Formation of Phosphoglycosides in Caenorhabditis elegans: A Novel Biotransformation Pathway

Background

Caenorhabditis elegans (C. elegans) has become a widely used model to explore the effect of food constituents on health as well as on life-span extension. The results imply that besides essential nutrients several flavonoids are able to impact the aging process. What is less investigated is the bioavailability and biotransformation of these compounds in C. elegans. In the present study, we focused on the soy isoflavone genistein and its metabolism in the nematode as a basis for assessing whether this model system mimics the mammalian condition.

Principal Findings

C. elegans was exposed to 100 µM genistein for 48 hours. The worm homogenate was extracted and analyzed by liquid chromatography (LC). 11 metabolites of genistein were detected and characterized using LC electrospray ionization mass spectrometry. All genistein metabolites formed by C. elegans were found to be sugar conjugates, primarily genistein-O-glucosides. The dominant metabolite was identified as genistein-7-O-phosphoglucoside. Further interesting metabolites include two genistein-di-O-glycosides, a genistein-O-disaccharide as well as a genistein-O-phosphodisaccharide.

Conclusions/Significance

Our study provides evidence for a novel biotransformation pathway in C. elegans leading to conjugative metabolites which are not known for mammals. The metabolism of genistein in mammals and in C. elegans differs widely which may greatly impact the bioactivity. These differences need to be appropriately taken into consideration when C. elegans is used as a model to assess possible health or aging effects.     

Simultaneous Simulations of Uptake in Plants and Leaching to Groundwater of Cadmium and Lead for Arable Land Amended with Compost or Farmyard Manure

The water budget of soil, the uptake in plants and the leaching to groundwater of cadmium (Cd) and lead (Pb) were simulated simultaneously using a physiological plant uptake model and a tipping buckets water and solute transport model for soil. Simulations were compared to results from a ten-year experimental field study, where four organic amendments were applied every second year. Predicted concentrations slightly decreased (Cd) or stagnated (Pb) in control soils, but increased in amended soils by about 10% (Cd) and 6% to 18% (Pb). Estimated plant uptake was lower in amended plots, due to an increase of K_d (dry soil to water partition coefficient). Predicted concentrations in plants were close to measured levels in plant residues (straw), but higher than measured concentrations in grains. Initially, Pb was mainly predicted to deposit from air into plants (82% in 1998); the next years, uptake from soil became dominating (30% from air in 2006), because of decreasing levels in air. For Cd, predicted uptake from air into plants was negligible (1–5%).     

Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses

The TLRs 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. U-rich RNA sequences were recently discovered that stimulate human TLR7/8-mediated or murine TLR7-mediated immune effects. In this study we identified single-stranded RNA sequences containing defined sequence motifs that either preferentially activate human TLR8-mediated as opposed to TLR7- or TLR7/8-mediated immune responses. The identified TLR8 RNA motifs signal via TLR8 and fail to induce IFN-alpha from TLR7-expressing plasmacytoid dendritic cells but induce the secretion of Th1-like and proinflammatory cytokines from TLR8-expressing immune cells such as monocytes or myeloid dendritic cells. In contrast, RNA sequences containing the TLR7/8 motif signal via TLR7 and TLR8 and stimulate cytokine secretion from both TLR7- and TLR8-positive immunocytes. The TLR8-specific RNA sequences are able to trigger cytokine responses from human and bovine but not from mouse, rat, and porcine immune cells, suggesting that these species lack the capability to respond properly to TLR8 RNA ligands. In summary, we describe two classes of single-stranded TLR7/8 and TLR8 RNA agonists with diverse target cell and species specificities and immune response profiles.     

Lipopolysaccharide-activated IL-10-secreting dendritic cells suppress experimental autoimmune uveoretinitis by MHCII-dependent activation of CD62L-expressing regulatory T cells

Dendritic cells (DC) are key regulators of immune responses. Mature DC are traditionally considered to be immunogenic, although there is accumulating evidence that they can also be tolerogenic and induce Ag-specific regulatory T cells (Tregs). However, the mechanism of this Treg induction and the site of Treg action in vivo are yet to be defined. In this study, using the experimental model of interphotoreceptor retinoid-binding protein peptide (1-20)-induced experimental autoimmune uveoretinitis, we show that s.c. inoculation of IRBP-peptide-pulsed IL-10-producing LPS-activated mature DC (IL-10-DC) at one site (the cervical region) suppresses autoimmunity induced at a separate site (the inguinal region). Our data show that s.c. IL-10-DC correlates with an increase in the number of CD4(+)CD25(+)Foxp3(+) Tregs at the DC-draining lymph nodes (DC-dLN). However, although MHCII(-/-) IL-10-DC also induces Treg expansion at this DC-dLN, they failed to suppress experimental autoimmune uveoretinitis. Furthermore, unlike wild-type IL-10-DC, MHCII(-/-) IL-10-DC did not correlate with an increase in the percentage of Tregs expressing CD62L at the DC-dLN, nor did they associate with an increase in Treg number at a distal site. Similar effects were also observed after s.c. hen egg lysozyme-pulsed IL-10-DC, which produced a strong reduction in the number and activation of proliferating Ag-specific CD4(+) 3A9 T effector cells. We therefore propose that IL-10-DC require MHCII-dependent Ag presentation, and hence TCR ligation, to promote CD62L-mediated trafficking of Tregs to the site of T effector cell priming, where they suppress autoimmunity.     

