Comparison of D-alanine and diaminopimelic acid as bacterial markers in young calves

D-alanine (DAL) and diaminopimelic acid (DAP) were compared as markers to estimate proportion of bacterial N in total N reaching the abomasum of young calves. Sixteen Holstein bull calves fed complete pelleted starter or unpelleted starter plus hay and weaned at 4 or 8 wk of age were fitted with ruminal and abomasal cannulas and sampled twice weekly from 2 to 11 wk of age. Isolated ruminal bacterial cells contained more DAL than DAP at all weeks and averaged 7.0 and 5.4 mg N/g N, respectively. Weekly mean marker concentrations were highly correlated (.89) in ruminal bacteria, except at 3 wk of age. Concentration of DAL in abomasal digesta was greater than that of DAP at all weeks and averaged 5.2 and 2.4 mg N/g N, respectively. Weekly mean DAL correlated with DAP .61 in abomasal digesta and correlated .57 and .89 with starter intake, respectively. The proportion of bacterial N in total abomasal N was greater at all weeks when estimated by DAL than by DAP and averaged 77% and 46%, respectively. Estimates by DAL exceeded 100% in several cases and reflected large variation in analytical estimates. Estimates by DAL and DAP correlated .33 and .92 with starter intake. D-alanine was not an acceptable bacterial marker in this study.     

Immunisation against ovine caseous lymphadenitis: efficacy of monocomponent Corynebacterium pseudotuberculosis toxoid vaccine and combined clostridial-corynebacterial vaccines

Sheep were immunised with Corynebacterium pseudotuberculosis toxoid formulated as a monocomponent vaccine with aluminium adjuvant or in combination with 5 clostridial antigens, and also in the combined form with sodium selenate. Immunised and control sheep were experimentally infected 16 days after vaccination and slaughtered and inspected after a further 3 months to determine their resistance to infection. All 3 vaccines afforded an equal and high level of protection; 91% of vaccinated sheep exhibiting no lesions of caseous lymphadenitis compared with 51.5% affected sheep in the control group. Average lesion counts were 1.2 per affected vaccinated sheep and 4.5 per affected control sheep. Antitoxin responses to the clostridial toxoids incorporated in the combined vaccines were not affected by inclusion of the C pseudotuberculosis toxoid or the sodium selenate.     

Immunisation against ovine caseous lymphadenitis: comparison of Corynebacterium pseudotuberculosis vaccines with and without bacterial cells

Sheep were immunised with Corynebacterium pseudotuberculosis vaccines prepared from cell-free toxoid or from toxoid with formalin-killed cells of C pseudotuberculosis added. Resistance of sheep to infection was tested 6 months after immunisation by inoculation with caseous lymphadenitis pus. The outcome was assessed 3 months later by slaughter and inspection of the sheep for lesions of caseous lymphadenitis. immunised sheep were adequately protected against infection as shown by a significant reduction in the number of sheep exhibiting lesions compared with control sheep, and by fewer abscesses in affected vaccinated sheep than in affected control sheep. The protective potency of the vaccines was not improved by the inclusion of cells of C pseudotuberculosis.     

Untersuchungen über den Einfluß von Gibberellinsäurespritzungen auf den Ertrag,die Anatomie der Wurzel und die Karotinverteilung bei Karotten

Investigations on the Influence of Gibberellic Acid Treatments on Yield, Root Anatomy and Carotene Distribution of Carrot Plants . Application of GA₃ onto the shoots of carrot plants increases shoot growth whereas root growth was reduced. Furthermore, due to the GA₃-application carotene content (Table 2) in the shoots was increased and decreased in the roots. Anatomical investigations of the roots revealed that apparently the reduction of root growth after GA₃-application was due to a decrease of phloem oriented cambial activity (Table 3).     

