Investigation of S<Subscript>N</Subscript>2 [<Superscript>11</Superscript>C]cyanation for base-sensitive substrates: an improved radiosynthesis of <Emphasis Type="SmallCaps">l</Emphasis>-[5-<Superscript>11</Superscript>C]-glutamine

Carbon-11 (β⁺ emitter, t _1/2 = 20.4 min) radiolabeled l-glutamine is a potentially useful molecular imaging agent that can be utilized with positron emission tomography for both human oncological diagnosis and plant imaging research. Based upon a previously reported [¹¹C]cyanide end-capping labeling method, a systematic investigation of nucleophilic cyanation reactions and acidic hydrolysis reaction parameters, including base, metal ion source, phase transfer catalyst, solvent, reaction temperature and reaction time, was conducted. The result was a milder, more reliable, two-step method which provides l-[5-¹¹C]-glutamine with a radiochemical yield of 63.8 ± 8.7 % (range from 51 to 74 %, n = 10) with >90 % radiochemical purity and >90 % enantiomeric purity. The total synthesis time was 40–50 min from the end of bombardment. In addition, an Fmoc derivatization method was developed to measure the specific activity of this radiotracer.     

A horizontally transferred nuclear gene is associated with microhabitat variation in a natural plant population

Horizontal gene transfer involves the non-sexual interspecific transmission of genetic material. Even if they are initially functional, horizontally transferred genes are expected to deteriorate into non-expressed pseudogenes, unless they become adaptively relevant in the recipient organism. However, little is known about the distributions of natural transgenes within wild species or the adaptive significance of natural transgenes within wild populations. Here, we examine the distribution of a natural plant-to-plant nuclear transgene in relation to environmental variation within a wild population. Festuca ovina is polymorphic for an extra (second) expressed copy of the nuclear gene (PgiC) encoding cytosolic phosphoglucose isomerase, with the extra PgiC locus having been acquired horizontally from the distantly related grass genus Poa. We investigated variation at PgiC in samples of F. ovina from a fine-scale, repeating patchwork of grassland microhabitats, replicated within spatially separated sites. Even after accounting for spatial effects, the distributions of F. ovina individuals carrying the additional PgiC locus, and one of the enzyme products encoded by the locus, are significantly associated with fine-scale habitat variation. Our results suggest that the PgiC transgene contributes, together with the unlinked ‘native’ PgiC locus, to local adaptation to a fine-scale mosaic of edaphic and biotic grassland microhabitats.     

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.     

Anti-type II collagen immune complex-induced granulocyte reactivity is associated with joint erosions in RA patients with anti-collagen antibodies

IntroductionRheumatoid arthritis (RA) patients with autoantibodies against collagen type II (CII) are characterized by acute RA onset with elevated inflammatory measures and early joint erosions as well as increased production of tumor necrosis factor-α (ΤΝF-α) by peripheral blood mononuclear cells (PBMC) stimulated by anti-CII immune complexes (IC) in vitro. Polymorphonuclear granulocytes (PMN) are abundant in RA synovial fluids, where they might interact directly with anti-CII IC in the articular cartilage, but no studies have investigated PMN responses towards anti-CII IC. The aim was to investigate whether PMN react towards anti-CII IC, and to what extent such reactivity might relate to the clinical acute onset RA phenotype associated with elevated levels of anti-CII.MethodsPMN and PBMC isolated from healthy donors were stimulated with IC made with a set of 72 baseline patient sera (24 anti-CII positive, 48 anti-CII negative) chosen from a clinically well-characterized RA cohort with two-year radiological follow-up with Larsen scoring. PMN expression of cluster of differentiation (CD)11b, CD66b, CD16 and CD32 was measured by flow cytometry, whereas PMN production of myeloperoxidase (MPO) and interleukin (IL)-17, and PBMC production of ΤΝF-α was measured with enzyme linked immunosorbent assay.ResultsPMN expression of CD11b, CD66b and MPO, and PBMC production of ΤΝF-α were upregulated whereas PMN expression of CD16 and CD32 were downregulated by anti-CII IC. CD16, CD66b, and MPO production correlated to serum anti-CII levels (Spearman’s ρ = 0.315, 0.675 and 0.253, respectively). CD16 was associated with early joint erosions (P = 0.024, 0.034, 0.046 at baseline, one and two years) and CD66b was associated with changes in joint erosions (P = 0.017 and 0.016, at one and two years compared to baseline, respectively). CD66b was associated with baseline C-reactive protein and PBMC production of ΤΝF-α was associated with baseline erythrocyte sedimentation rate, in accordance with our earlier findings. No clinical associations were observed for MPO or IL-17.ConclusionPMN responses against anti-CII IC are more closely associated with early joint erosions than are PBMC cytokine responses. PMN reactivity against anti-CII IC may contribute to joint destruction in newly diagnosed RA patients with high levels of anti-CII.     

Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin) included in rosin-based coatings

Rosin-based coatings loaded with 0.1% (w/v) ivermectin were found to be effective in preventing colonization by barnacles (Balanus improvisus) both on test panels as well as on yachts for at least two fouling seasons. The leaching rate of ivermectin was determined by mass-spectroscopy (LC/MS-MS) to be 0.7?ng cm(-2) day(-1). This low leaching rate, as deduced from the Higuchi model, is a result of the low loading, low water solubility, high affinity to the matrix and high molar volume of the model biocide. Comparison of ivermectin and control areas of panels immersed in the field showed undisturbed colonisation of barnacles after immersion for 35 days. After 73 days the mean barnacle base plate area on the controls was 13?mm(2), while on the ivermectin coating it was 3?mm(2). After 388 days, no barnacles were observed on the ivermectin coating while the barnacles on the control coating had reached a mean of 60?mm(2). In another series of coated panels, ivermectin was dissolved in a cosolvent mixture of propylene glycol and glycerol formal prior to the addition to the paint base. This method further improved the anti-barnacle performance of the coatings. An increased release rate (3?ng cm(-2) day(-1)) and dispersion of ivermectin, determined by fluorescence microscopy, and decreased hardness of the coatings were the consequences of the cosolvent mixture in the paint. The antifouling mechanism of macrocyclic lactones, such as avermectins, needs to be clarified in further studies. Beside chronic intoxication as ivermectin is slowly released from the paint film even contact intoxication occurring inside the coatings, triggered by penetration of the coating by barnacles, is a possible explanation for the mode of action and this is under investigation.     

Inhibition of thioredoxin reductase 1 by porphyrins and other small molecules identified by a high-throughput screening assay

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K_i = 2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k_inact) of (0.73 ± 0.07) × 10^? 3 μM^? 1 min^? 1. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.