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Novel steroid glycosides, acanthasterosides A1, B1, and B3, have been isolated from the crown-of-thorns starfish Acanthaster planci. Acanthasterosides B1 and B3 having two separated xyloses induced neurite outgrowth as like as nerve growth factor (NGF) in the rat pheochromocytoma cell line PC12, whereas acanthasteroside A1, having one xylose, did not induce neurite outgrowth. The acanthasteroside B3 induced neuritogenesis via the significant activation of p38 mitogen-activated protein kinase after the activation of the small G-protein Cdc42 rather than via Ras-MEK-ERK pathway that is predominantly activated by NGF. Following subcutaneous administration, acanthasteroside B3 attenuated cognitive impairment of senescence-accelerated mice (SAMP8) in two different cognitive tests. Liquid chromatography-mass spectrometry-assisted quantitative analysis demonstrated that acanthasteroside B3 could be transported into the brain via the circulatory system in mice. Thus, acanthasteroside B3 (and possibly B1) are a novel class of potential drug candidates for neurodegenerative diseases.

Phytophthora are plant pathogens that damage agricultural products. Lycosides (1a–d), found in vegetable juice, have the potential to curb the rapid outbreak and crop damage caused by the asexual reproduction of Phytophthora. Here, aglycones 2a, b with slightly higher activity than lycosides were synthesized as a diastereomeric mixture (mix-2) possessing activity (IC50 = 4.1 µm) comparable with that of lycosides. The importance of the cyclohexanone structure and side-chain length was demonstrated via structure–activity relationship analysis using synthetic intermediates. In addition, the action mechanism of lycosides was investigated using transcriptome analysis, which revealed a contribution to proline biosynthesis inhibition, a process crucial for the asexual reproduction of Phytophthora. These findings indicate that lycosides (and aglycone) are environmentally benign agents that can be used for protecting agricultural products from Phytophthora pathogens.

Botrytis cinerea, a generalist fungal pathogen of economically important crop species, has been shown to exhibit reduced sensitivity to fungicides and plant toxins. Specifically, previous reports indicateB. cinerea’s efficacy in tolerating a wide array of phytoalexins, toxic plant metabolites that play key role in plant immune defense strategies. Previously, we have shown that a distinct set of genes was induced inB. cinereawhen treated with phytoalexins derived from different plant species such as rishitin (tomato and potato), capsidiol (tobacco and bell pepper) or resveratrol (grape and blueberry). In this study, we focused on the functional analyses ofB. cinereagenes induced by rishitin treatment.B. cinereacan metabolize rishitin to at least 4 oxidized forms. Heterologous expression of rishitin-inducedB. cinereagenes in the plant symbiotic fungus,Epichloë festucae, revealed that oxidoreductase (Bcin08g04910) and cytochrome P450 (Bcin16g01490) genes are involved in the oxidation of rishitin. BcatrB is an exporter of structurally unrelated anti-microbial compounds such as resveratrol, camalexin and fungicide fenpicionil. Expression ofBcatrBis upregulated by rishitin, but not by structurally resembling capsidiol.BcatrBknock out transformants (ΔbcatrB) showed enhanced sensitivity to rishitin, but not to capsidiol. Likewise,ΔbcatrBshowed reduced virulence on tomato fruits (which produce rishitin), but showed full virulence onNicotiana benthamiana(which mainly produces capsidiol), suggesting thatB. cinereadistinguishes phytoalexins and activates expression of appropriate transporter genes during the infection. Activation ofBcatrBpromoter was detected using P_BcatrB:GFPtransformant during theB. cinereainfection in plant tissues. Surveying of 26 plant species across 13 families revealed that theBcatrBpromoter is mainly activated during the infection of plants belonging to the Solanaceae, Fabaceae and Brassicaceae families. TheBcatrBpromoter is activated by the treatment with Fabaceae phytoalexins medicarpin and glyceollin, andΔbcatrBshowed reduced virulence on red clover, which produces medicarpin. These results suggest thatBcatrBplays a critical role in the strategy employed byB. cinereato bypass the plant innate immune responses in a wide variety of important crops belonging to the Solanaceae, Brassicaceae and Fabaceae families.

The gray mold pathogen Botrytis cinerea has a broad host range, causing disease in >400 plant species, but it is not known how this pathogen evolved this polyxenous nature. Botrytis cinerea can metabolize a wide range of phytoalexins, including the stilbenoid resveratrol in grape, and the sesquiterpenoids capsidiol in tobacco and rishitin in potato and tomato. In this study, we analyzed the metabolism of sesquiterpenoid phytoalexins by B. cinerea. Capsidiol was dehydrogenated to capsenone, which was then further oxidized, while rishitin was directly oxidized to epoxy- or hydroxyrishitins, indicating that B. cinerea has separate mechanisms to detoxify structurally similar sesquiterpenoid phytoalexins. RNA-seq analysis revealed that a distinct set of genes were induced in B. cinerea when treated with capsidiol or rishitin, suggesting that B. cinerea can distinguish structurally similar phytoalexins to activate appropriate detoxification mechanisms. The gene most highly upregulated by capsidiol treatment encoded a dehydrogenase, designated Bccpdh. Heterologous expression of Bccpdh in a capsidiol-sensitive plant symbiotic fungus, Epichloë festucae, resulted in an acquired tolerance of capsidiol and the ability to metabolize capsidiol to capsenone, while B. cinerea Δbccpdh mutants became relatively sensitive to capsidiol. The Δbccpdh mutant showed reduced virulence on the capsidiol producing Nicotiana and Capsicum species but remained fully pathogenic on potato and tomato. Homologs of Bccpdh are found in taxonomically distant Ascomycota fungi but not in related Leotiomycetes species, suggesting that B. cinerea acquired the ancestral Bccpdh by horizontal gene transfer, thereby extending the pathogenic host range of this polyxenous pathogen to capsidiol-producing plant species.

