Search

The down-regulation of a tobacco (Nicotiana tabacum) plasma membrane-localized nicotine uptake permease, NUP1, was previously reported to reduce total alkaloid levels in tobacco plants. However, it was unclear how this nicotine transporter affected the biosynthesis of the alkaloid nicotine. When NUP1 expression was suppressed in cultured tobacco cells treated with jasmonate, which induces nicotine biosynthesis, the NICOTINE2-locus transcription factor gene ETHYLENE RESPONSE FACTOR189 (ERF189) and its target structural genes, which function in nicotine biosynthesis and transport, were strongly suppressed, resulting in decreased total alkaloid levels. Conversely, NUP1 overexpression had the opposite effect. In these experiments, the expression levels of the MYC2 transcription factor gene and its jasmonate-inducible target gene were not altered. Inhibiting tobacco alkaloid biosynthesis by suppressing the expression of genes encoding enzymes in the nicotine pathway did not affect the expression of ERF189 and other nicotine pathway genes, indicating that ERF189 is not regulated by cellular alkaloid levels. Suppressing the expression of jasmonate signaling components in cultured tobacco cells showed that NUP1 acts downstream of the CORONATINE INSENSITIVE1 receptor and MYC2, but upstream of ERF189. These results suggest that although jasmonate-activated expression of MYC2 induces the expression of both NUP1 and ERF189, expression of ERF189 may actually be mediated by NUP1. Furthermore, NUP1 overexpression in tobacco plants inhibited the long-range transport of nicotine from the roots to the aerial parts. Thus, NUP1 not only mediates the uptake of tobacco alkaloids into root cells, but also positively controls the expression of ERF189, a key gene in the biosynthesis of these alkaloids., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Plant pathogens deploy an array of virulence factors to suppress host defense and promote pathogenicity. Numerous strains of Pseudomonas syringae produce the phytotoxin coronatine (COR). A major aspect of COR function is its ability to mimic a bioactive jasmonic acid (JA) conjugate and thus target the JA-receptor COR-insensitive 1 (COI1). Biological activities of COR include stimulation of JA-signaling and consequent suppression of SA-dependent defense through antagonistic crosstalk, antagonism of stomatal closure to allow bacterial entry into the interior of plant leaves, contribution to chlorotic symptoms in infected plants, and suppression of plant cell wall defense through perturbation of secondary metabolism. Here, we review the virulence function of COR, including updates on these established activities as well as more recent findings revealing COI1-independent activity of COR and shedding light on cooperative or redundant defense suppression between COR and type III effector proteins.

Brazilein, a natural small molecule, shows a variety of pharmacological activities, especially on nervous system and immune system. As a potential multifunctional drug, we studied the distribution and the transport behavior and metabolic behavior of brazilein in vivo and in vitro. Brazilein was found to be able to distribute in the mouse brain and transport into neural cells. A metabolite was found in the brain and in the cells. Positive and negative mode-MS/MS and Q-TOF were used to identify the metabolite. MS/MS fragmentation mechanisms showed the methylation occurred at the 10-hydroxyl of brazilein (10-O-methylbrazilein). Further, catechol-O- methyltransferase (COMT) was confirmed as a crucial enzyme correlated with the methylated metabolite generation by molecular docking and pharmacological experiment.

Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-L-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study., (© 2014 American Society of Plant Biologists. All rights reserved.)

(+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile) regulates developmental and stress responses in plants. Its perception involves the formation of a ternary complex with the F-box COI1 and a member of the JAZ family of co-repressors and leads to JAZ degradation. Coronatine (COR) is a bacterial phytotoxin that functionally mimics JA-Ile and interacts with the COI1-JAZ co-receptor with higher affinity than JA-Ile. On the basis of the co-receptor structure, we designed ligand derivatives that spatially impede the interaction of the co-receptor proteins and, therefore, should act as competitive antagonists. One derivative, coronatine-O-methyloxime (COR-MO), has strong activity in preventing the COI1-JAZ interaction, JAZ degradation and the effects of JA-Ile or COR on several JA-mediated responses in Arabidopsis thaliana. Moreover, it potentiates plant resistance, preventing the effect of bacterially produced COR during Pseudomonas syringae infections in different plant species. In addition to the utility of COR-MO for plant biology research, our results underscore its biotechnological potential for safer and sustainable agriculture.

To restrict pathogen entry, plants close stomata as an integral part of innate immunity. To counteract this defense, Pseudomonas syringae pv tomato produces coronatine (COR), which mimics jasmonic acid (JA), to reopen stomata for bacterial entry. It is believed that abscisic acid (ABA) plays a central role in regulating bacteria-triggered stomatal closure and that stomatal reopening requires the JA/COR pathway, but the downstream signaling events remain unclear. We studied the stomatal immunity of tomato (Solanum lycopersicum) and report here the distinct roles of two homologous NAC (for NAM, ATAF1,2, and CUC2) transcription factors, JA2 (for jasmonic acid2) and JA2L (for JA2-like), in regulating pathogen-triggered stomatal movement. ABA activates JA2 expression, and genetic manipulation of JA2 revealed its positive role in ABA-mediated stomatal closure. We show that JA2 exerts this effect by regulating the expression of an ABA biosynthetic gene. By contrast, JA and COR activate JA2L expression, and genetic manipulation of JA2L revealed its positive role in JA/COR-mediated stomatal reopening. We show that JA2L executes this effect by regulating the expression of genes involved in the metabolism of salicylic acid. Thus, these closely related NAC proteins differentially regulate pathogen-induced stomatal closure and reopening through distinct mechanisms., (© 2014 American Society of Plant Biologists. All rights reserved.)

