Rhizobial polysaccharide-degrading enzymes from roots of legumes

The surface structure and chemistry of symbiotic bacteria from the genus Rhizobium are probably important for the outcome of the infection of legume hosts. Exopolysaccharide, capsular polysaccharide, lipopolysaccharide and a low-molecular-weight polysaccharide were isolated from R. trifolii UTC 110-1 and R. leguminosarum UTC 114-5 and partially characterized. No or only minor differences in sugar composition could be found for the corresponding fractions from the two organisms. A general method to measure low activities of polymer-degrading enzymes was developed, and used to determine enzyme activities in root extracts of Trifolium repens L. cv. Lena and Pisum xativiini L. cv. Little Marvel against the isolated rhizobial polysaccharides. An enzyme preparation from T. repens partially degraded all polysaccharides isolated from its symbiont R. trifolii while polysaccharides from R. leguminosarum , symbiont of P. sativum , were degraded to a much lesser extent. Correspondingly, an enzyme preparation from P. sativum degraded all polysaccharides isolated from both its symbiont R. leguminosarum and its non-symbiont R. trifolii. The amount of symbiont polysaccharides degraded was larger than the amount of polysaccharides degraded from the non-symbiont R. trifolii.     

Shift Work Disorder in a Random Population Sample – Prevalence and Comorbidities

Few studies have investigated the presence of shift work disorder (SWD) in the general community. We addressed many of the limitations in this literature and present new findings. SWD has been treated as an ‘all or none’ construct but we propose the need to consider the ‘severity’ of the disorder. Using random digit dialling, we randomly recruited 1163 participants. Participants completed an extensive battery of scales and questions concerning work, health and individual differences. Three questions based on the criteria from the International Classification for Sleep Disorders were used to categorise participants with SWD (n = 176). In addition, we asked participants whether SWD interfered with aspects of their life and high ratings were used to define severe shift work disorder (SSWD). The prevalence of SWD was 32.1% among night workers and 10.1% in day workers (p<.001). SSWD was present in 9.1% of night workers and 1.3% of day workers (p<.001). Adjusted logistic regression analyses found significant associations between SWD and night work (OR  = 3.35, CI 2.19-5.12), weekly work hours (OR  = 1.02, CI 1.00–1.04), short sleep (≤6 h; OR  = 2.93, CI 1.94–4.41), languidity (OR  = 1.11, CI 1.06–1.16) and resilience (OR  = 0.56, CI 0.43–0.81). Night work, short sleep, languidity, and hypertension were significantly associated with SSWD. Overall, participants with SSWD slept 0.80 h less than other participants (p<.001). Night work, short sleep and languidity were associated with both SWD and SSWD. Day workers with SWD symptoms reported significantly shorter sleep duration, higher levels of languidity and worked longer working hours compared to day workers without SWD.     

Algal Turbidity Hampers Ornament Perception,but Not Expression,in a Sex‐Role‐Reversed Pipefish

Sexual ornaments are used both in intra‐ and intersexual contexts, and these signals have evolved to function in the particular habitat the animal is adapted to. Habitat characteristics may, however, change rapidly due to anthropogenic effects, sometimes at rates too fast for many organisms to adaptively respond. In aquatic ecosystems, eutrophication is currently changing chemical as well as visual properties of the environment. Algae blooms increase water turbidity, and the reduction of water transparency thus has the potential to alter visual ornaments and their perception. However, results are not congruent. Rather, algae turbidity may decrease, increase, or leave ornaments unaffected. The effect seems to depend on exposure time, condition, population and species. Here, we found that the perception of sexual signals, but not their expression, was hampered by turbidity in the sex‐role‐reversed pipefish Nerophis ophidion. In a laboratory experiment we found that female sexual ornaments (i.e., blue color markings and a skinfold) and fecundity was unaffected by turbidity. Male adaptive mate choice for larger females with large ornament was, however, hampered under turbid conditions, whereas in clear water males choose larger, more ornamented females. Thus, we show that water turbidity had no effect on signal expression but did hamper ornament perception and consequently randomized mate choice.     

