Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths

Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.     

Turning the 'mustard oil bomb' into a 'cyanide bomb': aromatic glucosinolate metabolism in a specialist insect herbivore

Plants have evolved a variety of mechanisms for dealing with insect herbivory among which chemical defense through secondary metabolites plays a prominent role. Physiological, behavioural and sensorical adaptations to these chemicals provide herbivores with selective advantages allowing them to diversify within the newly occupied ecological niche. In turn, this may influence the evolution of plant metabolism giving rise to e.g. new chemical defenses. The association of Pierid butterflies and plants of the Brassicales has been cited as an illustrative example of this adaptive process known as 'coevolutionary armsrace'. All plants of the Brassicales are defended by the glucosinolate-myrosinase system to which larvae of cabbage white butterflies and related species are biochemically adapted through a gut nitrile-specifier protein. Here, we provide evidence by metabolite profiling and enzyme assays that metabolism of benzylglucosinolate in Pieris rapae results in release of equimolar amounts of cyanide, a potent inhibitor of cellular respiration. We further demonstrate that P. rapae larvae develop on transgenic Arabidopsis plants with ectopic production of the cyanogenic glucoside dhurrin without ill effects. Metabolite analyses and fumigation experiments indicate that cyanide is detoxified by β-cyanoalanine synthase and rhodanese in the larvae. Based on these results as well as on the facts that benzylglucosinolate was one of the predominant glucosinolates in ancient Brassicales and that ancient Brassicales lack nitrilases involved in alternative pathways, we propose that the ability of Pierid species to safely handle cyanide contributed to the primary host shift from Fabales to Brassicales that occured about 75 million years ago and was followed by Pierid species diversification.     

Free fatty acid palmitate impairs the vitality and function of cultured human bladder smooth muscle cells

Background

Incidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6) in cultured human bladder smooth muscle cells (hBSMC). Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways.

Methodology/Principal Findings

HBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-κB, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i) palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii) direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii) in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv) further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v) increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1.

Conclusions/Significance

Saturated free fatty acids (e.g., palmitate) cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby, certain cytokines might counteract the palmitate-induced downregulation of cell proliferation and vitality. This could be an important link to clinical findings of increased risk of metabolic related bladder diseases such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).     

Serum Level of CC-Chemokine Ligand 18 Is Increased in Patients with Non-Small-Cell Lung Cancer and Correlates with Survival Time in Adenocarcinomas

CC-chemokine ligand 18 (CCL18) is mainly expressed by alternatively activated macrophages and DCs and plays an important role in lung fibrosis, arthritis and other diseases. Here CCL18 was measured in sera of 31 healthy volunteers and 170 patients with lung cancer and correlated these data with histology, tumor stage and clinical parameters. Mean CCL18 serum level of the patients with non-small-cell lung cancer was 150(857) ng/ml vs. 32(61) ng/ml in the healthy control group. Patient groups differ significantly according their histology (adenocarcinoma 143(528) ng/ml vs squamous cell carcinoma 187(857) ng/ml, p<0.02). In addition, we found a significant difference between patients with lower versus higher T-stage (p<0.003). Receiver operating characteristic (ROC) analyses revealed a cutoff point of 83 ng/ml (area under the curve (AUC): 0.968; p<0.0001) to discriminate between healthy controls and non-small-cell lung cancer patients. ROC analyses to discriminate between patients, who died because of cancer related death and those who died for other reasons did not lead to a valid AUC. To stratify the tumor patients, a criterion value plot was performed leading to a point of equal sensitivity and specificity (54%) of 162 ng/ml. Patients with a CCL18 serum level higher than 160 ng/ml had a mean survival time of 623 days. In contrast, those in patients with a baseline level between 83 ng/ml and 160 ng/ml the mean survival time was 984 days (p<0.005). Survival-analysis revealed in adenocarcinoma a mean survival of 1152 days in the group below 83 ng/ml. In the median group the mean survival time was 788 days and in the group with the highest levels the mean survival time was 388 days (p<0.001). In contrast, we found no correlation between the FEV1 and the CCL18 baseline level. In conclusion, in patients suffering from adenocarcinoma increased serum CCL18 levels predict a diminished survival time.     

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis,Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P₂. In N. crassa hyphae, PtdIns(4,5)P₂ localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P₂ that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.     

Induction,rapid fixation and retention of mutations in vegetatively propagated banana

Mutation discovery technologies have enabled the development of reverse genetics for many plant species and allowed sophisticated evaluation of the consequences of mutagenesis. Such methods are relatively straightforward for seed‐propagated plants. To develop a platform suitable for vegetatively propagated species, we treated isolated banana shoot apical meristems with the chemical mutagen ethyl methanesulphonate, recovered plantlets and screened for induced mutations. A high density of GC‐AT transition mutations were recovered, similar to that reported in seed‐propagated polyploids. Through analysis of the inheritance of mutations, we observed that genotypically heterogeneous stem cells resulting from mutagenic treatment are rapidly sorted to fix a single genotype in the meristem. Further, mutant genotypes are stably inherited in subsequent generations. Evaluation of natural nucleotide variation showed the accumulation of potentially deleterious heterozygous alleles, suggesting that mutation induction may uncover recessive traits. This work therefore provides genotypic insights into the fate of totipotent cells after mutagenesis and suggests rapid approaches for mutation‐based functional genomics and improvement of vegetatively propagated crops.     

Surface Plasmon Polariton Emission Prompted by Organic Nanofibers on Thin Gold Films

The excitation of surface plasmon polaritons (SPP) at a gold?Cvacuum interface by femtosecond light pulses mediated by organic nanofiber-induced dielectric perturbations is observed using interferometric time-resolved photoemission electron microscopy. The experimental data are quantitatively reproduced by analytic simulations, where the nanofibers are considered as superior source of the SPP emission. The flexibility and tuneability of phenylene-based nanofibers in their morphology and intrinsic optical properties open up future applications to fabricate custom-designed nanoscale sources of SPP.