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 μg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 μg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 μg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 μg/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.

The duration of action of melatonin may be important for improvements in sleep efficiency in insomniacs. Ramelteon, a selective melatonin agonist, is primarily metabolized to the active metabolite M-II, which has a longer half-life and greater systemic exposure than ramelteon. Hence, M-II may contribute significantly to the hypnotic benefits of ramelteon. We assessed the ramelteon-like activity of M-II in vitro and in vivo using cats. Binding and functional studies in Chinese hamster ovary cells expressing human melatonin receptors (MT1 or MT2) revealed that M-II binds melatonin receptors with lower affinity (Ki: 114 and 566 pmol/l for MT1 and MT2, respectively) and has lower potency (IC50: 208 and 1,470 pmol/l for MT1 and MT2, respectively) compared with ramelteon. However, higher M-II doses significantly improved sleep in cats. Thus, M-II may contribute to the clinical efficacy of ramelteon., (© 2014 S. Karger AG, Basel.)

In a recent Proc. Natl. Acad. Sci. USA paper by Wiemann et al., a gene supercluster for secondary metabolite production in Aspergillus fumigatus is genetically dissected. The analysis demonstrates that gene regulation takes place at different layers and that genes belonging to specific clusters are intertwined., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the control of the master regulator of secondary metabolism, LaeA, contains, in its entirety, the genetic machinery for three natural products (fumitremorgin, fumagillin, and pseurotin), where genes for fumagillin and pseurotin are physically intertwined in a single supercluster. Deletions of 29 adjoining genes revealed that fumagillin and pseurotin are coregulated by the supercluster-embedded regulatory gene with biosynthetic genes belonging to one of the two metabolic pathways in a noncontiguous manner. Comparative genomics indicates the fumagillin/pseurotin supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This blended design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution.

Gram-negative bacterial pathogens deliver a variety of virulence proteins through the type III secretion system (T3SS) directly into the host cytoplasm. These type III secreted effectors (T3SEs) play an essential role in bacterial infection, mainly by targeting host immunity. However, the molecular basis of their functionalities remains largely enigmatic. Here, we show that the Pseudomonas syringae T3SE HopZ1a, a member of the widely distributed YopJ effector family, directly interacts with jasmonate ZIM-domain (JAZ) proteins through the conserved Jas domain in plant hosts. JAZs are transcription repressors of jasmonate (JA)-responsive genes and major components of the jasmonate receptor complex. Upon interaction, JAZs can be acetylated by HopZ1a through a putative acetyltransferase activity. Importantly, P. syringae producing the wild-type, but not a catalytic mutant of HopZ1a, promotes the degradation of HopZ1-interacting JAZs and activates JA signaling during bacterial infection. Furthermore, HopZ1a could partially rescue the virulence defect of a P. syringae mutant that lacks the production of coronatine, a JA-mimicking phytotoxin produced by a few P. syringae strains. These results highlight a novel example by which a bacterial effector directly manipulates the core regulators of phytohormone signaling to facilitate infection. The targeting of JAZ repressors by both coronatine toxin and HopZ1 effector suggests that the JA receptor complex is potentially a major hub of host targets for bacterial pathogens.

The nonhost-specific phytotoxin coronatine (COR) produced by several pathovars of Pseudomonas syringae functions as a jasmonic acid-isoleucine (JA-Ile) mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1) is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators for JA signaling in Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR) cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000), and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS), we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP), INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.

Background: Anticoagulants are the only available compounds in the EU to control rat populations. Resistance to anticoagulant rodenticides (antivitamin K or AVK) is described and widespread across Europe. The present objective was to determine whether resistance was associated with an increased potential for bioaccumulation of AVK in the liver. Rats were selected from three major resistant genetically identified strains across Europe: Y139C (Germany), Y139F (France) and L120Q (United Kingdom). The rats were housed in individual cages and fed chlorophacinone wheat bait (50 mg kg(-1) ). Animals were assigned to groups for euthanasia either on day 1, 4, 9 or 14 (resistant rats) or on days 1 and 4 (susceptible rats)., Results: Chlorophacinone accumulated from day 1 to day 4 in all strains (maximum 160 µg liver(-1)) and remained stable thereafter. There was no significant difference between strains. Extensive metabolism of chlorophacinone was also found, and was similar (in nature and proportion of metabolites) across strains (3 OH-metabolites identified). Only the survival time differed significantly (L120Q > Y139C = Y139F > susceptible)., Conclusions: Accumulation of chlorophacinone occurs from day 1 to day 4, and an equilibrium is reached, suggesting rapid elimination. Resistant and susceptible rats accumulate chlorophacinone to the same extent and only differ in terms of survival times. Resistant rats may then be a threat for non-target species for prolonged periods of time., (Copyright © 2012 Society of Chemical Industry.)

Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14mg/L in control conditions to 21.48mg/L (day 12) with MeJA and 77.46mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA- or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Pseudomonas syringae pv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plant Nicotiana benthamiana in a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed in N. benthamiana with cfa, cma, and cmaL mutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either the cma operon or cmaL accumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered in cmaL mutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaL mutant when it is supplemented with 50 μg/ml l-allo-isoleucine, the starting unit for CMA production. cmaL is found in all other sequenced P. syringae strains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family.

Strigolactones are plant signaling molecules that induce germination of parasitic plant seeds, initiate host plant - arbuscular mycorrhizal fungus symbiosis and act as plant hormones controlling shoot branching and root architecture. To date four unique strigolactones (e.g., orobanchol, didehydroorobanchol isomers 1 and 2 and the aromatic strigolactone solanacol) have been reported in the root exudates and extracts of tomato (Solanum lycopersicum). Here we report on the presence of several additional strigolactones in tomato root exudates and extracts, orobanchyl acetate, two 7-hydroxyorobanchol isomers, 7-oxoorobanchol and two additional didehydroorobanchol isomers and discuss their possible biological relevance.