Molecules and morphology in concert. II. The Festuca brachyphylla complex (Poaceae) in Svalbard

We used a combined molecular and morphological approach to unravel variation in the autogamous Festuca brachyphylla polyploid complex in the arctic archipelago of Svalbard. Forty populations were analyzed for random amplified polymorphic DNAs (RAPDs) and 46 morphological characters. Eighteen RAPD multilocus phenotypes were observed in the 86 plants analyzed, based on 30 polymorphic markers. Multivariate analyses of the RAPD data revealed four distinct groups of multilocus phenotypes; in contrast, the variation was more or less continuous in multivariate analyses of the morphological data. However, we identified several individual morphological characters that unambiguously discriminated among the four groups of RAPD multilocus phenotypes. Analysis of type material suggests that the four groups in Svalbard can be referred to Festuca baffinensis, F. brachyphylla, F. hyperborea, and F. edlundiae. This study shows that concerted analysis of molecules and morphology is a powerful tool in low-level taxonomy.     

Interactive effects of lettuce (Lactuca sativa L.), irradiance, and ferulic acid in axenic, hydroponic culture

Ferulic acid (FA) is released by living roots and by decaying plant material and is involved in chemical interactions between plants. Effects of FA on plant growth and root development of lettuce (Lactuca sativa L. cv. Grand Rapids) cultivated in axenic nutrient solution were studied in two factorial experiments. Root and shoot growth was impeded when 200 M trans-FA was added to the nutrient solution and the light intensity was in the range of 250–380 mol m^-2 s^-1. Root growth showed a stronger response to FA than did shoot growth. At 200 M, FA strongly inhibited root hair formation and reduced mean lengths of primary, secondary and tertiary roots, but stimulated primary and secondary root branching. Both isomerization to the cis isomer and the presence of the plant reduced the concentration of trans-FA in the nutrient solution during the two weeks exposure period. A third experiment was conducted to assess the influence of irradiance on the phytotoxicity of FA. At a light intensity of 489 mol m^-2 s^-1, or in the presence of microorganisms, the concentration of FA in the nutrient solution was lowered and the phytotoxic effects were reduced.     

Automated large-volume sample stacking procedure to detect labeled peptides at picomolar concentration using capillary electrophoresis and laser-induced fluorescence detection

We have developed an automated large-volume sample stacking (LVSS) procedure to detect fluorescein isothiocyanate-labeled peptides in the picomolar range. The injection duration is 10 min at 50 mbar to fill 62% of the capillary volume to the detection cell. The calculated limit of detection (S/N=3), filling 1% of the capillary volume, is 74 pM for bradykinin and 45 pM for L-enkephalin with samples diluted in water and analyzed in a 50 mM borate buffer, pH 9.2. With the automated LVSS system, the limits of detection are 7 pM for bradykinin, 3 pM for L-enkephalin and 2 pM for substance P. LVSS is shown to be quantitative from 500 to 10 pM.     

Favorable Alteration of Tumor Microenvironment by Immunomodulatory Cytokines for Efficient T-Cell Therapy in Solid Tumors

Unfavorable ratios between the number and activation status of effector and suppressor immune cells infiltrating the tumor contribute to resistance of solid tumors to T-cell based therapies. Here, we studied the capacity of FDA and EMA approved recombinant cytokines to manipulate this balance in favor of efficient anti-tumor responses in B16.OVA melanoma bearing C57BL/6 mice. Intratumoral administration of IFN-α2, IFN-γ, TNF-α, and IL-2 significantly enhanced the anti-tumor effect of ovalbumin-specific CD8+ T-cell (OT-I) therapy, whereas GM-CSF increased tumor growth in association with an increase in immunosuppressive cell populations. None of the cytokines augmented tumor trafficking of OT-I cells significantly, but injections of IFN-α2, IFN-γ and IL-2 increased intratumoral cytokine secretion and recruitment of endogenous immune cells capable of stimulating T-cells, such as natural killer and maturated CD11c+ antigen-presenting cells. Moreover, IFN-α2 and IL-2 increased the levels of activated tumor-infiltrating CD8+ T-cells concomitant with reduction in the CD8+ T-cell expression of anergy markers CTLA-4 and PD-1. In conclusion, intratumoral administration of IFN-α2, IFN-γ and IL-2 can lead to immune sensitization of the established tumor, whereas GM-CSF may contribute to tumor-associated immunosuppression. The results described here provide rationale for including local administration of immunostimulatory cytokines into T-cell therapy regimens. One appealing embodiment of this would be vectored delivery which could be advantageous over direct injection of recombinant molecules with regard to efficacy, cost, persistence and convenience.     

The immunomodulatory Pseudomonas aeruginosa signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone enters mammalian cells in an unregulated fashion

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.