Pradimicins (PRMs) are a unique family of natural products that exhibit antifungal activity via binding to cell wall mannans of fungi. Although their mannan-targeted antifungal action has attracted...

The gray mold pathogen Botrytis cinerea has a broad host range, causing disease in over 400 plant species, but it is not known how this pathogen evolved this polyxenous nature. B. cinerea can metabolize a wide range of phytoalexins, including the stilbenoid, resveratrol, and the sesquiterpenoids capsidiol in tobacco, and rishitin in potato and tomato. In this study, we analyzed the metabolism of sesquiterpenoid phytoalexins by B. cinerea. Capsidiol was dehydrogenated to capsenone which was then further oxidized, while rishitin was directly oxidized to epoxy-or hydroxy-rishitins indicating that B. cinerea has separate mechanisms to detoxify structurally similar sesquiterpenoid phytoalexins. RNAseq analysis revealed that a distinct set of genes were induced in B. cinerea when treated with capsidiol or rishitin, suggesting that B. cinerea can distinguish structurally similar phytoalexins to activate appropriate detoxification mechanisms. The gene most highly upregulated by capsidiol treatment encoded a dehydrogenase, designated Bccpdh. Heterologous expression of Bccpdh in a capsidiol-sensitive plant symbiotic fungus, Epichloë festucae, resulted in an acquired tolerance of capsidiol and the ability to metabolize capsidiol to capsenone, while B. cinerea Δbccpdh mutants became relatively sensitive to capsidiol. The Δbccpdh mutant showed reduced virulence on the capsidiol producing Nicotiana and Capsicum species but remained fully pathogenic on potato and tomato. Homologs of Bccpdh are not found in taxonomically distant Ascomycota fungi but not in related Leotiomycete species, suggesting that B. cinerea acquired the ancestral Bccpdh by horizontal gene transfer, thereby extending the pathogenic host range of this polyxenous pathogen to capsidiol-producing plant species.Significance StatementB. cinerea can metabolize a wide range of phytoalexins, however, the extent to which phytoalexin detoxification contributes to pathogenicity is largely unknown. In this study, we have shown that B. cinerea recognizes structurally resembling sesquiterpenoid phytoalexins, rishitin and capsidiol, to activate appropriate detoxification mechanisms. We identify Bccpdh, encoding a dehydrogenase for capsidiol detoxification, which is upregulated in B. cinerea exclusively during the infection of capsidiol producing plant species, and is required to exert full virulence. Analysis of the Bccpdh locus implicates that the gene was acquired via horizontal gene transfer. This work highlights that the polyxenous plant pathogen B. cinerea can distinguish its host plants by its anti-microbial compounds, to activate appropriate mechanisms for enhanced virulence.

The hard-to-culture slightly halophilic myxobacterium “Paraliomyxa miuraensis” SMH-27-4 produces antifungal cyclodepsipeptide miuraenamide A (1). Herein, the region (85.9 kbp) containing the biosynthetic gene cluster (BGC) coding the assembly of 1 was identified and heterologously expressed in Myxococcus xanthus. A biosynthetic pathway proposed using in silico analysis was verified through the gene disruption of the heterologous transformant. In addition to the core polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) genes, tyrosine halogenase and O-methyltransferase genes participated in the biosynthesis of 1 as their gene-disrupted mutants produced a new congener, debromomiuraenamide A (4), and a previously isolated congener, miuraenamide E (3), respectively. Multigene disruption provided a heterologous mutant that produced 1 with the highest yield among the prepared mutants. When fed on 3-bromo-L-tyrosine, this mutant produced more 1 in the yield of 1.21 mg/L, which was 20 times higher than that produced by the initially prepared heterologous transformant. Although this yield was comparable to that of the original producer SMH-27-4 (1 mg/L), the culture time was 4.5 times shorter than that of SMH-27-4, indicating a five-fold efficiency in productivity. The results indicate the great potential of the miuraenamide BGC for the future contribution to drug development through logical gene manipulation.

Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including Phytophthora and Pythium. Treatment of INF1 (an elicitin produced by P. infestans) induces a series of defense responses in Nicotiana species, including reactive oxygen species (ROS) production, transient induction of ethylene production, hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin capsidiol. In this study, we analyzed the expression profiles of N. benthamiana genes after INF1 treatment by RNAseq analysis. Based on their expression patterns, N. benthamiana genes were categorized into 20 clusters and 4,761 (8.3%) out of 57,140 genes were assigned to the clusters for INF1-induced genes. All genes encoding enzymes dedicated to capsidiol production, 5-epi-aristolochene (EA) synthase (NbEAS, 10 copies) and EA dehydrogenase (NbEAH, 6 copies), and some genes for ethylene production, such as 1-aminocyclopropane 1-carboxylate (ACC) synthase (NbACS) and ACC oxidase (NbACO), were significantly upregulated by INF1 treatment. Analysis of NbEAS1 and NbEAS4 promoters revealed that AGACGCC (GCC box-like motif) is the essential cis-element required for INF1-induced expression of NbEAS genes. Given that the GCC box is known to be targeted by ERF (ethylene-responsive factor) transcription factors, we created a complete list of N. benthamiana genes encoding AP2/ERF family transcription factors, and identified 45 out of 337 AP2/ERF genes in the clusters for INF1-induced genes. Among INF1-induced NbERF genes, silencing of NbERF-IX-33 compromised resistance against P. infestans and INF1-induced production of capsidiol. Recombinant NbERF-IX-33 protein can bind to the promoter sequence of NbEAS4, suggesting that NbERF-IX-33 is a transcription factor directly regulating the expression of genes for phytoalexin production.