Hydroxylated analogues of the anticancer topoisomerase I (Top1) inhibitors indotecan (LMP400) and indimitecan (LMP776) have been prepared because (1) a variety of potent Top1 poisons are known that contain strategically placed hydroxyl groups, which provides a clear rationale for incorporating them in the present case, and (2) the hydroxylated compounds could conceivably serve as synthetic standards for the identification of metabolites. Indeed, incubating LMP400 and LMP776 with human liver microsomes resulted in two major metabolites of each drug, which had HPLC retention times and mass fragmentation patterns identical to those of the synthetic standards. The hydroxylated indotecan and indimitecan metabolites and analogues were tested as Top1 poisons and for antiproliferative activity in a variety of human cancer cell cultures and in general were found to be very potent. Differences in activity resulting from the placement of the hydroxyl group are explained by molecular modeling analyses.

Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin-type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O-prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His(8)-FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. K(M) values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 μM, respectively. Average turnover number (k(cat)) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s(-1). FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13-dihydroxyfumitremorgin C, as substrates and catalyses their prenylation., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The phytotoxin coronatine (COR) promotes various aspects of Pseudomonas syringae virulence, including invasion through stomata, growth in the apoplast, and induction of disease symptoms. COR is a structural mimic of active jasmonic acid (JA) conjugates. Known activities of COR are mediated through its binding to the F-box-containing JA coreceptor CORONATINE INSENSITIVE1. By analyzing the interaction of P. syringae mutants with Arabidopsis thaliana mutants, we demonstrate that, in the apoplastic space of Arabidopsis, COR is a multifunctional defense suppressor. COR and the critical P. syringae type III effector HopM1 target distinct signaling steps to suppress callose deposition. In addition to its well-documented ability to suppress salicylic acid (SA) signaling, COR suppresses an SA-independent pathway contributing to callose deposition by reducing accumulation of an indole glucosinolate upstream of the activity of the PEN2 myrosinase. COR also suppresses callose deposition and promotes bacterial growth in coi1 mutant plants, indicating that COR may have multiple targets inside plant cells.

Jasmonates are lipid-derived compounds that act as signals in plant stress responses and developmental processes. Enzymes participating in biosynthesis of jasmonic acid (JA) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants have helped to define the pathway for synthesis of jasmonoyl-L-isoleucine (JA-Ile), the bioactive form of JA, and to identify the F-box protein COI1 as central regulatory unit. Details on the molecular mechanism of JA signaling were recently unraveled by the discovery of JAZ proteins that together with the adaptor protein NINJA and the general co-repressor TOPLESS form a transcriptional repressor complex. The current model of JA perception and signaling implies the SCF(COI1) complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ proteins for degradation by the 26S proteasome pathway, thereby allowing MYC2 and other transcription factors to activate gene expression. Chemical strategies, as integral part of jasmonate research, have helped the establishment of structure-activity relationships and the discovery of (+)-7-iso-JA-L-Ile as the major bioactive form of the hormone. The transient nature of its accumulation highlights the need to understand catabolism and inactivation of JA-Ile and recent studies indicate that oxidation of JA-Ile by cytochrome P450 monooxygenase is the major mechanism for turning JA signaling off. Plants contain numerous JA metabolites, which may have pronounced and differential bioactivity. A major challenge in the field of plant lipid signaling is to identify the cognate receptors and modes of action of these bioactive jasmonates/oxylipins.

Jasmonates (JAs) regulate various stress responses and development processes in plants, and the JA pathway is tightly controlled. In this study, we report the functional characterization of two novel RING-type ubiquitin ligases, RING DOMAIN LIGASE3 (RGLG3) and RGLG4, in modulating JA signaling. Both RGLG3 and RGLG4 possessed ubiquitin ligase activities and were widely distributed in Arabidopsis (Arabidopsis thaliana) tissues. Altered expression of RGLG3 and RGLG4 affected methyl JA-inhibited root growth and JA-inductive gene expression, which could be suppressed by the coronatine insensitive1 (coi1) mutant. rglg3 rglg4 also attenuated the inhibitory effect of JA-isoleucine-mimicking coronatine on root elongation, and consistently, rglg3 rglg4 was resistant to the coronatine-secreting pathogen Pseudomonas syringae pv tomato DC3000, suggesting that RGLG3 and RGLG4 acted in response to the coronatine and promoted JA-mediated pathogen susceptibility. In addition, rglg3 rglg4 repressed wound-stunted plant growth, wound-stimulated expression of JA-responsive genes, and wound-induced JA biosynthesis, indicating their roles in JA-dependent wound response. Furthermore, both RGLG3 and RGLG4 responded to methyl JA, P. syringae pv tomato DC3000, and wounding in a COI1-dependent manner. Taken together, these results indicate that the ubiquitin ligases RGLG3 and RGLG4 are essential upstream modulators of JA signaling in response to various stimuli.

