A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction

ABSTRACT: BACKGROUND: We have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high pressure liquid chromatography-fast scanning tandem mass spectrometry. RESULTS: Plant tissue samples (1-5 mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2-microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. CONCLUSIONS: The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents.     

Enterotoxigenic Escherichia coli (ETEC) and Klebsiella pneumoniae isolated from dogs with diarrhoea

Faecal samples from 148 dogs with diarrhoea and from 15 healthy dogs were cultured for bacterial pathogens with enterotoxigenic properties. The aim of the study was to define the toxin profile (production of heat-labile [LT] and heat-stable [ST] toxins) and possible surface fimbrial antigens. Enterotoxigenic bacteria were isolated from 6 (4.1%) dogs with diarrhoea, four of these were Escherichia coli and two were Klebsiella pneumoniae. The E. coli strains and K. pneumoniae strains were producing both LT and ST toxins. The LT toxin from these strains was not neutralized by human anti-LT serum or anti-choleragen and did not cause coagglutination with Staphylococcus aureus coated with anti-human-LT. This suggests that the LT toxin produced by these canine isolates is non-identical to LT toxin from human strains.

Three of the ETEC strains were haemagglutinating and showed surface hydrophobic properties. Electron microscopy showed that canine ETEC isolates possessed fimbriae of two different types: thick (5–5.5 nm) and thin (2–3 nm).     

Spatial and temporal variation of muddy floods in central Belgium, off-site impacts and potential control measures

Numerous villages in the European loess belt are confronted with floods caused by runoff from agricultural land. Seventy-nine percent of the municipalities in central Belgium experienced at least one muddy flood during the last decade. Of these flooded municipalities, 22% have been affected more than 10 times during this period. Twenty municipalities have been selected for a detailed analysis. A database of 367 locations affected by muddy floods has been compiled, and the connectivity between cultivated areas and inhabited zones could be assessed for 100 flooded locations. Roads and drainage network facilitate runoff transfer between cultivated and inhabited areas in 64% of cases. Three types of areas producing muddy floods have been identified: hillslopes (1–30 ha) without thalweg where runoff is generally dominated by sheet flow; small catchments (10–300 ha) characterised by runoff concentration in the thalweg and medium catchments (100–300 ha) with multiple thalwegs dominated by concentrated runoff. About 90% of muddy floods are generated on hillslopes and in small catchments. A critical area–slope threshold for triggering muddy floods has been computed for hillslopes. A logistic regression shows that muddy floods are generated in small and medium catchments with 99% probability after 43 mm rainfall. Rainfall depths required to trigger muddy floods are lower in May and June (25 ±12 mm) than between July and September (46 ± 20 mm), because of different surface conditions (crusting, roughness and crop cover). Each year, muddy floods lead to a total societal cost of 16 × 10⁶–172 × 10⁶ € in central Belgium, depending on the extent and intensity of thunderstorms and monetary values damaged. Recent datasets suggest that the phenomenon is becoming more frequent in central Belgium, because of land consolidation, urban sprawl and expansion of row crops, sown in spring, at the expense of winter cereals. The huge costs induced by muddy floods justify the installation of erosion control measures. It is suggested to install a grassed buffer strip at the downslope edge of cultivated hillslopes to protect houses and roads. In small and medium catchments, it is preferred to install a grassed waterway and earthen dams in the thalweg.     

Genetic analysis of a duck circovirus detected in commercial Pekin ducks in New York

The genetic organization of the duck circovirus (DuCV) 33753-52 detected in commercial Pekin duck flocks from Long Island, NY, is described. The nucleotide sequence of virus 33753-52 exhibited high similarity with DuCVs previously detected in Germany and Hungary. It is possible that this DuCV from New York shares the same ancestor with the European counterparts. The virus 33753-52 exhibited genetic features characteristic of other circoviruses, such as the presence of two major open reading frames (rep and cap), two intergenic regions, one stem-loop structure, four intergenic direct repeats, and the conserved motifs for the rolling circle replication and for the dNTP binding domain in the Rep protein. This report is the first report of the presence of DuCV in commercial Pekin duck farms in the United States. The clinical and pathologic significance of DuCV in the duck farms located on Long Island needs to be clarified. DuCv was detected in culled birds, due to low body development, leg deformities, or arthritis. Staphylococcus aureus and Riemerella anatipestifer serotype 4 were isolated from some of the DuCV-positive birds. The apparent low prevalence of the virus suggests that at this time, this infection is not a significant problem for the duck industry in New York. However, the immunosuppressive properties of this virus need to be clarified as well as its role as a predisposing agent for other diseases.     