Halophilic/halotolerant myxobacteria are extremely rare bacteria but an important source of novel bioactive secondary metabolites as drug leads. A slightly halophilic myxobacterium, “Paraliomyxa miuraensis” SMH-27-4, the producer of the antifungal antibiotic miuraenamide A, was considered to represent a novel genus. This study aimed to use the whole-genome sequence of this difficult-to-culture bacterium to provide genomic evidence supporting its taxonomy and to explore its potential as a novel secondary metabolite producer and its predicted gene functions. The draft genome was sequenced and de novo assembled into 164 contigs (11.8 Mbp). The 16S rRNA gene sequence-based and genome sequence-based phylogenetic analyses supported that this strain represents a novel genus of the family Nannocystaceae. Seventeen biosynthetic gene clusters (BGCs) were identified, and only five of them show some degree of similarity with the previously annotated BGCs, suggesting the great potential of producing novel secondary metabolites. The comparative genomic analysis within the family Nannocystaceae revealed the distribution of its members’ gene functions. This study unveiled the novel genomic features and potential of the secondary metabolite production of this myxobacterium.

Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including

Plants recognize a variety of external signals and induce appropriate mechanisms to increase their tolerance to biotic and abiotic stresses. Precise recognition of attacking pathogens and induction of effective resistance mechanisms are critical functions for plant survival. Some molecular patterns unique to a certain group of microbes (MAMPs, microbe-associated molecular patterns) are sensed by plant cells as non-self molecules via pattern recognition receptors. While a variety of MAMPs of bacterial and fungal origin have been identified, reports on MAMPs of oomycete pathogens are relatively limited. This study aimed to identify unique MAMP elicitors from the oomycete pathogen Phytophthora infestans, the causal agent of potato late blight. Using reactive oxygen species (ROS) production and phytoalexin production in potato as markers for the purification of oomycete elicitors, we identified two structurally different groups of elicitors, namely ceramides and diacylglycerols. P. infestans ceramide (Pi-Cer) elicitors induced ROS production, while diacylglycerol (Pi-DAG) elicitors, containing eicosapentaenoic acid (EPA) as a substructure, induced the formation of phytoalexins in potato. Pi-Cer and Pi-DAG are also contained in the mycelia of another oomycete pathogen Pythium aphanidermatum, indicating that they are MAMPs of oomycetes. When Arabidopsis was treated with Pi-Cer and EPA, partially overlapping but different sets of genes were induced. Furthermore, simultaneous treatment with Pi-Cer and EPA did not have a cumulative effect on induced genes, but rather the expression of some genes induced by EPA was attenuated by the co-treatment with Pi-Cer. These results indicate that plants may combine the signals from simultaneously recognized MAMP elicitors to specifically adapt the defense response to a particular pathogen.

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors (PRRs), inducing defense responses; yet how PRRs perceive pathogen sphingolipids remains unclear. Here, we show that the ceramide Pi-Cer D from a plant pathogenic oomycetePhytophthora infestanstriggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NCER2, and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RDA2. Importantly, 9-methyl-branched sphingoid base, which is unique to microbes, induces plant immune responses by interacting with RDA2. Loss ofRDA2orNCER2function compromised Arabidopsis resistance against an oomycete pathogen, indicating that these are crucial for defense. We provide new insights that help elucidate the recognition mechanisms of pathogen-derived lipid molecules in plants.One Sentence SummaryOomycete-derived ceramide is cleaved into sphingoid base by ceramidase and recognized by an Arabidopsis receptor kinase.

Seven new cyclic depsipeptides, clavariopsins C-I (3-9), together with two known congeners, clavariopsins A and B (1 and 2), were isolated from the aquatic hyphomycete Clavariopsis aquatica. Their planar structures, which consist of nine amino acids and one α-hydroxy acid, were elucidated by NMR spectroscopy and HRESIMS. The absolute configurations were established by the advanced Marfey's method and chiral-phase HPLC analysis. Their antifungal and cytotoxic activities were evaluated against six plant pathogenic fungi (Botrytis cinerea, Magnaporthe oryzae, Colletotrichum orbiculare, Fusarium oxysporum, Alternaria alternata, and Aspergillus niger) and a cancer cell line (HeLa-S3), respectively. The majority of the compounds exhibited potent antifungal activity against the fungi tested (minimum inhibition dose = 0.01-10 μg/disk) and induced hyphal swelling in A. niger (minimum effective dose = 0.3-3 μg/disk), whereas the compounds exhibited no cytotoxicity toward the cancer cell line. The results suggest that the clavariopsins could be a promising class of antifungal agents.

The heterothallic group of the plant pathogen Phytophthora can sexually reproduce between the cross-compatible mating types A1 and A2. The mating hormone α2, produced by A2 mating type and utilized to promote the sexual reproduction of the partner A1 type, is known to be biosynthesized from phytol. In this study, we identified 2 biosynthetic intermediates, 11- and 16-hydroxyphytols (1 and 2), for α2 by administering the synthetic intermediates to an A2-type strain to produce α2 and by administering phytol to A2 strains to detect the intermediates in the mycelia. The results suggest that α2 is biosynthesized by possibly 2 cytochrome P450 oxygenases via 2 hydroxyphytol intermediates (1 and 2) in A2 hyphae and secreted outside.