The CNS is both source and target of melatonin. This methoxyindole formed in the pineal gland is also produced in other CNS regions and additionally enters the brain by uptake from the circulation as well as via the pineal recess. The mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN), not only controls the pineal, but also receives a feedback information on darkness. Two G protein-coupled melatonin receptors, MT1 and MT2, are responsible for the transduction of many melatonergic actions. High receptor densities are especially found in the SCN, but their presence at lower expression levels in other areas is functionally important. Various metabolites and analogs are formed in the CNS, such as N-acetylserotonin, 5-methoxytryptamine, 5-methoxytryptophol, 5-methoxylated kynuramines, and even 6-sulfatoxymelatonin. The chronobiological effects of melatonin go beyond the resetting of a single circadian oscillator. They contribute to phase relationships between oscillatory subsets and are required for robust rhythm amplitudes. CNS effects of melatonin comprise sleep initiation, antiexcitatory, antiepileptic, antinociceptive, anxiolytic, proneurotrophic, antiinflammatory, antioxidant and other neuroprotective actions. The role as a sleep-promoting compound, which is limited by its short half-life in the circulation, has led to the development of controlled-release formulations and of various synthetic agonists, such as ramelteon, agomelatine, tasimelteon, TIK-301, UCM765 and UCM924. Their differences concerning receptor affinities, preferences for receptor subtypes, and pharmacokinetics are discussed, as well as additional antidepressive actions of agomelatine and TIK-301 based on properties as antagonists of the serotonergic 5-HT2C receptor. Indirect antidepressive effects by melatonergic drugs are largely explained by circadian readjustments.

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (K(d) range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.

Activation of the retinoid X receptor (RXR), which is involved in cell proliferation, differentiation, and apoptosis, is a strategy for cancer chemotherapy and chemoprevention, and 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36) (3) is among the few RXR ligands known. The presently reported studies of 3 include its binding to human plasma proteins, metabolic stability using human liver microsomes, metabolism by human liver microsomes and hepatocytes, and in vivo disposition in rat serum, liver, and mammary tissue. Compound 3 was 75% bound to human plasma proteins, and its metabolic stability was much greater than propranolol. One phase I metabolite was formed by human liver microsomes, seven phase I and II metabolites were formed by human hepatocytes, and five metabolites were detected in rat serum and liver after oral administration. The putative metabolites predicted using LC-MS-MS were synthesized to confirm their structures and to provide sufficient material for investigation of induction of RXRE transcriptional activity and inhibition of NFκB.

Coronatine (COR)-producing pathovars of Pseudomonas syringae, including pvs. tomato, maculicola, and glycinea, cause important diseases on tomato, crucifers, and soybean, respectively, and produce symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to COR. However, the significance of COR-induced chlorosis in localized lesion development and the molecular basis of disease-associated cell death is largely unknown. To identify host (chloroplast) genes that play a role in COR-mediated chlorosis, we used a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing and identified a gene which encodes 2-Cys peroxiredoxin (Prxs) that, when silenced, produced a spreading hypersensitive or necrosis-like phenotype instead of chlorosis after COR application in a COI1-dependent manner. Loss-of-function analysis of Prx and NADPH-dependent thioredoxin reductase C (NTRC), the central players of a chloroplast redox detoxification system, resulted in spreading accelerated P. syringae pv. tomato DC3000 disease-associated cell death with enhanced reactive oxygen species (ROS) accumulation in a COR-dependent manner in tomato and Arabidopsis. Consistent with these results, virulent strain DC3000 suppressed the expression of Prx and NTRC in Arabidopsis and tomato during pathogenesis. However, interestingly, authentic COR suppressed the expression of Prx and NTRC in tomato but not in Arabidopsis, suggesting that COR in conjunction with other effectors may modulate ROS and cell death in different host species. Taken together, these results indicated that NTRC or Prx function as a negative regulator of pathogen-induced cell death in the healthy tissues that surround the lesions, and COR-induced chloroplast-localized ROS play a role in enhancing the disease-associated cell death.

Indaziflam {N-[(1R, 2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-[(1RS)-1fluoroethyl]-1,3,5-triazine-2,4-diamine} is a new pre-emergence herbicide recently registered for a broad spectrum weed control in Florida citrus. Experiments were conducted to evaluate leaching of indaziflam applied at 73 and 145 g ai ha(-1) in Florida Candler soil under simulated rainfall of 5, 10, and 15 cm ha(-1). Indaziflam leached the least (12.6 ± 0.6 cm) when applied at 73 g ai ha(-1) under 5 cm ha(-1) rainfall. Indaziflam leached furthest (30.2 ± 0.9 cm) when applied at 145 g ai ha(-1) under 15 cm ha(-1) rainfall. The visual control ratings of a bio-indicator species ryegrass (Lolium multiflorum L.) was 97% at 15 cm ha(-1) rainfall when indaziflam applied at 145 g ai ha(-1) in the 26 to 30 cm horizon indicating the maximum movement and activity of indaziflam. A dose response experiment was conducted to determine the sensitivity of ryegrass to various doses of indaziflam that confirmed that application of indaziflam at 29.20 g ai ha(-1) was sufficient to prevent germination of ryegrass. There was no mortality of ryegrass plants beyond the 30 cm and the biomass of ryegrass was comparable with untreated control indicating that indaziflam did not leach beyond this distance even under 15 cm ha(-1) rainfall.

Long-duration comparative molecular dynamics simulations of the DNA-topoisomerase binary and DNA-topoisomerase-indenoisoquinoline ternary complexes have been carried out. The analyses demonstrated the role of the drug in conformationally stabilizing the protein-DNA interaction. In detail, the protein lips, clamping the DNA substrate, interact more tightly in the ternary complex than in the binary one. The drug also reduces the conformational space sampled by the protein linker domain through an increased interaction with the helix bundle proximal to the active site. A similar alteration of linker domain dynamics has been observed in a precedent work for topotecan but the molecular mechanisms were different if compared to those described in this work. Finally, the indenoisoquinoline keeps Lys532 far from the DNA, making it unable to participate in the religation reaction, indicating that both short- and long-range interactions contribute to the drug poisoning effect.