Functional characterization of enone oxidoreductases from strawberry and tomato fruit

Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase, is a ripening-induced, negatively auxin-regulated enzyme that catalyzes the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), the key flavor compound in strawberry fruit by the reduction of the alpha,beta-unsaturated bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF). Here we show that recombinant FaEO does not reduce the double bond of straight-chain 2-alkenals or 2-alkenones but rather hydrogenates previously unknown HMMF derivatives substituted at the methylene functional group. The furanones were prepared from 4-hydroxy-5-methyl-3(2H)-furanone with a number of aldehydes and a ketone. The kinetic data for the newly synthesized aroma-active substrates and products are similar to the values obtained for an enone oxidoreductase from Arabidopsis thaliana catalyzing the alpha,beta-hydrogenation of 2-alkenals. HMMF, the substrate of FaEO that is formed during strawberry fruit ripening, was also detected in tomato and pineapple fruit by HPLC-ESI-MSn and became 13C-labeled when d-[6-13C]-glucose was applied to the fruits, which suggested that a similar HDMF biosynthetic pathway occurs in the different plant species. With a database search (http://ted.bti.cornell.edu/ and http://genet.imb.uq.edu.au/Pineapple/), we identified a tomato and pineapple expressed sequence tag that shows significant homology to FaEO. Solanum lycopersicon EO (SlEO) was cloned from cDNA, and the protein was expressed in Escherichia coli and purified. Biochemical studies confirmed the involvement of SlEO in the biosynthesis of HDMF in tomato fruit.     

Large volume TENAX® extraction of the bioaccessible fraction of sediment-associated organic compounds for a subsequent effect-directed analysis

Background, Aims and Scope

Effect-directed analysis (EDA) is a powerful tool for the identification of key toxicants in complex environmental samples. In most cases, EDA is based on total extraction of organic contaminants, which may lead to an erroneous prioritisation with regard to hazard and risk. Bioaccessibility-directed extraction aims to discriminate between contaminants that take part in partitioning between sediment and biota in a relevant time frame and those that are enclosed in structures that do not allow rapid desorption. Standard protocols of targeted extraction of the rapidly desorbing, and thus bioaccessible, fraction using TENAX® are based only on small amounts of sediment. In order to obtain sufficient extract for subsequent biotesting, fractionation and structure elucidation, a large volume extraction technique needs to be developed applying one selected extraction time and excluding toxic procedural blanks.

Methods

Desorption behaviour of sediment contaminants was determined by combining consecutive extraction of sediment using TENAX® with a three-compartment desorption model. Time needed to remove the rapidly desorbing fraction, t_rap, was calculated to select a fixed extraction time for single extraction procedures. Up-scaling by about a factor of 125 provided a large volume extraction technique for EDA. Reproducibility and comparability to the small volume approach were analysed. TENAX® blanks and sediment extracts were tested for toxicity using Scenedesmus vacuolatus and Artemia salina as test organisms.

Results and Discussion

Desorption kinetics showed that 12 to 30% of sediment-associated pollutants were available for rapid desorption, while 70 to 90% of PAHs found in the sediment belong to the slowly and very slowly desorbing pool with very limited bioavailability. t_rap is compound dependent and covers a range of 2 to 18 h. A fixed extraction time of 24 h was selected as a time at which even the rapidly desorbing fraction of big hydrophobic compounds should be fully desorbed. High reproducibility of the large volume approach and good agreement with the small consecutive approach were found. Significant toxicity of procedural TENAX® blanks was found with Scenedesmus vacuolatus, which is in agreement with chemical analysis and could be reduced by pre-cleaning of TENAX® with Accelerated Solvent Extraction (ASE). Toxicity of blanks prior to ASE-clean up was about three orders of magnitude below the toxicity of sediment extracts.

Conclusions

For consideration of bioaccessibility in EDA, a large volume TENAX® extraction method was presented. Although several other solid phases can be used to extract the bioaccessible fraction, TENAX® has unique properties for depletive extraction of the rapidly desorbing fraction from large amounts of sediment. Toxicity and chemical blanks due to production residues are shortcomings of the method that can be overcome by accurate pre-cleaning, e.g. with ASE.