Lipid mapping in human dystrophic muscle by cluster-time-of-flight secondary ion mass spectrometry imaging

Human striated muscle samples, from male control and Duchenne muscular dystrophy-affected children, were subjected to cluster-time-of-flight secondary ion mass spectrometry (cluster-ToF-SIMS) imaging using a 25 keV Bi(3)(+) liquid metal ion gun under static SIMS conditions. Spectra and ion density maps, or secondary ion images, were acquired in both positive and negative ion mode over several areas of 500 x 500 microm(2) (image resolution, 256 x 256 pixels). Characteristic distributions of various lipids were observed. Vitamin E and phosphatidylinositols were found to concentrate within the cells, whereas intact phosphocholines accumulated over the most damaged areas of the dystrophic muscles, together with cholesterol and sphingomyelin species. Fatty acyl chain composition varied depending on the region, allowing estimation of the local damage extent.     

The GENIPOL European flounder Platichthys flesus L. toxicogenomics microarray: application for investigation of the response to furunculosis vaccination

The GENIPOL cDNA microarray, comprising some 14 000 elements representing 3336 unique expressed sequence tag clusters, was developed for studies on pollutant chemical effects on hepatic gene expression in the sentinel flatfish species Platichthys flesus , the European flounder. This array derived from probes obtained by targeted cloning of xenobiotic metabolising and responsive genes, subtractive suppressive hybridization of pollutant-treated fish and subtracted normalized cDNA libraries of treated fish has been successfully utilized for diagnosing effects of prototypical pollutants, endocrine disruptors and analysis of environmental effects. In the latter context, it has been recommended by the International Council for Exploration of the Sea that the array is tested in European monitoring programmes for assessment of the biological effects of contaminants. Sequence homology is sufficiently great between P. flesus and other pleuronectid flatfish to enable satisfactory use with other species, including American winter flounder and Japanese flounder, plaice, Atlantic halibut and sole. Elucidation of the effects of pollutants requires knowledge of other affectors of gene expression, and in this context the observed effects of a bacterial infection have been studied. The 'infection' was experimentally applied by challenge with a vaccine containing Aeromonas salmonicida , the bacterial agent causing furunculosis in flounder and salmonids. These results show that, despite its currently limited gene coverage, application of this array is not confined to toxicology.     

Stromal cells confer lymph node-specific properties by shaping a unique microenvironment influencing local immune responses

Lymph nodes (LN) consist not only of highly motile immune cells coming from the draining area or from the systemic circulation, but also of resident stromal cells building the backbone of the LN. These two cell types form a unique microenvironment which is important for initiating an optimal immune response. The present study asked how the unique microenvironment of the mesenteric lymph node (mLN) is influenced by highly motile cells and/or by the stromal cells. A transplantation model in rats and mice was established. After resecting the mLN, fragments of peripheral lymph node (pLN) or mLN were inserted into the mesentery. The pLN and mLN have LN-specific properties, resulting in differences of, for example, the CD103(+) dendritic cell subset, the adhesion molecule mucosal addressin cell adhesion molecule 1, the chemokine receptor CCR9, the cytokine IL-4, and the enzyme retinal dehydrogenase 2. This new model clearly showed that during regeneration stromal cells survived and immune cells were replaced. Surviving high endothelial venules retained their site-specific expression (mucosal addressin cell adhesion molecule 1). In addition, the low expression of retinal dehydrogenase 2 and CCR9 persisted in the transplanted pLN, suggesting that stromal cells influence the lymph node-specific properties. To examine the functional relevance of this different expression pattern in transplanted animals, an immune response against orally applied cholera toxin was initiated. The data showed that the IgA response against cholera toxin is significantly diminished in animals transplanted with pLN. This model documents that stromal cells of the LN are active players in shaping a unique microenvironment and influencing immune responses in the drained area.     

Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.