Production of disease symptoms represents the final phase of infectious diseases and is a main cause of crop loss and/or marketability. However, little is known about the molecular basis of disease symptom development. In this study, a genetic screening was conducted to identify Arabidopsis (Arabidopsis thaliana) mutants that are impaired specifically in the development of disease symptoms (leaf chlorosis and/or necrosis) after infection with the bacterial pathogen Pseudomonas syringae pv tomato (Pst) DC3000. An ethyl methanesulfonate-induced Arabidopsis mutant (no chlorosis1 [noc1]) was identified. In wild-type plants, the abundance of chlorophylls decreased markedly after Pst DC3000 infection, whereas the total amount of chlorophylls remained relatively unchanged in the noc1 mutant. Interestingly, noc1 mutant plants also exhibited reduced disease symptoms in response to the fungal pathogen Alternaria brassicicola. Genetic and molecular analyses showed that the nuclear gene STAYGREEN (SGR; or Mendel's I locus) is mutated (resulting in the aspartic acid to tyrosine substitution at amino acid position 88) in noc1 plants. Transforming wild-type SGR cDNA into the noc1 mutant rescued the chlorosis phenotype in response to Pst DC3000 infection. The SGR transcript was highly induced by Pst DC3000, A. brassicicola, or coronatine (COR), a bacterial phytotoxin that promotes chlorosis. The induction of SGR expression by COR is dependent on COI1, a principal component of the jasmonate receptor complex. These results suggest that pathogen/COR-induced expression of SGR is a critical step underlying the development of plant disease chlorosis.

Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.

Alzheimer's disease and schizophrenia are characterized by expression of psychotic, affective, and cognitive symptoms. Currently, there is a lack of adequate treatment for the cognitive symptoms associated with these diseases. Cholinergic signaling and, in particular, M1 muscarinic acetylcholine receptor (m1AChR) signaling have been implicated in the regulation of multiple cognitive domains. Thus, the M1AChR has been identified as a therapeutic drug target for diseases, such as schizophrenia and Alzheimer's disease, that exhibit marked cognitive dysfunction as part of their clinical manifestation. Unfortunately, the development of selective M1 agonist medications has not been successful, mostly because of the highly conserved orthosteric acetylcholine binding site among the five muscarinic receptor subtypes. More recent efforts have focused on the development of allosteric M1AChR modulators that target regions of the receptor distinct from the orthosteric site that are less conserved between family members. However, orthosteric and allosteric ligands may differentially modulate receptor function and ultimately downstream signaling pathways. Thus, the need for highly selective M1AChR orthosteric agonists still exists, not only as a potential therapeutic but also as a pharmacological tool to better understand the physiologic consequences of M1AChR orthosteric activation. Here, we describe the novel, potent and selective M1AChR orthosteric partial agonist LY593093 [N-[(1R,2R)-6-({(1E)-1-[(4-fluorobenzyl)(methyl)amino]ethylidene})amino)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]biphenyl-4-carboxamide]. This compound demonstrates modest to no activity at the other muscarinic receptor subtypes, stimulates Gα(q)-coupled signaling events as well as β-arrestin recruitment, and displays significant efficacy in in vivo models of cognition.

• Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) causes an economically important bacterial speck disease on tomato and produces symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to a jasmonic acid (JA)-isoleucine analogue, coronatine (COR), produced by Pst DC3000. However, the molecular processes underlying lesion development and COR-induced chlorosis are poorly understood. • In this study, we took advantage of a chlorotic phenotype elicited by COR on Nicotiana benthamiana leaves and virus-induced gene silencing (VIGS) as a rapid reverse genetic screening tool and identified a role for SGT1 (suppressor of G2 allele of skp1) in COR-induced chlorosis. • Silencing of SGT1 in tomato resulted in reduction of disease-associated symptoms (cell death and chlorosis), suggesting a molecular connection between COR-induced chlorosis and cell death. In Arabidopsis, AtSGT1b but not AtSGT1a was required for COR responses, including root growth inhibition and Pst DC3000 symptom (water soaked lesion) development. Notably, overexpression of AtSGT1b did not alter Pst DC3000 symptoms or sensitivity to COR. • Taken together, our results demonstrate that SGT1/SGT1b is required for COR-induced chlorosis and subsequent necrotic disease development in tomato and Arabidopsis. SGT1 is therefore a component of the COR/JA-mediated signal transduction pathway., (© The Authors (2010). Journal compilation © New Phytologist Trust (2010).)

White rot fungi of the genus Phlebia have demonstrated a high capacity to degrade organic pollutants, including polychlorinated dibenzo-p-dioxins and polychlorinated biphenyls. In this study, we evaluated the ability of 18 white rot fungi species of genus Phlebia to degrade heptachlor and heptachlor epoxide, and described the metabolic pathways by selected white rot fungi. Phlebia tremellosa, Phlebia brevispora and Phlebia acanthocystis removed about 71%, 74% and 90% of heptachlor, respectively, after 14 days of incubation. A large amount of heptachlor epoxide and a small amount of 1-hydroxychlordene and 1-hydroxy-2,3-epoxychlordene were detected as metabolic products of heptachlor from most fungal cultures. The screening of heptachlor epoxide-degrading fungi revealed that several fungi are capable of degrading heptachlor epoxide, which is a recalcitrant metabolite of heptachlor. Phlebia acanthocystis, P. brevispora, Phlebia lindtneri and Phlebia aurea removed about 16%, 16%, 22% and 25% of heptachlor epoxide, respectively, after 14 days of incubation. Heptachlor diol and 1-hydroxy-2,3-epoxychlordene were produced in these fungal cultures as metabolites, suggesting that the hydrolysis and hydroxylation reaction occur in the epoxide ring and in position 1 of heptachlor epoxide, respectively., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

