A multiplex GC-MS/MS technique for the sensitive and quantitative single-run analysis of acidic phytohormones and related compounds,and its application to Arabidopsis thaliana

A highly sensitive and accurate multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique is reported for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid. The optimized setup allows the routine processing and analysis of up to 60 plant samples of between 20 and 200 mg of fresh weight per day. The protocol was designed and the equipment used was chosen to facilitate implementation of the method into other laboratories and to provide access to state-of-the-art analytical tools for the acidic phytohormones and related signalling molecules. Whole-plant organ-distribution maps for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid were generated for Arabidopsis thaliana (L.) Heynh. For leaves of A. thaliana, a spatial resolution of hormone quantitation down to approximately 2 mm(2) was achieved.     

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Following chromatography on hydroxyapatite, the elution profile of the thiolase activity of the glyoxysomal fraction from sunflower (Helianthus annuus L.) cotyledons exhibited two peaks when the enzyme activity was assayed with acetoacetyl-CoA as substrate. Only one of these two activity peaks was detectable when a long-chain thiolase substrate was used in the activity assay. The proteins (thiolase I and thiolase II) underlying the two activity peaks detected with acetoacetyl-CoA were of glyoxysomal origin. They were purified using glyoxysomal matrices as starting material, and biochemically characterized. Thiolase I is an acetoacetyl-CoA thiolase (EC 2.3.1.9) exhibiting activity only towards acetoacetyl-CoA (Km = 11 microM). Its contribution to the total glyoxysomal thiolytic activity towards acetoacetyl-CoA amounted to about 15%. Thiolase II is a 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The activity of the enzyme towards 3-oxoacyl-CoAs increased with increasing chain length of the substrate. Thiolase II exhibited a Km value of 27 microM with acetoacetyl-CoA as substrate. and Km values between 3 and 7 microM with substrates having a carbon chain length from 6 to 16 carbon atoms. The thiolase activity of the glyoxysomes towards acetoacetyl-CoA and 3-oxopalmitoyl-CoA exceeded the glyoxysomal butyryl-CoA and palmitoyl-CoA beta-oxidation rates, respectively, by about 10-fold at all substrate concentrations employed (1-15 microM).     

Shape analysis of symmetric structures: quantifying variation among individuals and asymmetry

Abstract.— Morphometric studies often consider parts with internal left-right symmetry, for instance, the vertebrate skull. This type of symmetry is called object symmetry and is distinguished from matching symmetry, in which two separate structures exist as mirror images of each other, one on each body side. We explain a method for partitioning the total shape variation of landmark configurations with object symmetry into components of symmetric variation among individuals and asymmetry. This method is based on the Procrustes superimposition of the original and a reflected copy of each landmark configuration and is compatible with the two-factor ANOVA model customary in studies of fluctuating asymmetry. We show a fully multivariate framework for testing the effects in the two-factor model with MANOVA statistics, which also applies to shapes with matching symmetry. We apply the new methods in a small case study of pharyngeal jaws of the Neotropical cichlid fish Amphilophus citrinellus . The analysis revealed that the symmetric component of variation in the pharyngeal jaws is dominated by the contrast between two alternative trophic morphs in this species and that there is subtle but statistically significant directional asymmetry. Finally, we provide some general recommendations for morphometric studies of symmetric shapes.     

Spatial representation of the glomerular map in the Drosophila protocerebrum

In the fruit fly, Drosophila, olfactory sensory neurons expressing a given receptor project to spatially invariant loci in the antennal lobe to create a topographic map of receptor activation. We have asked how the map in the antennal lobe is represented in higher sensory centers in the brain. Random labeling of individual projection neurons using the FLP-out technique reveals that projection neurons that innervate the same glomerulus exhibit strikingly similar axonal topography, whereas neurons from different glomeruli display very different patterns of projection in the protocerebrum. These results demonstrate that a topographic map of olfactory information is retained in higher brain centers, but the character of the map differs from that of the antennal lobe, affording an opportunity for integration of olfactory sensory input.     

Suppression of plant defence in rhizobia-legume symbiosis

The symbiosis between rhizobia and legumes is characterized by the formation of dinitrogen-fixing root nodules. Although rhizobia colonize roots in a way that is reminiscent of pathogenic microorganisms, no host plant defence reactions are triggered during successful symbioses. Nevertheless, the plants obviously control the invading bacteria; failure in effective nodule formation or infections with rhizobia defective in surface polysaccharides often result in pathogenic responses. This article focuses on whether and how defence responses in effective symbiosis might be suppressed. Recent results suggest a central role for rhizobial polysaccharides acting as antagonists in the negative regulation of defence induction.     

