Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.     

The aminergic control of cockroach salivary glands

The acinar salivary glands of cockroaches receive a dual innervation from the subesophageal ganglion and the stomatogastric nervous system. Acinar cells are surrounded by a plexus of dopaminergic and serotonergic varicose fibers. In addition, serotonergic terminals lie deep in the extracellular spaces between acinar cells. Excitation-secretion coupling in cockroach salivary glands is stimulated by both dopamine and serotonin. These monoamines cause increases in the intracellular concentrations of cAMP and Ca(2+). Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Thus, two elementary secretory processes, namely electrolyte/water secretion and protein secretion, are triggered by different aminergic transmitters. Because of its simplicity and experimental accessibility, cockroach salivary glands have been used extensively as a model system to study the cellular actions of biogenic amines and to examine the pharmacological properties of biogenic amine receptors. In this review, we summarize current knowledge concerning the aminergic control of cockroach salivary glands and discuss our efforts to characterize Periplaneta biogenic amine receptors molecularly.     

Excited state properties of dinaphthyl ditelluride

Di-1-naphthyl ditelluride (Te₂naphthyl₂) is characterized by two low-energy excited states. The corresponding electronic transitions nTe → σ¹ Te–Te and nTe → π¹ naphthyl CT give rise to absorptions at λ_max = 403 and 311 nm, respectively. In solution nTe → σ¹ excitation leads to the cleavage of the Te–Te bond. In contrast to Te₂naphthyl₂ in the dissolved state the solid compound shows a luminescence (λ_max = 576 nm) which originates from nTe → π¹ naphthyl CT triplet.     

A comparative Raman and CARS imaging study of colon tissue

An experimental evaluation of the information content of two complimentary techniques, linear Raman and coherent anti‐Stokes Raman scattering (CARS) microscopy, is presented. CARS is a nonlinear variant of Raman spectroscopy that enables rapid acquisition of images within seconds in combination with laser scanning microscopes. CARS images were recorded from thin colon tissue sections at 2850, 1660, 1450 and 1000 cm^–1 and compared with Raman images. Raman images were obtained from univariate and multivariate (k‐means clustering) methods, whereas all CARS images represent univariate results. Variances within tissue sections could be visualized in chemical maps of CARS and Raman images. However, identification of tissue types and characterization of variances between different tissue sections were only possible by analysis of cluster mean spectra, obtained from k‐means cluster analysis. This first comparison establishes the foundation for further development of the CARS technology to assess tissue. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning,Sugar Signaling,and Seed Yield in Arabidopsis