The oomycete Phytophthora infestans is the causal agent of potato late blight, one of the most destructive and historically significant pathogens in agricultural production. A virus-induced gene silencing-based screening of the solanaceous model plant N. benthamiana resulted in revealing a wide range of resistance mechanisms of solanaceous plants against this pathogen. In this article, we present an overview of the various pathways involved in the N. benthamiana–P. infestans pathosystem, including some of the follow-up work that was triggered by these findings. The purpose of this review is to assemble these findings and integrate them into our current understanding of plant pathogen defense mechanisms and discuss their potential application for the development of potato resistance to P. infestans.

Neurogenerative diseases, such as Parkinson’s disease, are associated, not only with the selective loss of dopamine (DA), but also with the accumulation of reactive catechol-aldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is formed as the immediate oxidation product of cytoplasmic DA by monoamine oxidase. DOPAL is well known to exhibit toxic effects on neuronal cells. Both catecholic and aldehyde groups seem to be associated with the neurotoxicity of DOPAL. However, the exact cause of toxicity caused by this compound remains unknown. Since the reactivity of DOPAL could be attributed to its immediate oxidation product, DOPAL-quinone, we examined the potential reactions of this toxic metabolite. The oxidation of DOPAL by mushroom tyrosinase at pH 5.3 produced conventional DOPAL-quinone, but oxidation at pH 7.4 produced the tautomeric quinone-methide, which gave rise to 3,4-dihydroxyphenylglycolaldehyde and 3,4-dihydroxybenzaldehyde as products through a series of reactions. When the oxidation reaction was performed in the presence of ascorbic acid, two additional products were detected, which were tentatively identified as the cyclized products, 5,6-dihydroxybenzofuran and 3,5,6-trihydroxybenzofuran. Physiological concentrations of Cu(II) ions could also cause the oxidation of DOPAL to DOPAL-quinone. DOPAL-quinone exhibited reactivity towards the cysteine residues of serum albumin. DOPAL-oligomer, the oxidation product of DOPAL, exhibited pro-oxidant activity oxidizing GSH to GSSG and producing hydrogen peroxide. These results indicate that DOPAL-quinone generates several toxic compounds that could augment the neurotoxicity of DOPAL.

Vegetable juices, typical culture media for the plant pathogen Phytophthora, effectively induce its asexual reproduction (zoosporangia formation). However, some chromatographic fractions from a vegetable juice were found to inhibit asexual reproduction. Bioassay-guided chromatographic steps led to the isolation of four novel compounds, named lycosides A-D, 1-4, that could be metabolic products from a carotenoid. They showed 50% inhibitory activity against the asexual reproduction of P. capsici at 2.1-7.6 μM. The structure-activity relationship and the universality of the inhibitory activity within the Phytophthora genus were also investigated. In addition, the quantitative analysis of lycosides in fresh vegetables and vegetable juices revealed that tomato is the source of these active substances. These food-derived chemicals could help provide safe agents to control the outbreak of the agricultural pest Phytophthora in fields.

Seven new cyclic depsipeptides, clavariopsins C-I (

Neuromelanin (NM) is a dark brown pigment found in dopaminergic neurons of the substantia nigra (SN) and in norepinephrinergic neurons of the locus coeruleus (LC). Although NM is thought to be involved in the etiology of Parkinson&rsquo, s disease (PD) because its content decreases in neurodegenerative diseases such as PD, details are still unknown. In this study, we characterized the biosynthetic pathway of the oxidation of dopamine (DA) by tyrosinase in the presence of thiol peptides and proteins using spectroscopic and high-performance liquid chromatography (HPLC) methods and we assessed the binding of DA via cysteine residues in proteins by oxidation catalyzed by redox-active metal ions. To examine whether the protein-bound DA conjugates exhibit pro-oxidant activities, we measured the depletion of glutathione (GSH) with the concomitant production of hydrogen peroxide. The results suggest that the fate of protein-bound DA conjugates depends on the structural features of the proteins and that DA-protein conjugates produced in the brain possess pro-oxidant activities, which may cause neurodegeneration due to the generation of reactive oxygen species (ROS) and the depletion of antioxidants.

Linckosides are members of the steroid glycoside family isolated from the starfish Linckia laevigata. These natural compounds have notable neuritogenic activity and synergistic effects on NGF-induced neuronal differentiation of PC12 cells. Neurogenic factors or molecules that are able to mimic their activities are known to be involved in the survival, proliferation and migration of neurons and glial cells; however how glial cells respond to specific neurogenic molecules such as linckosides has not been investigated. This study aimed to examine the effect of three different linckosides (linckoside A, B and granulatoside A) on the morphological properties, proliferation and migration of human olfactory ensheathing cells (hOECs). The proliferation rate after all the treatments was higher than control as detected by MTS assay. Additionally, hOECs displayed dramatic morphological changes characterized by a higher number of processes after linckoside treatment. Interestingly changes in microtubule organization and expression levels of some early neuronal markers (GAP43 and βIII-tubulin) were also observed. An increase in the phosphorylation of ERK 1/2 after addition of the compounds suggests that this pathway may be involved in the linckoside-mediated effects particularly those related to morphological changes. These results are the first description of the stimulating effects of linckosides on hOECs and raise the potential for this natural compound or its derivatives to be used to regulate and enhance the therapeutic properties of OECs, particularly for cell transplantation therapies.

