Antifeedant and Antifungal Activities of Metabolites Isolated from the Coculture of Endophytic Fungus Aspergillus tubingensis S1120 with Red Ginseng.

A new globoscinic acid derivative, aspertubin A (1) along with four known compounds, were obtained from the co-culture of Aspergillus tubingensis S1120 with red ginseng. The chemical structures of compounds were characterized by using spectroscopic methods, the calculated and experimental electronic circular dichroism. Panaxytriol (2) from red ginseng, and asperic acid (4) showed significant antifeedant effect with the antifeedant rates of 75 % and 80 % at the concentrations of 50 µg/cm2 . Monomeric carviolin (3) and asperazine (5) displayed weak attractant activity on silkworm. All compounds were assayed for antifungal activities against phytopathogens A. tubingensis, Nigrospora oryzae and Phoma herbarum and the results indicated that autotoxic aspertubin A (1) and panaxytriol (2) possessed selective inhibition against A. tubingensis with MIC values at 8 µg/mL. The co-culture extract showed higher antifeedant and antifungal activities against P. herbarum than those of monoculture of A. tubingensis in ordinary medium. So the medicinal plant and endophyte showed synergistic effect on the plant disease resistance by active compounds from the coculture of A. tubingensis S1120 and red ginseng.

Inhibitory effect of panaxytriol on BV-2 microglial cell activation.

Activated microglia induce brain inflammation and neuronal death. Panaxytriol, ((3R,9R,10R)-Heptadec-1-en-4,6-diyne-3,9,10-triol), is a component of Panax ginseng C. A. Meyer extracts and activates the Nrf2-ARE signaling pathway. However, little is known about its effects on activated microglia in the brain. In this study, we investigated the effect of panaxytriol on lipopolysaccharide (LPS)-induced activated microglia in BV-2 cells. Panaxytriol suppressed LPS-induced NO production and inhibited the increase in iNOS protein expression in BV-2 cells. Besides, panaxytriol inhibited the mRNA expression of proinflammatory cytokines such as TNF-α, IL-1ß, and IL-6. The inhibitory effect of panaxytriol on microglia activation did not affect the Nrf2-ARE pathway and the MAPK pathway. However, panaxytriol suppressed LPS-induced NF-κB nuclear translocation. These results suggest that panaxytriol inhibits the LPS-induced activation of microglia via the inhibition of NF-κB signaling pathway.

When the nine-membered enediynes play hide and seek.

The lack of stability of the 9-membered enediynes not associated with an apoprotein may explain the low number of isolated natural compounds containing this core. To overcome such a problem, particular attention should be paid during the process of extraction and isolation of secondary metabolites, especially from microorganisms such as actinomycetes in order to identify the non-cycloaromatized derivatives.

Improvement of the enediyne antitumor antibiotic C-1027 production by manipulating its biosynthetic pathway regulation in Streptomyces globisporus.

The production of C-1027 in Streptomyces globisporus was previously increased 2- to 3-fold by manipulating three pathway-specific activators, SgcR1, SgcR2, and SgcR3. In this study, we have further characterized two putative C-1027 regulatory genes, sgcE1 and sgcR, by in vivo inactivation. The HxlR family DNA-binding protein SgcE1 was not essential for C-1027 biosynthesis, since inactivation of sgcE1 showed no effect on C-1027 production. In contrast, the proposed repressive role of the sgcR gene was confirmed by a 3-fold increase in C-1027 production in the ΔsgcR mutant S. globisporus SB1022 strain relative to the wild-type strain. Considering SgcR shows no significant similarity to any protein of known function, it may be representative of a new family of regulatory proteins. Finally, overexpression of the previously characterized activator sgcR1 in S. globisporus SB1022 increased the C-1027 yield to 37.5 ± 7.7 mg/L, which is about 7-fold higher than the wild-type strain.

Determination of the geometry of acetoxyendiynes and acetoxyenynes by NMR heteronuclear (13)C-(1)H scalar couplings and (13)C NMR chemical shifts. Structural assignment of the oxylipin natural products peyssonenynes A and B.

Solution NMR methods were used for the structural characterization of the acetoxyendiyne E/Z configuration of the marine natural products peyssonenynes A and B and their synthetic analogs derived from palmitic acid. The scarcity of protons in the proximity of the olefin precluded the determination of the double bond geometry using (1)H NMR methods that rely on proton-proton scalar couplings or experiments such as NOESY or ROESY. Long range (1)H-(13)C heteronuclear scalar couplings, (n)J(CH), measured with the 2D excitation sculptured indirect detection experiment (EXSIDE) proved useful and highly reliable for the analysis of the enol acetate geometry. In addition, it was found that the chemical shift of some carbon atoms in the proximity of the olefin was also sensitive to the double bond configuration of these molecules providing an even simpler way to determine their geometry. This protocol showed its robustness by similar analysis of simpler silyl-protected acetoxyenynes derived from fatty acids. These NMR experimental results and stereochemical predictions were rationalized by DFT calculations.

Polyacetylenes from sardinian Oenanthe fistulosa: a molecular clue to risus sardonicus.