The extent to which vacuolar sugar transport activity affects molecular, cellular, and developmental processes in Arabidopsis (Arabidopsis thaliana) is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton-coupled monosaccharide import into isolated mesophyll vacuoles in comparison with wild-type vacuoles. TMT1 overexpressor mutants grew faster than wild-type plants on soil and in high-glucose (Glc)-containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by nonaqueous fractionation and by chlorophyll_ab-binding protein1 and nitrate reductase1 gene expression studies. Soil-grown TMT1 overexpressor plants respired less Glc than wild-type plants and only about half the amount of Glc respired by tmt1-2::tDNA mutants. In sum, these data show that TMT activity in wild-type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO₂ release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as Glc repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic Glc levels than wild-type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased the biomass of Arabidopsis seeds, and accelerated early plant development.Sugars fulfill an extraordinarily wide range of functions in plants as well as in other organisms. They serve as valuable energy resources that are easy to store and remobilize. Sugars are required for the synthesis of cell walls and carbohydrate polymers. They are also necessary for starch accumulation and serve as precursors for a range of primary and secondary plant intermediates. From a chemical point of view, sugars represent a large class of metabolites. Among the prominent members in higher plants are the monosaccharides Glc and Fru and the disaccharide Suc (ap Rees, 1994).In contrast to heterotrophic organisms, plants are able to synthesize sugars de novo and to degrade them via oxidative or fermentative metabolism (Heldt, 2005). Net sugar accumulation in plants takes place during the day, whereas net degradation of stored carbohydrate reserves takes place the following night. In higher plants, autotrophic and heterotrophic organs appear to be interconnected by phloem for long-distance transport of sugars (Ruiz-Medrano et al., 2001). Accordingly, sugars must be transported within cells, between cells, and between plant organs. Given these factors, along with the outstanding importance of sugars, it is not surprising that plants sense intracellular sugar availability and use this information to coordinate the expression of many genes (Koch, 1996; Moore et al., 2003).In Arabidopsis (Arabidopsis thaliana), about 60 genes code for putative monosaccharide transport proteins and about 10 genes encode predicted disaccharide carriers (Lalonde et al., 2004). Transport of neutral sugars has been monitored across the plasma membrane, the chloroplast envelope, and the vacuolar membrane (Weber et al., 2000; Niittylä et al., 2004; Martinoia et al., 2007). So far, all sugar carriers residing in the plant plasma membrane have been characterized to catalyze proton-coupled sugar movement (Sauer, 1992; Büttner and Sauer, 2000; Carpaneto et al., 2005). In contrast, both facilitated diffusion and proton-driven antiport mechanisms have been described for monosaccharide and Suc transport across the vacuolar membrane (Thom and Komor, 1984; Daie and Wilusz, 1987; Martinoia et al., 1987; Shiratake et al., 1997; Neuhaus, 2007).In plants, vacuoles fulfill critical functions in the long-term and temporary storage of sugars, sugar alcohols, and other primary metabolites such as carboxylates and amino acids (Dietz et al., 1990; Rentsch and Martinoia, 1991; Martinoia and Rentsch, 1992; Emmerlich et al., 2003). Recently, the first solute carriers responsible for vacuolar Suc and inositol transport have been identified (Endler et al., 2006; Schneider et al., 2008). In addition, TMT (for tonoplast monosaccharide transporter) and VGT (for vacuolar Glc transporter) were the first vacuolar carrier proteins proven to have transport capacity for both Glc and Fru (Wormit et al., 2006; Aluri and Büttner, 2007).TMT exists in three isoforms in Arabidopsis (TMT1–TMT3), and orthologs have been found in other plant species like grapevine (Vitis vinifera), barley (Hordeum vulgare), and rice (Oryza sativa; Wormit et al., 2006). In Arabidopsis, the genes TMT1 and TMT2 are expressed in various tissues, whereas TMT3 is hardly expressed throughout the entire plant life cycle (Wormit et al., 2006). Interestingly, TMT1 and TMT2 are induced by Glc, salt, drought, and cold stress (Wormit et al., 2006), and vacuoles isolated from a TMT1 loss-of-function (T-DNA) Arabidopsis mutant showed reduced Glc import capacity in comparison with corresponding wild-type organelles (Wormit et al., 2006). Moreover, after transfer into the cold, these mutant leaves showed impaired ability to accumulate Glc and Fru, underscoring the in vivo function of TMT under selected conditions (Wormit et al., 2006).However, it is unknown to what extent overexpression of a vacuolar sugar carrier affects subcellular sugar allocation in Arabidopsis. In addition, whether increased vacuolar sugar transport influences sugar signaling, plant development, or organ properties has not been determined. Thus, it is unknown how important controlled activity of vacuolar monosaccharide transport is to plant development or physiological properties. To reveal whether TMT activity affects these processes, we created TMT1-overexpressing Arabidopsis lines and analyzed their physiological and molecular feedbacks.     

Biochemical properties of heterologously expressed and native adenylyl cyclases from the honeybee brain (Apis mellifera L.)

Cyclic AMP is an important intracellular signaling molecule participating e.g. in sensory signal transduction, cardiac myocyte regulation, learning and memory. The formation of cAMP is catalyzed by adenylyl cyclases. A variety of factors can modulate the properties of these enzymes and lead to dynamic changes of the intracellular cAMP concentration. Here we determined the tissue distribution of a recently cloned adenylyl cyclase (AmAC3) in honeybee brain. The protein is present in all neuropils. Intensive immunoreactivity was found in parts of the proto- and deutocerebrum and in the suboesophageal ganglion. Biochemical and pharmacological properties of AmAC3 and of native adenylyl cyclases in subregions of the honeybee brain were examined. Values for half-maximal activation with NKH477 were in the low micromolar range with 10.2 μM for AmAC3 and 3.6–8.1 μM for native enzymes. Biosynthesis of cAMP was specifically blocked by P-site inhibitors. Adenylyl cyclases in antennal lobes and AmAC3 share the inhibitory profile with 2′,5′dd3′ATP > 3′AMP > 2′deoxyadenosine. In addition to P-site inhibitors AmAC3 activity was impaired by Ca²⁺/calmodulin. The results suggest that AmAC3 is a likely candidate to fulfill an integrative role in sensory, motor and higher-order information processing in the honeybee brain.     

Characterization of Phleum pratense pollen extracts by 2-D DIGE and allergen immunoreactivity

The allergen content of standardized pollen material is crucial for an effective diagnosis and treatment. However, variations in IgE reactivities of allergic patients to different preparations of Phleum pratense pollen have been reported. In order to define and directly compare the allergen composition of pollen preparations provided by different suppliers, a comprehensive proteome analysis of three different timothy grass pollen extracts was performed. More than 140 proteins were annotated comprising the pollen proteome/allergome in a global 2-D map. With regard to the individual pollen preparations, several major differences in the overall protein composition were detected that also affected known Phleum allergens and their isoforms. Importantly, these differences were also reflected at the level of antibody reactivities in 1-D and 2-D immunoblots. As a consequence, it is suggested that the observed differences should be taken into consideration aiming for a standardized diagnosis and therapy of grass pollen allergies as recommended by international medical agencies.