The crop destroyer Phytophthora uses mating hormones α1 and α2 to commence its sexual reproduction. The α1-induced sexual reproduction of the A2 mating type was unexpectedly found to be interfered with by the counterhormone α2 that the A2 type itself produces to induce the sexual reproduction of the A1 type. A plausible mechanism is proposed based on structure–activity relationships.

trans-Resveratrol (3,5,4'-trihydroxy-trans-stilbene, RES), a naturally occurring polyphenol, has recently attracted increased interest as a health-beneficial agent. However, based on its p-substituted phenol structure, RES is expected to be a substrate for tyrosinase and to produce a toxic o-quinone metabolite. The results of this study demonstrate that the oxidation of RES by tyrosinase produces 4-(3',5'-dihydroxy-trans-styrenyl)-1,2-benzoquinone (RES-quinone), which decays rapidly to an oligomeric product (RES-oligomer). RES-quinone was identified after reduction to its corresponding catechol, known as piceatannol. RES-quinone reacts with N-acetylcysteine, a small thiol, to form a diadduct and a triadduct, which were identified by NMR and MS analyses. The production of a triadduct is not common for o-quinones, suggesting a high reactivity of RES-quinone. RES-quinone also binds to bovine serum albumin through its cysteine residue. RES-oligomer can oxidize GSH to GSSG, indicating its pro-oxidant activity. These results suggest that RES could be cytotoxic to melanocytes due to the binding of RES-quinone to thiol proteins.

Solanaceous plants produce sesquiterpenoid phytoalexins to defend themselves against a variety of pathogens. These toxic compounds are not only harmful to the pathogen but also to the plant, and thus need to be detoxified by the plant after the threat has been eliminated. We report that the detoxification of rishitin, the major phytoalexin in potato tubers and tomato fruits, is mediated by a cytochrome P450 CYP76 family enzyme via the hydroxylation of the isopropenyl group resulting in the formation of 13-hydroxyrishitin, also known as rishitin-M1. We further observed hydroxylation of the potato phytoalexins solavetivone, lubimin and oxylubimin by the same enzyme. Constitutive expression of CYP76 in Nicotiana benthamiana also led to a reduction of the non-potato phytoalexins capsidiol and its derivative capsidiol 3-acetate. We therefore annotated this enzyme as sesquiterpenoid phytoalexins hydroxylase, SPH. This broad range of substrates indicates that SPH functions as a general phytoalexin detoxification enzyme in Solanaceae, and is therefore relevant for a better understanding of plant-pathogen interaction in solanaceous plants, which comprise many economically important crops, such as potato, tomato, eggplant and pepper.

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.

In order to elucidate the chemical structure of black to brown pigments, neuromelanins (NMs), in the substantia nigra (SN) and the locus coeruleus (LC) in the central nervous system of humans and other mammalian species during aging, chemical degradative methods are powerful tools. HPLC analysis after hydroiodic acid hydrolysis detected aminohydroxyphenylethylamines, aminohydroxyphenylacetic acids, and aminohydroxyethylbenzenes, which confirmed that SN‐NM and LC‐NM contain melanin derived not only from dopamine and norepinephrine (NE) but also from several other catecholic metabolites, such as 3,4‐dihydroxyphenylalanine, 3,4‐dihydroxyphenylacetic acid, 3,4‐dihydroxymandelic acid, 3,4‐dihydroxyphenylethanol, and 3,4‐dihydroxyphenylethylene glycol, in addition to the corresponding Cys‐derivatives in varying degrees. However, hydroiodic acid hydrolysis showed that LC‐NM produced the same degradation products as were detected in SN‐NM. Thus, we needed to develop a new chemical detection method to validate the existence of NE in LC‐NM. In the present study, we report that HCl hydrolysis of LC‐NM in the presence of thioglycolic acid yields new products arising from substitution of the hydroxyl group by thioglycolic acid at the benzyl position of NE and cysteinyl‐NE. This is the first chemical evidence showing that NE and cysteinyl‐NE are incorporated into LC‐NM. Using the chemical degradation methods for the determination of catechols in neuromelanin (NM), we have shown that dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), 3,4‐dihydroxyphenylethanol (DOPE), and 3,4‐dihydroxyphenylalanine (DOPA) are mainly responsible for the structure of NM from substantia nigra (SN), while norepinephrine (NE), 3,4‐dihydroxymandelic acid (DOMA), and 3,4‐dihydroxyphenylethylene glycol (DOPEG) are additionally responsible for the structure of NM from locus coeruleus (LC).

Phytophthora infestans is the most devastating pathogen of potato. For the biology study of P. infestans at the molecular level, one of the difficulties is the technique for the genetic transformation. In this study, the straightforward electro-transformation system was established for P. infestans with a green fluorescent protein expression vector and compared with the improved PEG/CaCl 2 mediated protoplast transformation system. The results showed that the straightforward electro-transformation could work in P. infestans and 32 positive transformants were obtained per about 1.10 × 10 6 zoospores. The transformants per μg of vector DNA were 1.08. The transformation efficiency of the straightforward electro-transformation was approximately 2 times higher than that of the improved PEG/CaCl 2 mediated protoplast transformation (17 positive transformants per about 1.05 × 10 6 protoplasts, 0.58 transformants per μg of vector DNA) according to the reported procedures. Furthermore, compared with the improved PEG/CaCl 2 transformation, the straightforward electroporation is simpler and requires less starting materials and operating time from collecting material to obtaining the resistant transformants. Our work will lay a foundation for the biology study of P. infestans in the future.

Four maleic anhydride derivatives, tricladolides A-D (1-4), and three alkylidene succinic acid derivatives, tricladic acids A-C (5-7), were isolated from the aquatic hyphomycete Tricladium castaneicola. The structures of these compounds were determined by spectroscopic analysis, and all were found to be novel. The compounds exhibited inhibitory activity against fungi, particularly Phytophthora sp., a plant pathogen of oomycetes. The inhibitory activity of these metabolites revealed the importance of the cyclic anhydride structure and the lipophilicity of the alkyl side chain. On the other hand, the cytotoxicity of the compounds against B16 melanoma cells indicated that the cyclic anhydride structure was not essential.

