Selective one-step extraction of Arabidopsis thaliana seed oleosins using organic solvents

Oleosins are hydrophobic proteins from oleaginous seeds, surrounding and stabilizing oil bodies. They are known to display interesting interfacial properties. Specific sera were raised against four different A. thaliana oleosins and used in dot-blot assays for oleosin quantification. These assays were used to set up extraction of oleosins from A. thaliana seeds. One mixture of chloroform/methanol gave optimal oleosin extraction. Extracted proteins represented 9% of seed proteins and were identified by immunoblot and proteomic analyses. Oleosins accounted for 79% of the extracted proteins. This simple one-step procedure allows selective extraction and concentration of oleosins from seeds without tedious oil body purification. Oleosin extract was indeed used to demonstrate the presence of the rare oleosin S5 in mature seeds. Moreover, this method will be useful to investigate the potential use of oleosins as emulsifier and to question their possible allergenicity.     

The detection of Chlamydophila psittaci genotype C infection in dogs

Reports of canine chlamydiosis are infrequent, possibly because the pathogen is rarely considered to be a cause of disease in dogs. This report presents details of Chlamydophila psittaci infection in four bitches with recurrent keratoconjunctivitis, severe respiratory distress and reduced litter size (up to 50% stillborn or non-viable puppies) in a small dog-breeding facility in Germany. Cell culture and immunofluorescence examination of conjunctival, nasal and pharyngeal swabs revealed chlamydial inclusions. PCR and sequencing of ompA amplification products confirmed the presence of Cp. psittaci genotype C. The zoonotic potential of the pathogen was illustrated by evidence of disease in two children that lived on the premises with the infected dogs. There was circumstantial evidence to suggest infection of dogs and humans may have followed the introduction of two canaries and a parrot to the household. The persistent nature of the chlamydial infection suggests that dogs may be reservoirs of Cp. psittaci, but this putative role and whether or not dogs shed the pathogen require further investigation.     

Soil organic carbon and nitrogen in aggregates in response to over seven decades of farmyard manure application

The study aimed to evaluate the effects of long-term fertilisation on soil aggregation and the associated changes in soil organic carbon (SOC) and nitrogen (N) pools in aggregates. The combined application of mineral fertiliser and manure improved soil aggregation, SOC and N content in aggregates, compared to manure or mineral fertiliser alone, and thus proved to be a suitable fertilisation strategy to increase C sequestration in agroecosystems.     

Comparison of soil phosphorus extraction methods regarding their suitability for organic farming systems

Background

Organic farmers frequently report sufficient yield levels despite low or even very low soil phosphorous (P) contents questioning the applicability of widely used laboratory methods for soil P testing for organic farming.

Aims

The aim of this study was to compare the validity of a broad range of different soil extraction methods on soils under organic management from South West Germany and to test the correlation of the measured soil P concentration with plant offtake.

Methods

Twenty-two soil samples of eight different organic farms were extracted with different solutions: (1) water, (2) CAL, (3) Olsen, (4) Mehlich 3, (5) Bray P1, (6) Bray P2, (7) NaOH+Na₂EDTA, and (8) total P. The results were then correlated with above ground plant P.

Results

Spearman's rank correlation coefficient (r_s) of correlations between above ground plant P and extractable soil P (Water-P, CAL-P, and Olsen-P [+active charcoal {+AC}]) determined with ICP-OES were strong (0.94, 0.90, and 0.93, respectively). Among the tested methods, above ground plant P showed a strong correlation with CAL-P as detected by ICP-OES (r_s = 0.90) and colorimetry (r_s = 0.91). The comparison of CAL-P data provided by farmers and CAL-P analyzed during this research showed discrepancies between the results.

Conclusions

The results of this study indicate that the CAL method can be used in organic farming despite a low extraction of organic P (P_org). Furthermore, it is recommended for farmers to take soil samples for analyses regularly and interpret changes in P in the long-term instead of interpreting individual samples.     