Ramelteon is a melatonin receptor agonist used as a treatment for insomnia. It is subject to a remarkably large drug-drug interaction (DDI) caused by fluvoxamine coadministration, resulting in a more than 100-fold increase in exposure. The objective of this study was to determine whether the DDI could be estimated using in vitro metabolism data. Ramelteon was shown to undergo hydroxylation in human liver microsomes to eight metabolites via six pathways. The main routes of metabolism included hydroxylation on the ethyl side chain and the benzylic position of the cyclopentyl ring, as assessed through enzyme kinetic measurements. Hydroxylation at the other benzylic position was observed in human intestinal microsomes. Ramelteon metabolism was catalyzed by CYP1A2, CYP2C19, and CYP3A4 as shown through the use of recombinant human cytochrome P450 enzymes and specific inhibitors. In liver, CYP1A2, CYP2C19, and CYP3A4 were estimated to contribute 49, 42, and 8.6%, respectively, whereas in intestine only CYP3A4 contributes. The in vitro data were used to estimate the magnitudes of DDI caused by ketoconazole, fluconazole, and fluvoxamine. The DDIs caused by the former were reliably estimated (1.82-fold estimated versus 1.82-fold actual for ketoconazole; 2.99-fold estimated versus 2.36-fold actual for fluconazole), whereas for fluvoxamine it was underestimated (11.4-fold estimated versus 128-fold actual). This suggests that there may be a limit on the magnitude of DDI that can be estimated from in vitro data. Nevertheless, the example of the fluvoxamine-ramelteon DDI offers a unique example wherein one drug can simultaneously inhibit multiple enzymatic pathways of a second drug.

The phyllosphere, i.e., the aerial parts of the plant, provides one of the most important niches for microbial colonization. This niche supports the survival and, often, proliferation of microbes such as fungi and bacteria with diverse lifestyles including epiphytes, saprophytes, and pathogens. Although most microbes may complete the life cycle on the leaf surface, pathogens must enter the leaf and multiply aggressively in the leaf interior. Natural surface openings, such as stomata, are important entry sites for bacteria. Stomata are known for their vital role in water transpiration and gas exchange between the plant and the environment that is essential for plant growth. Recent studies have shown that stomata can also play an active role in limiting bacterial invasion of both human and plant pathogenic bacteria as part of the plant innate immune system. As counter-defense, plant pathogens such as Pseudomonas syringae pv tomato (Pst) DC3000 use the virulence factor coronatine to suppress stomate-based defense. A novel and crucial early battleground in host-pathogen interaction in the phyllosphere has been discovered with broad implications in the study of bacterial pathogenesis, host immunity, and molecular ecology of bacterial diseases.

Jasmonates (JAs) are a class of oxylipin compounds that play diverse roles in plant defense and development. The F-box protein coronatine insensitive 1 (COI1) plays a crucial role in the JA signaling pathway. To determine whether COI1 binds directly to jasmonates, three biotin-tagged photoaffinity probes for JAs, a jasmonic acid photoaffinity probe (PAJA), a JAIle photoaffinity probe (PAJAIle), and a coronatine photoaffinity probe (PACOR), were designed and synthesized based on analysis of JA structure-activity relationships and molecular modeling of the interaction between COI1 and JAs. Among them, PACOR exhibited the most significant biological activity in inhibiting root growth, promoting accumulation of JA-responsive proteins, and triggering COI1-JAZ1 interaction in Arabidopsis seedlings. PACOR is an effective tool for elucidating the interaction between COI1 and JA., ((c) 2010 Elsevier Ltd. All rights reserved.)

In some medicinal plants a specific plant-fungus association, known as arbuscular mycorrhizal (AM) symbiosis, increases the levels of secondary plant metabolites and/or plant growth. In this study, the effects of three different AM treatments on biomass and sesquiterpenic acid concentrations in two IN VITRO propagated genotypes of valerian ( VALERIANA OFFICINALIS L., Valerianaceae) were investigated. Valerenic, acetoxyvalerenic and hydroxyvalerenic acid levels were analyzed in the rhizome and in two root fractions. Two of the AM treatments significantly increased the levels of sesquiterpenic acids in the underground parts of valerian. These treatments, however, influenced the biomass of rhizomes and roots negatively. Therefore this observed increase was not accompanied by an increase in yield of sesquiterpenic acids per plant. Furthermore, one of the two genotypes had remarkably high hydroxyvalerenic acid contents and can be regarded as a hydroxyvalerenic acid chemotype., (Copyright Georg Thieme Verlag KG Stuttgart New York.)

Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant-microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host-pathogen interactions.

The styAB genes from Pseudomonas putida CA-3, which encode styrene monooxygenase, were subjected to three rounds of in vitro evolution using error-prone polymerase chain reaction with a view to improving the rate of styrene oxide and indene oxide formation. Improvements in styrene monooxygenase activity were monitored using an indole to indigo conversion assay. Each round of random mutagenesis generated variants improved in indigo formation with third round variants improved nine- to 12-fold over the wild type enzyme. Each round of in vitro evolution resulted in two to three amino acid substitutions in styrene monooxygenase. While the majority of mutations occurred in styA (oxygenase), mutations were also observed in styB (reductase). A mutation resulting in the substitution of valine with isoleucine at amino acid residue 303 occurred near the styrene and flavin adenine dinucleotide binding site of styrene monooxygenase. One mutation caused a shift in the reading frame in styA and resulted in a StyA variant that is 19 amino acids longer than the wild-type protein. Whole cells expressing the best styrene monooxygenase variants (round 3) exhibited eight- and 12-fold improvements in styrene and indene oxidation rates compared to the wild-type enzyme. In all cases, a single enantiomer, (S)-styrene oxide, was formed from styrene while (1S,2R)-indene oxide was the predominant enantiomer (e.e. 97%) formed from indene. The average yield of styrene oxide and indene oxide from their respective alkene substrates was 65% and 90%, respectively.