Recommendations and Perspectives

Higher purity of TENAX® guaranteed by the manufacturers would significantly enhance the applicability of the method. Using TENAX® instead of total extraction may improve key toxicant prioritisation by considering exposure and effect rather than effect only.

     

Development of serological ovine field test (SOFT) by modified agar-gel immunodiffusion

A serological ovine field test (SOFT) has been developed for detection of lamb or sheep tissue in a wide variety of raw meat products. The test is an adaptation of previously developed field screening immunodiffusion tests for beef, poultry, and pork detection. The SOFT test was demonstrated to be specific, sensitive, and accurate in the analysis of 104 samples.     

4-hydroxy-2,5-dimethyl-3(2H)-furanone formation by Zygosaccharomyces rouxii: effect of the medium

The formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) by Zygosaccharomyces rouxii was studied in yeast-peptone-dextrose medium containing d-fructose 1,6-diphosphate under various culture conditions. Cell growth and HDMF production was heavily dependent on medium pH and sodium chloride concentration. Higher pH values of the nutrient medium had a positive effect on HDMF formation but retarded cell growth resulting in an optimal pH value of 5.1 with regard to the yield of HDMF. Salt stress stimulated HDMF formation by Z. rouxii as increasing sodium chloride concentration led to higher amounts of HDMF. The HDMF concentration in the culture supernatant and HDMF formation per yeast cell peaked at 20% sodium chloride in the nutrient medium. The nonutilizable carbohydrate d-xylose displayed a weak effect on HDMF formation, and the addition of glycerol to salt-stressed cells had no effect on the production of HDMF.     

Chemical characterization of heavy-metal contaminated soil in southeast Kansas

A tri-state mining region, including parts of Missouri, Oklahoma, and Kansas, was the site of intense lead and zinc mining and smelting activity until the 1950's. A study was initiated to characterize the heavy-metal contamination of soils in this area. Water-soluble, an index of plantavailable, total, and sequentially extractable metals; organic, and total carbon; and saturated paste pH were determined for mine tailings and soil samples. Mine tailings contained 81 to 89 mg kg^?1 total Cd, 1 150 to 1 370 mg kg^?1 total Pb, and 11 400 to 13 700 mg kg^?1 total Zn. Total concentrations in soil samples were 15 to 86 mg kg^?1 Cd, 35 to 1 620 mg kg^?1 Pb, and 99 to 18 500 mg kg^?1 Zn; and, DTPA extractable concentrations ranged from 0.6 to 10 mg kg^?1 Cd, 7.8 to 68 mg kg^?1 Pb, and 33 to 715 mg kg^?1 Zn. Samples were sequentially extracted to approximate the proportions of the metals in the sulfide, carbonate, organic, sorbed, and exchangeable fractions. For Zn and Cd, concentrations were greatest in the sulfide fraction followed by carbonate, organic, sorbed, and exchangeable. Lead followed the same pattern, except higher concentrations were observed in the sorbed than the organic fractions.     

Microbial transformation of aliphatic aldehydes by Bacillus megaterium to 2,3-dialkylacroleins

The biotransformation of a series of aliphatic aldehydes (C(8)-C(12)) by Bacillus megaterium isolated from strawberry leaf surfaces was investigated. Products were isolated by liquid/liquid extraction and analyzed by gas chromatography (GC) combined with mass spectrometry (MS). In addition to aliphatic alcohols and the remaining aldehydes, major transformation products included the corresponding acids as well as 2,3-dialkylacroleins, dehydrated aldol addition products, which were detected for the first time as biotransformation products. To verify the structures, 2,3-dialkylacroleins were chemically synthesized from the appropriate aldehydes by base-catalyzed aldol condensation reactions and characterized by (1)H and (13)C NMR spectroscopy. Time-course studies showed that the maximum yield of the acrolein derivatives was obtained after 6 days of incubation.