Tylosin detection in animal feed by liquid chromatography-tandem mass spectrometry with enzymatic hydrolysis of the tylosin urea adduct

When the use of tylosin as a feed additive was forbidden by Council Regulation 2821/98, the necessity of a chemical confirmation method for the monitoring of the ban was created. Recently a method was developed for the detection of tylosin in animal feed by means of LC-MS/MS. During the validation high deviating values for the decision limit, detection capability, and repeatability for tylosin in cattle feed were observed, and the presence of urea and the formation of a tylosin urea adduct (TUA) were suggested as possible explanations. In this study two hydrolysis approaches for the TUA adduct were compared, namely, a chemical hydrolysis and an enzymatic hydrolysis with urease. The latter yielded a more complete hydrolysis of urea and was used for further validation. The recovery increased by approximately 15-25% depending on the amount of urea present in the feed (0.5-2%). The decision limit and detection capability were hardly influenced by the enzymatic hydrolysis.     

Estimation of quantitative oxidation and fat retention from carbohydrate,protein and fat in growing pigs

Die Oxidation von Nährstoffen und Retention von Kohlenhydraten, Protein und Fett hei wachsenden Schweinen Die Oxidation von Nährstoffen sowie deren Beitrag zur Fettretention wurde an Hand von 185 individuellen Bilanzstudien mit wachsenden Schweinen (40–100 kg Lebendgewicht) beurteilt. Der Respirationskoeffizient ohne Berücksichtigung des Proteins (RQnp) lag entweder unter oder über 1. Die beschriebene Methode basierend auf Gasaustausch sowie Kohlenstoff-Stickstoffbilanz zur Berechnung der Oxidation von Nährstoffen und Lipogenese ist in beiden Fällen gültig (RQnp > 1 und RQnp < 1). Bei einer den Wachstumsbedarf deckenden Energiezufuhr (ME ≥ 1,2 MJ/kg^0.75) ist die Oxidation von Nährstoffen im Bezug auf die Proteinretention abhängig von der Menge und Qualität des verdauten Proteins. Weiterhin besteht eine Abhängigkeit von der Menge an verdaulichen Kohlenhydraten. Die Versorgung mit Fett spielt dagegen keine Rolle. Die Oxidation von Kohlenhydraten trägt mit 85%, die von Protein mit 15% zur anfallenden Wärmeenergie bei. Eine Fettoxidation findet auch bei hohem Fettgehalt der Nahrung nicht statt. Die Lipogenese aus Kohlenhydraten stellt die Hauptquelle für die Körperfettretention dar. Da keine Fettoxidation stattfindet, wird das gesamte Nahrungsfett im Körper reteniert. Bei einer für das Wachstum zu geringen Energiezufuhr werden Nahrungsfett und Körperfett oxidiert. Um Protein für die Retention zu erhalten, ist die Oxidation von Protein vermindert. Selbst bei Mobilisierung von Körperfett und Oxidation von Nahrungsfett wird ein Teil der Kohlenhydrate und des Proteins für die Lipogenese vermutlich zum Aufbau von Strukturfett verwendet.     

Response rates and survival times for cats with lymphoma treated with the University of Wisconsin-Madison chemotherapy protocol: 38 cases (1996-2003)

OBJECTIVE: To determine response rates and survival times for cats with lymphoma treated with the University of Wisconsin-Madison chemotherapy protocol. DESIGN: Retrospective study. ANIMALS: 38 cats with lymphoma. PROCEDURE: Medical records were reviewed, and information on age, sex, breed, FeLV and FIV infection status, anatomic form, clinical stage, and survival time was obtained. Immunophenotyping was not performed. RESULTS: Mean +/- SD age of the cats was 10.9 +/- 4.4 years. Overall median survival time was 210 days (interquartile range, 90 to 657 days), and overall duration of first remission was 156 days (interquartile range, 87 to 316 days). Age, sex, anatomic form, and clinical stage were not significantly associated with duration of first remission or survival time. Eighteen of the 38 (47%) cats had complete remission, 14 (37%) had partial remission, and 6 (16%) had no response. Duration of first remission was significantly longer for cats with complete remission (654 days) than for cats with partial remission (114 days). Median survival time for cats with complete remission (654 days) was significantly longer than median survival time for cats with partial remission (122 days) and for cats with no response (11 days). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that a high percentage of cats with lymphoma will respond to treatment with the University of Wisconsin-Madison chemotherapy protocol. Age, sex, anatomic form, and clinical stage were not significantly associated with duration of first response or survival time, but initial response to treatment was.     

Marked induction of IL-6, haptoglobin and IFNgamma following experimental BRSV infection in young calves

Bovine respiratory syncytial virus (BRSV) has been identified worldwide as an important pathogen associated with acute respiratory disease in calves. An infection model has been developed reflecting accurately the clinical course and the development of pathological signs during a natural BRSV-infection. In the experiments described in the present study, calves were infected at 13-21 weeks of age and reinfected 14 weeks later. Blood samples from the entire infection period were analysed for acute phase protein (haptoglobin) by ELISA and for expression (mRNA level in peripheral blood mononuclear cells) of the cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-gamma (IFNgamma) by quantitative real-time reverse transcribed polymerase chain reaction (RT-PCR). IFNgamma, interleukin-6 and haptoglobin were markedly induced together with development of clinical signs in response to the first infection with BRSV. The IFNgamma response was biphasic, with an early peak at day 1-3 post infection (p.i.) and a later increase between day 5 and 8 p.i. Reinfection also resulted in an induction of IFNgamma, but without induction of clinical signs, IL-6 and haptoglobin. These results indicate that early mediators connected with the innate responses are induced on a first encounter with the pathogen, but not on a second encounter (reinfection) where the adaptive immune system may act as the first line defence.     