Clinical Efficacy and Compatibility of Allogeneic Avital Tissue Transplants Sterilized with a Peracetic Acid/Ethanol Mixture

In the course of the past 20 years a quantity of approximately 60,000 allogeneic avital tissue grafts sterilized with the peracetic acid–ethanol method (PES) were transplanted successfully. Based on a retrospective report of clinical experience of the years 1997–2001 on the overall scope of tissue grafts manufactured by the Tissue Banks of the University Hospital Charité and the German Institute for Cell and Tissue Replacement, the clinical efficacy and side effects of 18.3% (3.087/16.823) of all transplants were studied. Cancellous (1.601/3.087) and cortical (291/3.087) bone transplants as well as amnion (1.027/3.087) constituted the greatest part. In 91% of the examined patients (2.369/2.592) tissue integration ratios ranging from good up to very well could be observed. The transplant function of defect replacement or of a spacer respectively could be obtained for all types of tissue. The clinical effect caused by the transplant resulted in more than 99% of the transplants in primary integration or in the desired aim of the therapy (defect replacement, stabilization in case of palliative operations, etc.). In less than 1% (9/2.592) of cases a secondary healing occurred for cancellous bone transplantations or, revisional operations became necessary. In all cases severe side effects, in particular transmission of infectious diseases or transplant rejections, were not observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Community cooperatives and insecticide-treated materials for malaria control: a new experience in Latin America

Background and objectives

Insecticide-treated materials (ITMs) are effective in substantially reducing the burden of malaria and other vector-borne diseases; but how can high coverage rates of ITMs be achieved and maintained? In south Mexico and on the Pacific and Atlantic coasts of Colombia 14 community-based cooperatives offering three different kinds of ITM services (sale of impregnation services; sale of impregnated nets; production of nets and sale of impregnated nets) were formed and supervised by a national health service (IMSS-SOLIDARIDAD, Mexico) and by an academic institution (the Colombian Institute of Tropical Medicine) along with local district health services. The objectives of this research were to analyse the processes and results of this approach and to identify the favourable and limiting factors.

Methods

The methods used for data collection and analysis were group discussions, individual and semi-structured interviews with users and non-users of ITMs, individual in-depth interviews with cooperative members and supervisors, checks of sales book and observation of impregnation services.

Results

Coverage with unimpregnated nets was above 50% in all study areas. The fastest increase of ITM coverage was achieved through the exclusive sale of impregnation services. Low-cost social marketing techniques were used to increase demand. The large-scale production of nets in two cooperatives was only possible with the aid of an international NGO which ordered impregnated bednets for their target group. A number of favourable and limiting factors relating to the success of ITM cooperatives were identified. Of particular importance for the more successful Mexican cooperatives were: a) support by health services, b) smaller size, c) lesser desire for quick returns and d) lower ITM unit costs.

Conclusions

ITM community cooperatives supported and supervised by the health services have good potential in the Latin American context for achieving and maintaining high impregnation rates.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related V_L chain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V_L chain genes caused by selection and clonal expansion of B cells expressing particular V_L genes. In addition, the data document an accumulation of B cells bearing mutated V_L gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

Molecular Regulation of β-Lactam Biosynthesis in Filamentous Fungi

The most commonly used β-lactam antibiotics for the therapy of infectious diseases are penicillin and cephalosporin. Penicillin is produced as an end product by some fungi, most notably by Aspergillus (Emericella) nidulans and Penicillium chrysogenum. Cephalosporins are synthesized by both bacteria and fungi, e.g., by the fungus Acremonium chrysogenum (Cephalosporium acremonium). The biosynthetic pathways leading to both secondary metabolites start from the same three amino acid precursors and have the first two enzymatic reactions in common. Penicillin biosynthesis is catalyzed by three enzymes encoded by acvA (pcbAB), ipnA (pcbC), and aatA (penDE). The genes are organized into a cluster. In A. chrysogenum, in addition to acvA and ipnA, a second cluster contains the genes encoding enzymes that catalyze the reactions of the later steps of the cephalosporin pathway (cefEF and cefG). Within the last few years, several studies have indicated that the fungal β-lactam biosynthesis genes are controlled by a complex regulatory network, e.g., by the ambient pH, carbon source, and amino acids. A comparison with the regulatory mechanisms (regulatory proteins and DNA elements) involved in the regulation of genes of primary metabolism in lower eukaryotes is thus of great interest. This has already led to the elucidation of new regulatory mechanisms. Furthermore, such investigations have contributed to the elucidation of signals leading to the production of β-lactams and their physiological meaning for the producing fungi, and they can be expected to have a major impact on rational strain improvement programs. The knowledge of biosynthesis genes has already been used to produce new compounds.