Summary RS-4-(4-Hydroxyphenyl)-2-butanol (rhododendrol, RD) was reported to induce leukoderma of the skin. To explore the mechanism underlying that effect, we previously showed that oxidation of RD with mushroom tyrosinase produces RD-quinone, which is converted to secondary quinone products, and we suggested that those quinones are cytotoxic because they bind to cellular proteins and produce reactive oxygen species. We then confirmed that human tyrosinase can oxidize both enantiomers of RD. In this study, we examined the metabolism of RD in B16F1 melanoma cells in vitro. Using 4-amino-3-hydroxy-n-butylbenzene as a specific indicator, we detected moderate levels of RD-pheomelanin in B16F1 cells exposed to 0.3 to 0.5 mM RD for 72 h. We also confirmed the covalent binding of RD-quinone to non-protein thiols and proteins through cysteinyl residues. The covalent binding of RD-quinone to proteins was 20- to 30-fold greater than dopaquinone. These results suggest that the tyrosinase-induced metabolism of RD causes melanocyte toxicity.

Cytotoxicity‐guided fractionation of the culture broth of Actinomadura sp. TP‐A0019 led to the isolation of quinocidin (1), a cytotoxic antibiotic with an unusual 3,4‐dihydroquinolizinium ring. The structural assignment was made on the basis of high‐field NMR experiments and chemical synthesis. Comparison of the spectral properties of 1 with those of its synthetic counterparts revealed that 1 is a racemic mixture of two enantiomers, which showed similar cytotoxicity against HeLa‐S3 cells. Nucleophile‐trapping experiments demonstrated that 1 captured 2‐mercaptoethanol and N‐acetyl‐l‐cysteine by means of a Michael addition‐type reaction, but was inert toward 2‐aminoethanol and glycolic acid. Notably, the addition of 1 to thiols proceeded smoothly in neutral aqueous media at room temperature. In view of the thiol‐trapping ability and the unusual structure, 1 provides a unique scaffold for designing drug leads and protein‐labeling probes., ファイル公開：2018-12-19

Sexual reproduction of the plant pest Phytophthora is regulated by two mating hormones α1 and α2, which are acyclic oxygenated diterpenes first isolated from P. nicotianae A1 and A2 mating types, respectively. A previous report suggested the universality of these factors within this genus. To confirm this concept, we investigated 80 strains (19 species) of Phytophthora and a related genus, not only for the responsiveness to mating hormones but also for their productivity. The results indicated that among the 55 heterothallic strains, 24 (44%) responded to a mating hormone and 40 (73%) produced one or both hormones. These findings demonstrate the interspecies universality of mating hormones within the genus Phytophthora. Hormone productivity was found to be highly diverse and dependent on the strains used. Although the A2 mating type has been regarded as the α2 producer, 19 (59%) of the 32 A2-type strains produced both the hormones and two A2-type strains exclusively produced α1 in high yields. These results indicate that hormone biosynthesis in Phytophthora is universal but highly diverse and complex, and varies with culture conditions, providing us valuable information for future studies on the mechanism of mating hormone biosynthesis of Phytophthora.

The endophytic fungus Epichlo&euml, festucae is known to produce bioactive metabolites, which consequently protect the host plants from biotic and abiotic stresses. We previously found that the overexpression of vibA (a gene for transcription factor) in E. festucae strain E437 resulted in the secretion of an unknown fungicide. In the present study, the active substance was purified and chemically identified as &epsilon, poly-L-lysine (&epsilon, PL), which consisted of 28-34 lysine units. The productivity was 3.7-fold compared with that of the wild type strain E437. The isolated &epsilon, PL showed inhibitory activity against the spore germination of the plant pathogens Drechslera erythrospila, Botrytis cinerea, and Phytophthora infestans at 1-10 &mu, g/mL. We also isolated the fungal gene &ldquo, epls&rdquo, encoding &epsilon, PL synthetase Epls. Overexpression of epls in the wild type strain E437 resulted in the enhanced production of &epsilon, PL by 6.7-fold. Interestingly, overexpression of epls in the different strain E. festucae Fl1 resulted in the production of shorter &epsilon, PL with 8-20 lysine, which exhibited a comparable antifungal activity to the longer one. The results demonstrate the first example of &epsilon, PL synthetase gene from the eukaryotic genomes and suggest the potential of enhanced expression of vibA or/and epls genes in the Epichlo&euml, endophyte for constructing pest-tolerant plants.

Methyl 5-thio-α-d-mannopyranoside (1) and six inositols were evaluated for their ability to bind to pradimicins (PRMs) via molecular modeling and three binding assays. In all the experiments, the binding affinity of 1 was slightly lower than that of methyl α-d-mannopyranoside (Man-OMe) and inositols hardly bound to PRMs. These results indicate that the endocyclic oxygen atom of Man-OMe is involved in its binding to PRMs., ファイル公開：2022-02-27

Mature Nicotiana benthamiana shows stable resistance to the oomycete pathogen Phytophthora infestans. Induction of phytoalexin (capsidiol) production is essential for the resistance, which is upregulated via a mitogen-activated protein kinase (MAPK) cascade (NbMEK2-WIPK/SIPK) followed by ethylene signaling. In this study, NbNup75 (encodes a nuclear pore protein Nucleoporin75) was identified as an essential gene for resistance of N. benthamiana to P. infestans. In NbNup75-silenced plants, initial events of elicitor-induced responses such as phosphorylation of MAPK and expression of defense-related genes were not affected, whereas induction of later defense responses such as capsidiol production and cell death induction was suppressed or delayed. Ethylene production induced by either INF1 or NbMEK2 was reduced in NbNup75-silenced plants, whereas the expression of NbEAS (a gene for capsidiol biosynthesis) induced by ethylene was not affected, indicating that Nup75 is required for the induction of ethylene production but not for ethylene signaling. Given that nuclear accumulation of polyA RNA was increased in NbNup75-silenced plants, efficient export of mRNA from nuclei via nuclear pores would be important for the timely upregulation of defense responses. Collectively, Nup75 is involved in the induction of a later stage of defense responses, including the ethylene-mediated production of phytoalexin for the resistance of N. benthamiana to P. infestans.