An investigation of Oenanthe fistulosa from Sardinia afforded oenanthotoxin (1a) and dihydrooenanthotoxin (1b) from the roots and the diacetylenic epoxydiol 2 from the seeds. The absolute configuration of 1a and 1b was established as R by the modified Mosher's method, and the structure of 2 by chemical correlation with (+)-(3R,8S)-falcarindiol. Oenanthotoxin (1a) and dihydrooenanthotoxin (1b) were found to potently block GABAergic responses, providing a molecular rationale for the symptoms of poisoning from water-dropwort (Oenanthe crocata) and related plants. These observations bear relevance for a series of historical and ethnopharmacological observations on the identification of the Sardonic herb and the molecular details of the facial muscular contraction caused by its ingestion (risus sardonicus).

Characterization of the two-component, FAD-dependent monooxygenase SgcC that requires carrier protein-tethered substrates for the biosynthesis of the enediyne antitumor antibiotic C-1027.

C-1027 is a potent antitumor antibiotic composed of an apoprotein (CagA) and a reactive enediyne chromophore. The chromophore has four distinct chemical moieties, including an ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety, the biosynthesis of which from l-alpha-tyrosine requires five proteins: SgcC, SgcC1, SgcC2, SgcC3, and SgcC4; a sixth protein, SgcC5, catalyzes the incorporation of this beta-amino acid moiety into C-1027. Biochemical characterization of SgcC has now revealed that (i) SgcC is a two-component, flavin adenine dinucleotide (FAD)-dependent monooxygenase, (ii) SgcC is only active with SgcC2 (peptidyl carrier protein)-tethered substrates, (iii) SgcC-catalyzed hydroxylation requires O 2 and FADH 2, the latter supplied by the C-1027 pathway-specific flavin reductase SgcE6 or Escherichia coli flavin reductase Fre, and (iv) SgcC efficiently catalyzes regioselective hydroxylation of 3-substituted beta-tyrosyl-S-SgcC2 analogues, including the chloro-, bromo-, iodo-, fluoro-, and methyl-substituted analogues, but does not accept 3-hydroxy-beta-tyrosyl-S-SgcC2 as a substrate. Together with the in vitro data for SgcC4, SgcC1, and SgcC3, the results establish that SgcC catalyzes the hydroxylation of ( S)-3-chloro-beta-tyrosyl-S-SgcC2 as the final step in the biosynthesis of the ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety prior to incorporation into C-1027. SgcC now represents the first biochemically characterized two-component, FAD-dependent monooxygenase that acts on a carrier-protein-tethered aromatic substrate.

Strain Prioritization and Genome Mining for Enediyne Natural Products.

The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. IMPORTANCE: Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family.

Poisoning due to water hemlock.

INTRODUCTION: Water hemlock, which encompasses a range of species divided across two genera (Cicuta and Oenanthe), are regarded as being among the most poisonous plants both in North America and in the United Kingdom. Despite their toxicity, the literature consists almost entirely of case reports. AIM: The aim of this review is to summarize this literature by covering all aspects of taxonomy and botanical characterization, principal toxins, basic pharmacology including mechanisms of toxicity, and the clinical features, diagnosis, and management of poisoning. MECHANISMS OF TOXICITY: The principal toxins, cicutoxin and oenanthotoxin, belong to a group of C17 conjugated polyacetylenes. They act as (noncompetitive) gamma-aminobutyric acid antagonists in the central nervous system (CNS), resulting in unabated neuronal depolarization that can lead to seizures. Ingestion of even a small amount of plant matter may result in severe intoxication. FEATURES: After ingestion, the patient is most likely to experience CNS stimulatory effects including seizures that, in the absence of aggressive supportive care, can result in death. Other features include nausea, vomiting, diarrhea, tachycardia, mydriasis, rhabdomyolysis, renal failure, coma, respiratory impairment, and cardiac dysrhythmias. MANAGEMENT: Treatment consists mainly of prompt airway management and seizure control, plus decontamination if achieved early and after stabilization. In the event of renal failure, the use of hemodialysis has been employed successfully. CONCLUSIONS: The ingestion of water hemlock can lead to serious complications that may be fatal. Prognosis is good, however, if prompt supportive care is provided.

Regiospecific chlorination of (S)-beta-tyrosyl-S-carrier protein catalyzed by SgcC3 in the biosynthesis of the enediyne antitumor antibiotic C-1027.

C-1027 is a potent antitumor antibiotic composed of an apo-protein and a reactive enediyne chromophore. The chromophore consists of four different chemical subunits including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety, the biosynthesis of which from l-alpha-tyrosine is catalyzed by six proteins, SgcC, SgcC1, SgcC2, SgcC3, SgcC4, and SgcC5. Biochemical characterization of SgcC3 unveiled the following: (i) SgcC3 is a flavin adenine dinucleotide (FAD)-dependent halogenase; (ii) SgcC3 acts only on the SgcC2 peptidyl carrier protein-tethered substrates; (iii) SgcC3-catalyzed halogenation requires O2 and reduced FAD and either the C-1027 pathway-specific flavin reductase SgcE6 or E. coli flavin reductase (Fre) can support the SgcC3 activity; (iv) SgcC3 also efficiently catalyzes bromination but not fluorination or iodination; (v) SgcC3 can utilize both (S)- and (R)-beta-tyrosyl-S-SgcC2 but not 3-hydroxy-beta-tyrosyl-S-SgcC2 as a substrate. These results establish that SgcC3 catalyzes the third enzymatic transformation during the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027 from l-alpha-tyrosine. SgcC3 now represents the second biochemically characterized flavin-dependent halogenase that acts on a carrier protein-tethered substrate. These findings will facilitate the engineering of new C-1027 analogs by combinatorial biosynthesis methods.