The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions

Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.     

Purified Brominated Indole Derivatives from Dicathais orbita Induce Apoptosis and Cell Cycle Arrest in Colorectal Cancer Cell Lines

Dicathais orbita is a large Australian marine gastropod known to produce bioactive compounds with anticancer properties. In this research, we used bioassay guided fractionation from the egg mass extract of D. orbita using flash column chromatography and identified fractions containing tyrindoleninone and 6-bromoisatin as the most active against colon cancer cells HT29 and Caco-2. Liquid chromatography coupled with mass spectrometry (LCMS) and ¹H NMR were used to characterize the purity and chemical composition of the isolated compounds. An MTT assay was used to determine effects on cell viability. Necrosis and apoptosis induction using caspase/LDH assay and flow cytometry (PI/Annexin-V) and cell cycle analysis were also investigated. Our results show that semi-purified 6-bromoisatin had the highest anti-cancer activity by inhibiting cell viability (IC₅₀ = ~100 µM) and increasing caspase 3/7 activity in both of the cell lines at low concentration. The fraction containing 6-bromoisatin induced 77.6% apoptosis and arrested 25.7% of the cells in G2/M phase of cell cycle in HT29 cells. Tyrindoleninone was less potent but significantly decreased the viability of HT29 cells at IC₅₀ = 390 µM and induced apoptosis at 195 µM by increasing caspase 3/7 activity in these cells. This research will facilitate the development of these molluscan natural products as novel complementary medicines for colorectal cancer.     

Nitrous oxide emissions from fertilized,irrigated cotton (Gossypium hirsutum L.) in the Aral Sea Basin,Uzbekistan: Influence of nitrogen applications and irrigation practices

Nitrous oxide emissions were monitored at three sites over a 2-year period in irrigated cotton fields in Khorezm, Uzbekistan, a region located in the arid deserts of the Aral Sea Basin. The fields were managed using different fertilizer management strategies and irrigation water regimes. N₂O emissions varied widely between years, within 1 year throughout the vegetation season, and between the sites. The amount of irrigation water applied, the amount and type of N fertilizer used, and topsoil temperature had the greatest effect on these emissions.Very high N₂O emissions of up to 3000 μg N₂O-N m^?2 h^?1 were measured in periods following N-fertilizer application in combination with irrigation events. These “emission pulses” accounted for 80–95% of the total N₂O emissions between April and September and varied from 0.9 to 6.5 kg N₂O-N ha^?1.. Emission factors (EF), uncorrected for background emission, ranged from 0.4% to 2.6% of total N applied, corresponding to an average EF of 1.48% of applied N fertilizer lost as N₂O-N. This is in line with the default global average value of 1.25% of applied N used in calculations of N₂O emissions by the Intergovernmental Panel on Climate Change.During the emission pulses, which were triggered by high soil moisture and high availability of mineral N, a clear diurnal pattern of N₂O emissions was observed, driven by daily changes in topsoil temperature. For these periods, air sampling from 8:00 to 10:00 and from 18:00 to 20:00 was found to best represent the mean daily N₂O flux rates. The wet topsoil conditions caused by irrigation favored the production of N₂O from NO₃^? fertilizers, but not from NH₄⁺ fertilizers, thus indicating that denitrification was the main process causing N₂O emissions. It is therefore argued that there is scope for reducing N₂O emission from irrigated cotton production; i.e. through the exclusive use of NH₄⁺ fertilizers. Advanced application and irrigation techniques such as subsurface fertilizer application, drip irrigation and fertigation may also minimize N₂O emission from this regionally dominant agro-ecosystem.     

Frost response in wheat and early detection using proximal sensors

Frost can significantly reduce production of field crops grown in Mediterranean‐type environments, where significant economic losses for Australian wheat occur annually. If non‐destructive sensors could make rapid, spatial assessment of frost damage, this could limit economic losses through timely management decisions. This paper reports on a methodology for imposing frost treatments to wheat under field conditions and the utility of canopy reflectance data for detecting early crop damage. Purpose‐built chambers using stepped additions of dry ice allowed for a range of frost scenarios to be tested when applied at wheat heading and anthesis. For frost treatments applied at anthesis, grain number and yield were reduced by 8.8% and 7.2%, respectively, for every degree Celsius below zero (down to ?4°C). This effect was additive over two consecutive nights. For cold load equivalent, there was a 2.2% and 1.9% reduction in grain number and yield, respectively, per °C hr (below 0°C). For wheat, spectral indices PRI and NDVI (reflectance) and FRF_G and SFR_G (fluorescence) showed significant relationships, with cold load applied for heading treatments. Next steps include targeting frost intensities equivalent to cold loads between 20 and 80°C hr and testing the utility of these proposed indices in a commercial paddock setting.