Biogenetic studies in Syringa vulgaris L.: synthesis and bioconversion of deuterium-labeled precursors into lilac aldehydes and lilac alcohols

Syringa vulgaris L. inflorescences were fed with aqueous solutions of regioselectively deuterated compounds assumed to be precursors of lilac aldehyde and lilac alcohol, respectively. Volatiles were extracted by stir bar sorptive extraction (SBSE) and analyzed using enantioselective multidimensional gas chromatography/mass spectrometry (enantio-MDGC/MS); deuterium-labeled lilac aldehydes and lilac alcohols were separated from unlabeled stereoisomers on a fused silica capillary column, coated with heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin (DIME-beta-CD) (30%) in SE 52 (70%), as the chiral stationary phase. Feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone 22 and [5,5-(2)H(2)]deoxy-d-xylose 23 indicate that the novel mevalonate independent 1-deoxy-d-xylose 5-phosphate/2C-methyl-d-erythritol 4-phosphate pathway is the dominant metabolic route for biosynthesis in lilac flowers. Additionally, bioconversion of deuterium-labeled d(5)-(R/S)-linalool 3, d(6)-(R)-linalool 21, d(5)-(R/S)-8-hydroxylinalool 6, d(5)-(R/S)-8-oxolinalool 7, d(5)-lilac aldehydes 8-11 and d(5)-lilac alcohols 12-15 into lilac during in vivo feeding experiments was investigated and the metabolic pathway is discussed. Incubation of petals with an aqueous solution of deuterated d(5)-(R/S)-linalool 3 indicates an autonomic terpene biosynthesis of lilac flavor compounds in the flower petals of lilac.     

Beschreibung des Verlaufs der Stickstoffaufnahme von Kohlrabi (Brassica oleracea convar. acephala var. gongylodes L.) mit Hilfe von Wachstumsstadien und eines empirischen Wachstumsmodells

Nitrogen uptake of kohlrabi, estimated by growth stages and an empirical growth model Some fertilizer recommendation systems for field vegetables are based on estimated nitrogen uptake curves. The estimation of nitrogen uptake should be as accurate as possible. Therefore 3 estimation methods and their accuracy (standard error of difference between measured and estimated values, sd) are compared. Comparisons were carried out with measured nitrogen uptake curves of kohlrabi (Brassica oleracea var. gongylodes L.) from 16 field experiments. The use of observed growth stages resulted in the smallest estimation error (sd = 14 kg N · ha^?1). This method is recommendable because it is accurate and easy to use. Estimations by applying an empirical nitrogen uptake model (sd = 16 kg N · ha^?1) are more complicated. The estimation based on time after planting is easy to use but showed the largest estimation error (sd = 28 kg N · ha^?1).     

Constituents in evening primrose oil with radical scavenging,cyclooxygenase, and neutrophil elastase inhibitory activities

Cold-pressed, non-raffinated evening primrose oil was found to contain lipophilic radical scavengers. A highly enriched fraction of these compounds could be obtained from the oil by extraction with aqueous ethanol and subsequent liquid-liquid partitioning with petroleum. LC-DAD-MS analysis revealed that the fraction contained three aromatic compounds with identical UV and ESI-MS spectra. The compounds were isolated by RP-HPLC and their structures established by chemical and spectroscopic means as 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid. The morolic acid derivative was a new compound. The three esters exhibited pronounced radical scavenging activity against the stable 2,2-diphenyl-1-picrylhydrazyl radical and were potent inhibitors of neutrophil elastase and cyclooxygenase-1 and -2 in vitro. Commercial samples of evening primrose oils contained only traces of these lipophilic antioxidants.     

Identification of transgenes from wood of genetically transformed poplar trees

We have extracted total DNA from different fractions of fresh wood as well as from cold-stored and air-dried wood harvested from transgenic aspen grown in the field. The highest amounts of DNA were obtained from bark/cambium tissue; the DNA quality, however, was poor. Best results in PCR and Southern blot analyses were obtained from DNA extracted from early wood. Using appropriate primer pairs, amplification products were obtained from both the foreign gene (transgene) and aspen genomic sequences. In Southern blot analyses transgene-specific hybridisation signals were obtained. This is the first report on the detection of foreign genes in wood sampled from genetically modified trees.     

Prevalence of Campylobacter spp. in poultry meat and raw milk using PCR, conventional cultural methods and Fourier transform infrared spectroscopy

The identification of thermotolerant campylobacters in official food control in the state of Baden-Wuerttemberg has been traditionally performed using the cultural procedure as described in the ISO-Norm 10272:1995. Analysis thus took 5-6 days to complete. Additionally diagnostic problems caused by the accompanying flora as well as the resistance to nalidixic acid occured. Within the scope of this study these problems could be solved by introducing a filtration step for the reduction of the accompanying flora and by performing the indoxyl acetate-hydrolysis-test in addition to the antibiotic-resistance-test. Besides various PCR protocols for the identification of thermotolerant campylobacters from food were established as an alternative to the cultural procedure, providing reliable results within two days. Furthermore, infrared spectroscopy was tested for the identification of Campylobacter isolates. Using this technique and with the help of a suitable data base, bacterial pure cultures can be differentiated within 2 hours. Among others 356 samples of raw poultry meat were tested with the newly established procedures as well as with the classical cultural method, showing that 32% of the samples were Campylobacter spp. positive. 37% of these isolates were resistant against nalidixic acid. This indicates that the development of resistances in Campylobacter spp. in Germany follows the same trend described for other European countries.     