Racemic RS-4-(4-hydroxyphenyl)-2-butanol (rhododendrol, RD) was used as a topical skin-whitening agent until it was recently reported to induce leukoderma. We then showed that oxidation of RD with mushroom tyrosinase rapidly produces RD-quinone, which is quickly converted to RD-cyclic quinone and RD-hydroxy-p-quinone. In this study, we examined whether either or both of the enantiomers of RD can be oxidized by human tyrosinase. Using a chiral HPLC column, racemic RD was resolved optically to R(-)-RD and S(+)-RD enantiomers. In the presence of a catalytic amount of l-dopa, human tyrosinase, which can oxidize l-tyrosine but not d-tyrosine, was found to oxidize both R(-)- and S(+)-RD to give RD-catechol and its oxidation products. S(+)-RD was more effectively oxidized than l-tyrosine, while R(-)-RD was less effective. These results support the notion that the melanocyte toxicity of RD depends on its tyrosinase-catalyzed conversion to toxic quinones and the concomitant production of reactive oxygen species.

Naturally occurring pradimicins (PRMs) show specific recognition of d-mannose (d-Man) in aqueous media, which has never been achieved by artificial small molecules. Although the Ca2+-mediated dimerization of PRMs is essential for their d-Man binding, the dimeric structure has yet to be elucidated, leaving the question open as to how PRMs recognize d-Man. Thus, we herein report the structural elucidation of the dimer by a combination of X-ray crystallography and solid-state NMR spectroscopy. Coupled with our previous knowledge regarding the d-Man binding geometry of PRMs, elucidation of the dimer allowed reliable estimation of the mode of d-Man binding. Based on the binding model, we further developed an azide-functionalized PRM derivative (PRM-Azide) with d-Man binding specificity. Notably, PRM-Azide stained Candida rugosa cells having mannans on their cell surface through conjugation with the tetramethylrhodamine fluorophore. The present study provides the practical demonstration that PRMs can serve as lectin mimics for use in glycobiological studies., ファイル公開：2020-07-18

The exposure of human skin to 4-(4-hydroxyphenyl)-2-butanone (raspberry ketone, RK) is known to cause chemical/occupational leukoderma. RK has a structure closely related to 4-(4-hydroxyphenyl)-2-butanol (rhododendrol), a skin whitening agent that was found to cause leukoderma in the skin of consumers in 2013. Rhododendrol is a good substrate for tyrosinase and causes a tyrosinase-dependent cytotoxicity to melanocytes, cells that are responsible for skin pigmentation. Therefore, it is expected that RK exerts its cytotoxicity to melanocytes through the tyrosinase-catalyzed oxidation to cytotoxic o-quinones. The results of this study demonstrate that the oxidation of RK by mushroom tyrosinase rapidly produces 4-(3-oxobutyl)-1,2-benzoquinone (RK-quinone), which is converted within 10-20 min to (E)-4-(3-oxo-1-butenyl)-1,2-benzoquinone (DBL-quinone). These quinones were identified as their corresponding catechols after reduction by ascorbic acid. RK-quinone and DBL-quinone quantitatively bind to the small thiol N-acetyl-l-cysteine to form thiol adducts and can also bind to the thiol protein bovine serum albumin through its cysteinyl residue. DBL-quinone is more reactive than RK-quinone, as judged by their half-lives (6.2 min vs 10.5 min, respectively), and decays rapidly to form an oligomeric pigment (RK-oligomer). The RK-oligomer can oxidize GSH to GSSG with a concomitant production of hydrogen peroxide, indicating its pro-oxidant activity, similar to that of the RD-oligomer. These results suggest that RK is cytotoxic to melanocytes through the binding of RK-derived quinones to thiol proteins and the pro-oxidant activity of the RK-oligomer.

From an extract of the Okinawan sponge Petrosia strongylata, five novel acetylenic metabolites, petrosiols A (1)–E (5), were isolated and chemically characterized. These compounds induced nerve growth factor (NGF)-like neuronal differentiation of PC12 cells. The structure–activity relationships of the petrosiols, their derivatives, and some related known compounds suggest the importance of the rare 2,4-diyne-1,6,7,8-tetraol fragment for the neurotrophic activity of the petrosiols.

Miuraenamides A and D, cyclodepsipeptides with antimicrobial and antitumor activity, were synthesized. The synthesis of an unsaturated hydroxycarboxylic acid moiety, starting from a chiral epoxide, was achieved by Suzuki-Miyaura coupling as a key step. As a result, the overall yield for miuraenamide A over the longest linear sequence is 3.2%, while the yield of the previously reported procedure is 1.9%. In addition, the cell growth-inhibitory activity and anti-Phytophthora activity of the synthesized compounds were evaluated.