Serotypes and anti-microbial susceptibility of Plesiomonas shigelloides isolates from humans, animals and aquatic environments in different countries

A total of 73 strains of Plesiomonas shigelloides isolated from humans (24 strains) animals (21 strains) and aquatic environment (28 strains) were determined for their O:H serotype and susceptibility to 18 anti-microbial substances and to the vibriostatic agent O/129. Of all strains, 86.3% were typeable by the O and 94.5% by the H anti-sera used. The serotype distribution was heterogeneous within a country and between the countries. Of the 57 different serotypes identified, O11:H2 (2 strains), O22:H3 (4 strains), O35:HH11 (2 strains), O52:H3 (2 strains) and O90:H6 (2 strains) were found among isolates from humans and animals (mainly in cats) in Finland and Cuba, and O23:H1a1b (3 strains) among isolates from environmental sources in Slovak Republic and Italy. Most (93-100%) of all strains were susceptible to all anti-microbials tested but resistant (92-96%) to the broad-spectrum penicillins (ampicillin, mezlocillin). No correlation between anti-microbial resistance patterns and serotypes was found.     

Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.     

Functional polymorphisms in Toll-like receptor genes for innate immunity in farm animals

The exploitation of the genetic factors affecting the health status of farm animals represents an alternative approach to controlling the diseases caused by microbial pathogens. The determination of innate immunity based on the genotype of the germplasm cells is a constraint for specificity but becomes an advantage in breeding schemes. The structural deviations among Toll-like receptors (TLRs), as the most frequently studied innate immunity components, have been documented at all levels, i.e., interspecific, inter- and intravarietal, in the main farm species. The current computational methods facilitate the prediction of the functional consequences of the observed mutations. Subsequently, these predictions can be verified through immunological responsiveness and population-wide association studies. The frequency and haplotype grouping of individual polymorphisms are used to track the origin and selection coefficient as independent indicators of functional changes. The Toll-like receptor variants associated with mastitis and mycobacterial infection have been identified in cattle, consequently, the targeting of these proteins in breeding could contribute to disease control. The range of infections affected by TLR polymorphisms suggests that the improvement of innate resistance is feasible in more species. Thus, the traditional breeds and wild populations should be regarded as the resources of genetic variability accessible for these purposes.     

Ecophysiology of the cave isopod Mesoniscus graniger (Frivaldszky, 1865) (Crustacea: Isopoda)

Feeding biology and thermal adaptations of the terrestrial isopod Mesoniscus graniger were studied. M. graniger is a depigmented isopod mainly inhabiting cave systems, although it has also been reported in endogeic (soil) habitats. Physiological adaptations are expected to reflect the unique environmental characteristics of caves, including restricted food sources, and stable microclimate with temperatures not exceeding about 10 °C and high relative humidity. The M. graniger from Domica and Ardovská caves (Slovakian Karst) were investigated. We identified organic deposits with associated microorganisms as sources exploited by M. graniger and assessed how these might supply essential polyunsaturated fatty acids (PUFA) in its nutrition. Algae, fungi and bat guano were found as the most important potential resources of PUFA for isopods. Digestive enzymes amylase, trehalase, saccharase and maltase were confirmed in the whole body homogenates of isopods; neither cellulolytic activity nor activities against xylan, laminaran and lichenan were observed. Amylase, maltase and cellobiase activities were also observed in bacterial strains isolated and cultured from isopod midgut, and may account for the measured whole-animal activities. In an artificial temperature gradient, M. graniger selected temperature 3.5 ± 5.4 °C with lower and upper extremes of –1.5 and 18.5 °C. Respiration, as measured by VO₂, was almost independent of temperature between 5 and 10 °C, then increased between 10 and 30 °C. These ecophysiological measures are consistent with adaptation to a stenothermal environment.