Mechanism of wine lactone formation: demonstration of stereoselective cyclization and 1,3-hydride shift

The cyclization mechanism of (E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid to wine lactone under acidic aqueous conditions was investigated using the two stereoselectively deuterium-labeled precursors (2E,6R,7Z)-[8-2H]-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid and (2E,7E)-(+/-)-[8-2H]-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid. A detailed analysis of the generated wine lactone isomers by enantioselective multidimensional gas chromatography (MDGC)/ion trap tandem mass spectrometry demonstrates that the formation of wine lactone proceeds via a nonenzymatic stereoselective cationic cyclization cascade that includes a 1,3-hydride shift. Usually, such mechanisms are features of cyclization reactions that are catalyzed by terpene cyclases. This nonenzymatic conversion of an acyclic precursor to a bicyclic monoterpene under relevant cationic cyclization conditions has rarely been observed and confirms recent suggestions that the precursor itself can provide the chemical functionality required for specific steps in the cyclization cascade.     

Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status

To learn more about the molecular biology of Pasteurella multocida 289 strains isolated from various clinically healthy and diseased hosts were examined for capsule biosynthesis genes (capA, B, D, E, and F) and 14 virulence associated genes by PCR and DNA-DNA-hybridization. As expected, capsule type A strains were highly adapted to bovines (92.3%) and poultry (85.7%) while we mainly found capA (34.9%)- and capD (58.1%)-positive strains in swine. A noticeable amount of capD-positive strains also originated from small ruminants (34.9%) and capF was detected in wild type strains from diseased cattle (2.2%) and cats (7.4%). None of the isolates harboured capE, while capB was exclusively found in all strains from buffaloes. Nearly all isolates showed a combination of genes encoding outer membrane proteins, colonization factors, iron aquisition factors and superoxid-dismutases without any clue for host specificity. In contrast, the transferrin binding protein encoding gene tbpA (31.5%) was limited to ruminant strains and only 37.0% of all P. multocida strains harboured pfhA, coding for a filamentous hemagglutinin, supposed to be a putative adhesion- und serum resistance factor. PfhA revealed a strong positive association to the outcome of disease in bovine hosts and in combination with toxA to that in swine. The dermonecrotoxin encoding toxA, present in 12.5% of all strains, was detected in isolates from swine, small ruminants, cattle, and poultry. A significant association to the disease status, however, was only existent in swine, although with 66.7% we found a notably high prevalence of the toxin gene among strains from small ruminants. The genes toxA, tbpA and pfhA as well as capsule biosynthesis genes are supposed to be important epidemiological marker genes for characterizing P. multocida field strains.     

The impact of daily Duddingtonia flagrans application to lactating ewes on gastrointestinal nematodes infections in their lambs in the Netherlands

Two experiments were performed in 2002 and 2003 to evaluate the effect of biological control of gastrointestinal nematodes in sheep through the daily feeding of 500,000 chlamydospores of Duddingtonia flagrans/kg bodyweight to lactating ewes during the first 9 weeks with their young lambs on pasture. In both experiments four groups of eight ewes and their April-borne lambs were used. They were turned out on four separate plots (plots A) at the beginning of May, moved to similar separate plots after 3 (plots B) and 6 weeks (plots C), respectively, and weaning occurred after 9 weeks. In both experiments, two groups were fed spores daily while the two other groups served as controls. The effect of D. flagrans application was evaluated through faecal egg counts of ewes and lambs, the yield of faecal cultures in ewes, pasture larval counts and worm counts of lambs and tracer lambs. The results demonstrated no effect of D. flagrans application during the first 5 (2002) or 4 (2003) weeks. Subsequently, fungus application strongly reduced the yield in faecal cultures of the ewes. This was, however, not reflected in the pasture larval counts, but lower worm burdens were observed in tracer lambs of 'treated' plots C in 2002 than on those of 'control' plots. In 2003 worm burdens in 'treated' lambs returned to plots B were lower than those of 'control' lambs and a tendency for the same was observed for plots C. However, in all groups, lambs and tracer lambs developed severe haemonchosis.     

Simple prediction of the survival of follicles in cryopreserved human ovarian tissue

This study examines the possible predictive value of the LIVE/DEAD fluorescence viability assay for evaluation of survival of cryopreserved human ovarian tissue. Ovarian tissue from ten patients was examined by LIVE/DEAD viability staining before and after cryopreservation and after freezing in a -20 C freezer (negative control). After cryopreservation with a slow freezing protocol and cryoprotectant the LIVE/DEAD assay showed 86% viable follicles (an intact oocyte and at least more than 50% of the granulosa cells alive), whereas after freezing at -20 C the survival rate was 67%. The healthy follicular loss after cryopreservation was 4%, whereas with freezing at -20 C, it was 25%. Although this assay overestimates the survival rate of cryopreserved primordial follicles, if the LIVE/DEAD assay yields greater than approximately 85% viable follicles, it can be assumed that the follicles in the cryopreserved tissue have maintained their developmental potential and that the tissue is suitable for retransplantation.