Establishment success in a forest biodiversity and ecosystem functioning experiment in subtropical China (BEF-China)

Experimental forest plantations to study biodiversity–ecosystem functioning (BEF) relationships have recently been established in different regions of the world, but subtropical biomes have not been covered so far. Here, we report about the initial survivorship of 26 tree species in the first such experiment in subtropical China. In the context of the joint Sino–German–Swiss Research Unit “BEF-China,” 271 experimental forest plots were established using 24 naturally occurring tree species and two native commercial conifers. Based on the survival inventories carried out in November 2009 and June 2010, the overall survival rate was 87 % after the first 14 months. Generalized mixed-effects models showed that survival rates of seedlings were significantly affected by species richness, the species’ leaf habit (deciduous or evergreen), species identity, planting date, and altitude. In the first survey, seedling establishment success decreased with increasing richness levels, a tendency that disappeared in the second survey after replanting. Though evergreen species performed less well than deciduous species with establishment rates of 84 versus 93 % in the second survey, their planting success exceeded the general expectation for subtropical broad-leaved evergreen species. These results have important implications for establishing mixed-species plantations for diversity conservation and improvement of ecosystem functioning in the Chinese subtropics and elsewhere. Additional costs associated with mixed-species plantations as compared to conventional plantations also demonstrate the potential of upscaling BEF experiments to large-scale afforestation projects.     

Strong resistance to the fungicide fenhexamid entails a fitness cost in Botrytis cinerea, as shown by comparisons of isogenic strains

BACKGROUND: Fenhexamid, a sterol biosynthesis inhibitor effective against Botrytis, inhibits the 3‐ketoreductase (Erg27) involved in C‐4 demethylation. Several fenhexamid‐resistant phenotypes have been detected in Botrytis cinerea populations from French vineyards. The field isolates with the highest resistance levels display amino acid changes in Erg27 (F412S, F412I or F412V). RESULTS: Fenhexamid‐resistant mutants were generated by site‐directed mutagenesis of the erg27 gene in a sensitive recipient strain to overcome the impact of different genetic backgrounds. The wild‐type erg27 allele was replaced by the three mutated alleles (erg27^F412S/I/V) by homologous recombination. These isogenic strains were shown to be fenhexamid‐resistant and were used to quantify the impact of F412 mutations on fungal fitness. Several parameters, including radial growth, the production of sclerotia and conidia, freezing resistance and aggressiveness, were quantified in laboratory conditions. Analysis of variance demonstrated significant differences between the mutant and parental strains for some characters. In particular, the mutants grew more slowly than the wild‐type strain and displayed variations in the production of sclerotia and conidia with temperature and susceptibility to freezing. CONCLUSIONS: The results highlight a moderate but significant impact of F412 mutations on the survival capacity of B. cinerea strains displaying high levels of resistance to fenhexamid in laboratory conditions, potentially limiting their dispersal and persistence, particularly in terms of overwintering, in field conditions. Copyright © 2011 Society of Chemical Industry     

Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine innate immune cells

ABSTRACT: Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM) and trehalose monomycolate (TMM), the apolar phthiocerol dimycocersates (PDIMs), triacyl glycerol (TAG), pentacyl trehalose (PAT), phenolic glycolipid (PGL), and mono-mycolyl glycerol (MMG). Polar lipids identified included glucose monomycolate (GMM), diphosphatidyl glycerol (DPG), phenylethanolamine (PE) and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs). These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1β, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.     

Comparison of isoflurane inhalation anaesthesia, injection anaesthesia and high volume caudal epidural anaesthesia for umbilical surgery in calves; metabolic, endocrine and cardiopulmonary effects

