidylcholines, a group of lipids with one nitrogen atom in the headgroup. To elucidate the cause for the ¹⁵N-depletion in the TLE, the δ¹⁵N of amino acids was determined, including serine because it is one of the main sources of nitrogen to N-containing lipids. Serine δ¹⁵N values differed by -7 to +2 ‰ from bulk biomass δ¹⁵N, and correlated well with the ¹⁵N depletion in TLEs. On average, serine was less depleted (-3‰) than the TLE (-7 ‰), possibly due to fractionation during biosynthesis of N-containing headgroups, or that other nitrogen-containing compounds, such as urea and choline, or recycled nitrogen contribute to the nitrogen isotopic composition of the TLE. The depletion in ¹⁵N of the TLE relative to biomass increased with the trophic level of the organisms.     

Antennal and behavioural responses of the spruce seed moth,Cydia strobilella,to floral volatiles of Norway spruce,Picea abies,and temporal variation in emission of active compounds

We investigated whether spruce seed moth, Cydia strobilella L. (Lepidoptera: Tortricidae, Grapholitini), one of the most damaging seed predators on Norway spruce, Picea abies (L.) H. Karst (Pinaceae), uses olfactory cues during host search. Analyses with coupled gas chromatography and electroantennography revealed that antennae of both sexes of moths responded consistently to three compounds in the headspace from female spruce flowers, i.e., α‐pinene, β‐pinene, and myrcene, but not to limonene as has been previously reported for this species. The amounts of these active volatiles released from flowers and cones of P. abies were quantified, and their diurnal and seasonal variation was monitored. The total release of the active volatiles correlated well with the diurnal and seasonal flight activity of C. strobilella as revealed by catches of males in pheromone‐baited traps. In field trapping experiments, where baits were loaded with proportions and enantiomeric ratios of α‐pinene, β‐pinene, and myrcene matching those of the female P. abies floral headspace, substantial catches of male C. strobilella were achieved, whereas few females were captured. These surprising results suggest that male C. strobilella make use of host volatiles to aid them in their search for females.     

Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose – an emerging prebiotic

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT‐29 cells increased three‐fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D‐DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress‐related protein increasing in a range of +2.1 ? +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to ?2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α‐galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β‐galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.     

Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines

The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly’s high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans.     

Assessing the taxonomic status of Osmoderma cristinae (Coleoptera: Scarabaeidae), endemic to Sicily,by genetic,morphological and pheromonal analyses

Resolving complexes of closely related and cryptic insect species can be challenging, especially when dealing with rare and protected taxa that are difficult to collect for genetic and morphological analyses. Until recently, populations of the genus Osmoderma (Scarabaeidae), widespread in Europe, were treated as a single species O. eremita (Scopoli, 1763) in spite of observed geographic variation in morphology. A previous survey using sequence data from the mtDNA cytochrome C oxidase I gene (COI) revealed the occurrence of at least two distinct lineages within this species complex: O. eremita in the west and O. barnabita Motschulsky, 1845, in the east. Interestingly, beetles confined to Sicily have been described as a distinct species, O. cristinae Sparacio, 1994, based on morphological traits. Only few Sicilian specimens were included in the former genetic analysis, and the results led to a still questionable taxonomic rank for these populations. To explore the robustness of the previous taxonomic arrangement for O. cristinae, a combination of genetic, morphological and pheromonal analyses was used. A 617‐bp fragment of the COI gene, aligned with O. cristinae and O. eremita sequences already available in GenBank, showed a clear genetic divergence between the two species (interspecific mean distance = 6.6%). Moreover, results from AFLP markers sustained the distinction of the two species. In addition, geometric morphometric analyses of the shape of male genitalia revealed a clear differentiation between the two species. Via scent analysis and field trapping, we demonstrated the production of the sex pheromone (R)‐(+)‐γ‐decalactone by males of O. cristinae, the attraction by conspecific individuals (mostly females) to this compound, and a lack of antagonistic effect of (S)‐(–)‐γ‐decalactone. The fact that O. eremita and O. eremita use the same compound for mate finding suggests that this sex pheromone has not undergone a differentiation and probably the allopatry of these two species compensates for the absence of a mechanism to avoid cross‐attraction. Our genetic and morphological data support the divergence of the two species and confirm the species status for O. cristinae, while sex pheromones are confirmed to be invariant among different species of the genus Osmoderma.     

Establishment of a Refined Oral Glucose Tolerance Test in Pigs,and Assessment of Insulin,Glucagon and Glucagon-Like Peptide-1 Responses

Diabetes mellitus is increasing worldwide and reliable animal models are important for progression of the research field. The pig is a commonly used large animal model in diabetes research and the present study aimed to refine a model for oral glucose tolerance test (OGTT) in young growing pigs, as well as describing intravenous glucose tolerance test (IVGTT) in the same age group. The refined porcine OGTT will reflect that used in children and adolescents. Eighteen pigs were obtained one week after weaning and trained for two weeks to bottle-feed glucose solution, mimicking the human OGTT. The pigs subsequently underwent OGTT (1.75 g/kg BW) and IVGTT (0.5 g/kg BW). Blood samples were collected from indwelling vein catheters for measurements of glucose and the diabetes related hormones insulin, glucagon and active glucagon-like peptide-1. The study confirmed that pigs can be trained to bottle-feed glucose dissolved in water and thereby undergo an OGTT more similar to the human standard OGTT than previously described methods in pigs. With the refined method for OGTT, oral intake only consists of glucose and water, which is an advantage over previously described methods in pigs where glucose is given together with feed which will affect glucose absorption. Patterns of hormonal secretion in response to oral and intravenous glucose were similar to those in humans; however, the pigs were more glucose tolerant with lower insulin levels than humans. In translational medicine, this refined OGTT and IVGTT methods provide important tools in diabetes research when pigs are used as models for children and adolescents in diabetes research.