Metastatic melanoma is resistant to conventional therapies. N-propionyl-4-S-cysteaminylphenol (NPrCAP), an N-protected sulfur-amine analog of tyrosine, is a good substrate for tyrosinase and is selectively incorporated into melanoma cells, causing cytotoxicity in vitro and in vivo. We have recently shown that intratumoral injections of NPrCAP suppress not only the growth of primary B16F1 melanoma tumors but also of secondary, re-challenged tumors. The participation of CD8(+) T cells has been suggested for the NPrCAP-mediated anti-B16 melanoma immunity. In this study, the molecular mechanism of the NPrCAP cytotoxicity and immunogenicity was examined. The phenol NPrCAP was shown to be activated by mushroom tyrosinase to the ortho-quinone N-propionyl-4-S-cysteaminyl-1,2-benzoquinone (NPrCAQ), and the structure was confirmed by reducing it to the corresponding catechol. NPrCAQ reacted rapidly with biologically relevant sulfhydryl compounds such as cysteine, glutathione and bovine serum albumin. The NPrCAQ-thiol adduct formation was proven with a model thiol N-acetylcysteine by spectroscopic methods. The production and release of NPrCAQ-protein adducts was verified in B16F1 melanoma cells in vitro and in B16F1 melanoma-bearing mice in vivo through the detection of 5-S-cysteaminyl-3-S-cysteinylcatechol after acid hydrolysis of the protein fraction. These results suggest that the phenol NPrCAP, acting as a prohapten, can be activated in melanoma cells by tyrosinase to the quinone-hapten NPrCAQ, which binds to melanosomal proteins through their cysteine residues to form possible neo-antigens, thus triggering the immunological response. NPrCAP thus represents a potential new approach to immunotherapy against metastatic melanoma.

A library of extracts established from hundreds of marine organisms was screened by a cytotoxicity test. The active organic extract of an Okinawan marine sponge of the genus Dysidea was subjected to bioassay-guided fractionation to give three new polyoxygenated steroids dysideasterols F-H (1-3), together with two known related compounds (4 and 5). Their structures were confirmed by NMR and mass spectroscopic analyses. A characteristic structural feature of 2, 4 and 5 is an allylic epoxide, whereas this epoxide undergoes ring-opening by a neighbouring hydroxyl group to give a tetrahydrofuran ring in 1 and 3. All compounds 1-5 exhibited a similar cytotoxic effect with IC50 values of 0.15-0.3 µM against human epidermoid carcinoma A431 cells, demonstrating that the allylic epoxide moiety was not responsible for this cytotoxic effect.

The mating hormones α1 and α2 induce sexual reproduction of the phytopathogenic genus Phytophthora. To demonstrate the structural elements responsible to hormonal activity, 17 derivatives of α1 and α2 were synthesized and their hormonal activity (oospore-inducing activity) was evaluated. The terminal ester derivatives of α1 (diacetate and dibenzoate) retained the hormonal activity, whereas a dicarbamate derivative completely suppressed the activity. Even monocarbamates showed weak activities; among them the 1-O-carbamate was less active than 16-O-carbamate, suggesting that the 1-OH group is a little more important than 16-OH. Dihydro, dehydro, and demethyl derivatives exhibited the minimum level of activity. Surviving activity of 15-epi-α1 suggested a less importance of this stereochemistry. Contrary to α1, not only the terminal diacetate derivative but also monoacetates of α2 exhibited no or little activity. Among the monoacetates, 1-O-acetyl-α2 exhibited little yet relatively better activity than the others. No activity was observed for mono- and dicarbamoyl derivatives of α2. Dihydro α2 with the saturated double bond lost most of the activity. These findings suggest that both the mating hormones α1 and α2 require most of the functional (hydroxyl, keto, and olefinic) groups they possess in their natural form for inducing the sexual reproduction of Phytophthora.

Five cytotoxic macrolides, aplyronines D–H (4–8), were isolated from the Japanese sea hare Aplysia kurodai. They are new congeners of the antitumor compound aplyronine A (1), which was previously isolated from the same organism. Their structures were determined by spectroscopic analysis (NMR and MS). The cytotoxicity of these new compounds was evaluated in comparison with that of aplyronines A–C (1–3), suggesting the importance of the 7-O-seryl ester group for mediating the potent cytotoxicity of aplyronines.

We have previously demonstrated that coffee and caffeine ameliorated hyperglycemia in spontaneously diabetic KK-A(y) mice. This present study evaluates the antidiabetic effects of coffee and caffeine on high-fat-diet-induced impaired glucose tolerance in C57BL/6J mice. C57BL/6J mice fed a high-fat diet were given regular drinking water (control group), or a 2.5-fold-diluted coffee or caffeine solution (200 mg/L) for 17 weeks. The ingestion of coffee or caffeine improved glucose tolerance, insulin sensitivity, and hyperinsulinemia when compared with mice in the control group. The adipose tissue mRNA levels of inflammatory adipocytokines (MCP-1 and IL-6) and the liver mRNA levels of genes related to fatty acid synthesis were lower in the coffee and caffeine groups than those in the control group. These results suggest that coffee and caffeine exerted an ameliorative effect on high-fat-diet-induced impaired glucose tolerance by improving insulin sensitivity. This effect might be attributable in part to the reduction of inflammatory adipocytokine expression.

Four stereoisomers of Phytophthora mating hormone α2 were synthesized using both enantiomers of citronellol as starting materials. The absolute configuration of the natural product was determined to be 7 S ,11 R ,15 R by oospore-inducing assays of the synthetic isomers. A concise synthetic procedure of α1 was also established using a common synthetic intermediate of α2.

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys(73) and, to the lesser extent, the active site Cys(32). More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys(73), whereas Cys(32) exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.