ObjectiveTo compare three anaesthetic protocols for umbilical surgery in calves regarding adequacy of analgesia, and cardiopulmonary and hormonal responses.Study designProspective, randomised experimental study.AnimalsThirty healthy German Holstein calves (7 female, 23 male) aged 45.9 ± 6.4 days.MethodsAll calves underwent umbilical surgery in dorsal recumbency. The anaesthetic protocols were as follows: group INH (n = 10), induction 0.1 mg kg^?1 xylazine IM and 2.0 mg kg^?1 ketamine IV, maintenance isoflurane in oxygen; Group INJ (n = 10), induction 0.2 mg kg^?1 xylazine IM and 5.0 mg kg^?1 ketamine IV, maintenance 2.5 mg kg^?1 ketamine IV every 15 minutes or as required; group EPI (n = 10), high volume caudal epidural anaesthesia with 0.2 mg kg^?1 xylazine diluted to 0.6 mL kg^?1 with procaine 2%. All calves received peri-umbilical infiltration of procaine and pre-operative IV flunixin (2.2 mg kg^?1). Cardiopulmonary variables were measured at preset intervals for up to 2 hours after surgery. The endocrine stress response was determined. Intra-operative nociception was assessed using a VAS scale. Data were compared between groups using appropriate statistical tests. A value of p < 0.05 was considered significant.ResultsAll three protocols provided adequate anaesthesia for surgery although, as judged by the VAS scale, intra-operative response was greatest with INJ. Lowest mean cortisol levels during surgery occurred in EPI. Heart rate and cardiac output did not differ between groups, but mean arterial blood pressure, systemic vascular resistance, and partial pressure of carbon dioxide were higher and arterial pH lower in groups INH and INJ than in Group EPI. Group INJ became hypoxaemic and had a significantly greater vascular shunt than did the other groups.Conclusion and clinical relevanceGroups INH and EPI both proved acceptable protocols for calves undergoing umbilical surgery, whilst INJ resulted in variable anti-nociception and in hypoxaemia. High volume caudal epidural anaesthesia provides a practical inexpensive method of anaesthesia for umbilical surgery.     

Sedative, cardiovascular, haematologic and biochemical effects of four different drug combinations administered intramuscularly in cats

ObjectiveTo compare effects of four drug combinations on sedation, echocardiographic, haematologic and biochemical variables and recovery in cats.Study designExperimental randomized ‘blinded’ cross-over study.AnimalsSix healthy cats.Materials and MethodsTreatments were administered intramuscularly: midazolam 0.4 mg kg^?1 and butorphanol 0.4 mg kg^?1 (MB); midazolam 0.4 mg kg^?1, butorphanol 0.4 mg kg^?1 and ketamine 3 mg kg^?1 (MBK); midazolam 0.4 mg kg^?1, butorphanol 0.4 mg kg^?1 and dexmedetomidine 5 μg kg^?1 (MBD); ketamine 3 mg kg^?1 and dexmedetomidine 5 μg kg^?1(KD). Sedation was evaluated at time-points over 10 minutes post injection. Echocardiography, systolic arterial blood pressure (SAP) measurement and blood sampling were performed at baseline and from 10 minutes after treatment. Quality of recovery was scored. Data were analysed by anova for repeated measures. p < 0.05 was considered significant.ResultsThe lowest sedation score was obtained by MB, (median 10.5 [7; 20]), highest by KD (36.5 [32; 38]). Quality of recovery was best with KD (0.5 [0; 2]), and worst with MB (7.5 [4; 11]). Relative to baseline measurements, treatments decreased SAP by 17%, 25%, 13%, 5% in MB, MBK, MBD and KD, respectively. Heart rate decreased (p < 0.05) after MBD (44%) and KD (34%). All treatments decreased stroke volume by 24%, 21%, 24%, 36%, and cardiac output by 23%, 34%, 54%, 53% in MB, MBK, MBD and KD, respectively. Packed cell volume was decreased (p < 0.05) by 20%, 31%, 29% in MBK, MBD and KD, respectively. Plasma glucose was increased after MBD (31%) and KD (52%) and lactate concentration was decreased (p < 0.05) after MBK (58%), MBD (72%) and KD (65%).Conclusions and clinical relevanceThe MB combination did not produce sedation in healthy cats. Treatment MBK led to acceptable sedation and minimal cardiovascular changes. Both treatments with dexmedetomidine produced excellent sedation and recovery but induced more cardiovascular depression and haematologic changes.     

Current knowledge on porcine regulatory T cells

Regulatory T cells (Tregs) are known in humans and mice from last 15 years, and several studies led to a detailed knowledge on their phenotype, functions, and role in various immune reactions. In swine, the existence of Tregs was first demonstrated in 2008 and research is still at the beginning. Nevertheless, basic information regarding phenotype, mechanisms and targets of suppression, as well as implications in transplantation and some diseases are available. Purpose of this review is to give a brief summary of the current knowledge about porcine Tregs.