Pseudomonas syringae pv tomato DC3000 (Pst DC3000), which causes disease in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana), produces coronatine (COR), a non-host-specific phytotoxin. COR, which functions as a jasmonate mimic, is required for full virulence of Pst DC3000 and for the induction of chlorosis in host plants. Previous genetic screens based on insensitivity to COR and/or methyl jasmonate identified several potential targets for COR and methyl jasmonate. In this study, we utilized Nicotiana benthamiana and virus-induced gene silencing to individually reduce the expression of over 4,000 genes. The silenced lines of N. benthamiana were then screened for altered responses to purified COR. Using this forward genetics approach, several genes were identified with altered responses to COR. These were designated as ALC (for altered COR response) genes. When silenced, one of the identified genes, ALC1, produced a hypersensitive/necrosis-like phenotype upon COR application in a Coronatine-Insensitive1 (COI1)-dependent manner. To understand the involvement of ALC1 during the Pst DC3000-host interaction, we used the nucleotide sequence of ALC1 and identified its ortholog in Arabidopsis (Thylakoid Formation1 [THF1]) and tomato (SlALC1). In pathogenicity assays performed on Arabidopsis thf1 mutant and SlALC1-silenced tomato plants, Pst DC3000 induced accelerated coalescing necrotic lesions. Furthermore, we showed that COR affects ALC1 localization in chloroplasts in a COI1-dependent manner. In conclusion, our results show that the virus-induced gene silencing-based forward genetic screen has the potential to identify new players in COR signaling and disease-associated necrotic cell death.

An estimated one-third of the general population is affected by insomnia, and this number is increasing due to more stressful working conditions and the progressive aging of society. However, current treatment of insomnia with hypnotics, gamma-aminobutyric acid A (GABA(A)) receptor modulators, induces various side effects, including cognitive impairment, motor disturbance, dependence, tolerance, hangover, and rebound insomnia. Ramelteon (Rozerem; Takeda Pharmaceutical Company Limited, Osaka, Japan) is an orally active, highly selective melatonin MT(1)/MT(2) receptor agonist. Unlike the sedative hypnotics that target GABA(A) receptor complexes, ramelteon is a chronohypnotic that acts on the melatonin MT(1) and MT(2) receptors, which are primarily located in the suprachiasmatic nucleus, the body's "master clock." As such, ramelteon possesses the first new therapeutic mechanism of action for a prescription insomnia medication in over three decades. Ramelteon has demonstrated sleep-promoting effects in clinical trials, and coupled with its favorable safety profile and lack of abuse potential or dependence, this chronohypnotic provides an important treatment option for insomnia.

Melatonin receptor agonists such as melatonin and ramelteon [(S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl]-propionamide; TAK-375] have sleep-promoting effects in humans. In preclinical models, these effects are more similar to those observed in monkeys than in other species. However, in contrast to the human melatonin receptors, the pharmacological characteristics of the monkey melatonin receptors have yet to be elucidated. In this study, we cloned the cynomolgus monkey MT(1) and MT(2) melatonin receptors based on rhesus monkey genome sequences and then characterized the monkey melatonin receptors and compared their pharmacological properties with those of the human homologs. The overall amino acid sequences of the monkey MT(1) and MT(2) melatonin receptors showed high homology to the human MT(1) (95%) and MT(2) (96%) receptors, respectively. Saturation binding experiments with 2-[(125)I]iodomelatonin revealed that the dissociation constants (K(d)) for the monkey MT(1) and MT(2) melatonin receptors were 19.9 and 70.4 pM, respectively. In ligand competition assays using 2-[(125)I]iodomelatonin, ramelteon displayed approximately 3- to 7-fold higher affinities than melatonin for the recombinant monkey MT(1) and MT(2) melatonin receptors and monkey suprachiasmatic nucleus membranes. This higher affinity of ramelteon compared with melatonin has also been observed in human melatonin receptors. Furthermore, ramelteon inhibited pituitary adenylate cyclase-activating polypeptide-27-stimulated cAMP production with higher potency than melatonin. In conclusion, this information will help us to understand the pharmacological effects of melatonin receptor agonists in monkeys.

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant-pathogen interactions. Recently it has been demonstrated that abscisic acid signalling pathways are utilized by the bacterial phytopathogen Pseudomonas syringae to promote pathogenesis. In this study, we examined the dynamics, inter-relationship and impact of three key acidic phytohormones, salicylic acid, abscisic acid and jasmonic acid, and the bacterial virulence factor, coronatine, during progression of P. syringae infection of Arabidopsis thaliana. We show that levels of SA and ABA, but not JA, appear to play important early roles in determining the outcome of the infection process. SA is required in order to mount a full innate immune responses, while bacterial effectors act rapidly to activate ABA biosynthesis. ABA suppresses inducible innate immune responses by down-regulating SA biosynthesis and SA-mediated defences. Mutant analyses indicated that endogenous ABA levels represent an important reservoir that is necessary for effector suppression of plant-inducible innate defence responses and SA synthesis prior to subsequent pathogen-induced increases in ABA. Enhanced susceptibility due to loss of SA-mediated basal resistance is epistatically dominant over acquired resistance due to ABA deficiency, although ABA also contributes to symptom development. We conclude that pathogen-modulated ABA signalling rapidly antagonizes SA-mediated defences. We predict that hormonal perturbations, either induced or as a result of environmental stress, have a marked impact on pathological outcomes, and we provide a mechanistic basis for understanding priming events in plant defence.

Jasmonates play a number of diverse roles in plant defense and development. CORONATINE INSENSITIVE1 (COI1), an F-box protein essential for all the jasmonate responses, interacts with multiple proteins to form the SCF(COI1) E3 ubiquitin ligase complex and recruits jasmonate ZIM-domain (JAZ) proteins for degradation by the 26S proteasome. To determine which protein directly binds to jasmonoyl-isoleucine (JA-Ile)/coronatine (COR) and serves as a receptor for jasmonate, we built a high-quality structural model of COI1 and performed molecular modeling of COI1-jasmonate interactions. Our results imply that COI1 has the structural traits for binding JA-Ile or COR. The direct binding of these molecules with COI1 was further examined using a combination of molecular and biochemical approaches. First, we used the immobilized jasmonate approach to show that the COI1 protein in crude leaf extracts can bind to the jasmonate moiety of JA-Ile. Second, we employed surface plasmon resonance technology with purified COI1 and JAZ1 protein to reveal the interaction among COI1, JA-Ile, and JAZ1. Finally, we used the photoaffinity labeling technology to show the direct binding of COR with purified insect-expressed COI1. Taken together, these results demonstrate that COI1 directly binds to JA-Ile and COR and serves as a receptor for jasmonate.

Sordarins are a class of natural antifungal agents which act by specifically inhibiting fungal protein synthesis through their interaction with the elongation factor 2, EF2. A number of natural sordarins produced by diverse fungi of different classes have been reported in the literature. We have run an exhaustive search of sordarin-producing fungi using two different approaches consecutively, the first one being a differential sensitivity screen using a sordarin-resistant mutant yeast strain run in parallel with a wild type strain, and the second one an empiric screen against Candida albicans followed by early detection of sordarins by LC-MS analysis. Using these two strategies we have detected as many as 22 new strains producing a number of different sordarin analogues, either known (sordarin, xylarin, zofimarin) or novel (isozofimarin and 4'-O-demethyl sordarin). Sordarin and xylarin were the most frequently found compounds in the class. The producing strains were subjected to sequencing of the ITS region to determine their phylogenetic affinities. All the strains were shown to belong to the Xylariales, being distributed across three families in this order, the Xylariaceae, the Amphisphaeriaceae, and the Diatrypaceae. Despite being screened in large numbers, we did not find sordarin production in any other fungal group, including those orders where sordarin producing fungi are known to exist (i.e., Sordariales, Eurotiales, and Microascales), suggesting that the production of sordarin is a trait more frequently associated to members of the Xylariales than to any other fungal order.

The AGPL1 (ADP-glucose pyrophosphorylase large subunit 1) promoter from watermelon (Citrullus vulgaris S.) has proved to exhibit fruit-specific expression patterns in tomato (Lycopersicon esculentum L.). A plant expression vector harboring sweet-taste protein, Brazzein, directed by AGPL1 promoter, was constructed and transferred into tomato plants through Agrobacterium-mediated transform methods. Histochemical staining assay, PCR screening, Southern blotting analysis and RT-PCR analysis showed that Brazzein gene was successfully integrated into the genome of transgenic tomato plants with stable expression. Sweet-taste fruits were produced under control of fruit-specific AGPL1 promoter, whereas other parameters of fruit quality were largely unchanged.

Systemic acquired resistance (SAR) develops in response to local microbial leaf inoculation and renders the whole plant more resistant to subsequent pathogen infection. Accumulation of salicylic acid (SA) in noninfected plant parts is required for SAR, and methyl salicylate (MeSA) and jasmonate (JA) are proposed to have critical roles during SAR long-distance signaling from inoculated to distant leaves. Here, we address the significance of MeSA and JA during SAR development in Arabidopsis thaliana. MeSA production increases in leaves inoculated with the SAR-inducing bacterial pathogen Pseudomonas syringae; however, most MeSA is emitted into the atmosphere, and only small amounts are retained. We show that in several Arabidopsis defense mutants, the abilities to produce MeSA and to establish SAR do not coincide. T-DNA insertion lines defective in expression of a pathogen-responsive SA methyltransferase gene are completely devoid of induced MeSA production but increase systemic SA levels and develop SAR upon local P. syringae inoculation. Therefore, MeSA is dispensable for SAR in Arabidopsis, and SA accumulation in distant leaves appears to occur by de novo synthesis via isochorismate synthase. We show that MeSA production induced by P. syringae depends on the JA pathway but that JA biosynthesis or downstream signaling is not required for SAR. In compatible interactions, MeSA production depends on the P. syringae virulence factor coronatine, suggesting that the phytopathogen uses coronatine-mediated volatilization of MeSA from leaves to attenuate the SA-based defense pathway.

Herein, the biogenesis of the hydrindane ring system within coronafacic acid (CFA) has been investigated. These studies reveal that in addition to the canonical polyketide chain elongation and functionalization encoded by type I polyketide synthase (PKSs), cascade reactions can take place during assembly line-like biosynthesis. Indeed, upon Cfa7-catalyzed Claisen condensation between enzyme-bound malonate and an N-acetylcysteamine (SNAC) thioester, latent reactivity within the elongated enzyme-bound intermediate is unveiled. This reactivity translates into an intramolecular cyclization, which can proceed in a facile manner as observed by the enzyme-independent cyclization of a linear beta-ketothioester intermediate.

A synthesis of the readily accessible glycinoeclepin A analogue 2 is reported.