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Guided by a probe-based molecular networking strategy, five undescribed cycloheptapeptides, phakefusins A-E (1-5), were isolated from the marine sponge Phakellia fusca. Compounds 1 and 2 contain the nonproteinogenic amino acid residues of dioxindolyalanine (Dioia) and β-3-oxindolylalanine (Oia), respectively. Compound 3 possesses a unique methionine sulfoxide, whereas compound 5 includes a glutamic acid ethyl ester unit. Their structures were elucidated through NMR spectroscopy, HR-MS/MS analysis, and the advanced Marfey's method. By synthesizing the (S, S/R)-Oia standard through tryptophan oxidation, we determined the configuration of this amino acid in compound 2 using the advanced Marfey's method. These cycloheptapeptides were evaluated for their antitumor, antibacterial, and antioxidant activities. Compound 1 showed moderate cytotoxicity against MCF-7 and PC9 cells, with IC 50 values of 6.8 and 9.6 μM, respectively, while compounds 2-5 demonstrated potential antioxidant effects by upregulating HO-1, NQO1, and SOD2 levels, as well as inducing Nrf2 activation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Ustiloxins I-M (1-5), five undescribed cyclopeptides bearing a 15-membered macrocyclic skeleton, were isolated from Cordyceps militaris. The structures of 1 and 5 were identified by spectroscopic and crystallographic methods, whereas the structures of 2-4 were assigned by spectroscopic and computational approaches. Biological evaluation of all the compounds toward human triple-negative breast cancer cells revealed that compounds 4 and 5 are toxic with IC 50 values of 64.29 μM and 28.89 μM, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pseudostellaria heterophylla (Miq.) Pax. (Taizishen, TZS) contains a variety of natural active cyclic-peptide compounds (CP). In this article, four kinds of CP monomers were isolated by HPLC and the structures were identified by mass spectrometry. The in vivo absorption of CP was detected by UPLC-MS/MS. The interaction between CP and membrane receptor was analyzed by SPR. As a result, the relative absorption rate of CP was Pesudostellarin B > Heterophyllin B > Pesudostellarin C > Pesudostellarin E. The difference in absorption rate of CP in vivo was related to its interaction with membrane receptors. The absorption mechanism of CP might be different. This is the first report that in vivo absorption study of different CP from TZS and explore its absorption mechanism, laying a theoretical foundation for the research and development of its oral drugs, and providing new ideas for the study of the absorption mechanism of CP from traditional Chinese medicine., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Suntamide A (1), a new cyclic peptide, was isolated from Cicadidae Periostracum. The gross structure of 1 was elucidated by detailed analysis of HRMS and 1D/2D NMR spectra, and the absolute configuration was established by C3 Marfey's method. We extended our study to examine biological activity of 1, and found that 1 protected SH-SY5Y cells against rotenone-induced neurotoxicity. This effect of 1 seemed to be attributed to antioxidant induction and protection of mitochondria from rotenone-caused injury. Along with augmentation of the antioxidant system by 1, there was an evident activation of Nrf2, a transcription factor involved in the activation of the antioxidant system. These results indicate that 1 rescued the cells from rotenone-mediated neurotoxicity by enhancing antioxidant capacity via induction of Nrf2, suggesting that the compound could be used as a therapeutic intervention in neurodegenerative diseases such as Parkinson's disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tunicyclin L (1), cyclo (L-Pro 1 -Gly-L-Phe 1 -L-Ile-L-Pro 2 -L-Phe 2 -L-Thr-L-Val), and 11 known compounds, including one cyclic peptide (2), eight carboline alkaloids (3 -10), one lignan (11) and one flavone (12) were isolated from the roots of Psammosilene tunicoides. Their structures were elucidated on the basis of extensive UV, IR, MS, NMR spectroscopic data and comparison with literature. Single-crystal X-ray diffraction results revealed the stereochemistry of the 24-membered ring cyclic peptide (1). Among these known compounds, compound 6 was found to be a new natural product, and compounds 3, 4, and 11 were isolated from this plant for the first time. Five compounds (1, 3, 4, 7, and 9) showed moderate anti-acetylcholinesterase (AChE) activity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A new cyclopeptide, pulvpeptin A (1), two pairs of norlignan lignanosides, tamariscinosides G and H (2, 3), together with five known compounds (4-8) were isolated from Selaginella pulvinata. The structures were elucidated by spectroscopic data and chemical degradation. The activity of tamariscinoside G (2) was evaluated by stimulating glucose uptake in 3T3-L1 adipocytes. The results showed 1.49-fold increase in glucose uptake in 3T3-L1 cells relative to basal., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

A variety of physiological functions such as exopolysaccharide synthesis, antibiotic production, and primary metabolism are tightly controlled by quorum sensing in microorganisms. In this study, a marine-derived bacterium Sphingomonas sp. WG was found to possess a cyclo(L-Pro-L-Phe)-mediated quorum sensing mechanism. The cyclo(L-Pro-L-Phe) produced by Sphingomonas sp. WG functions as a signalling molecule in this quorum sensing system. It is the first attempt to characterize cyclic dipeptides as quorum sensing signalling molecules in Sphingomonas sp. and the results effectively make the classical quorum sensing theory more perfect. Furthermore, the supplementation with isolated cyclo(L-Pro-L-Phe) resulted in a 15% increase in the production of welan gum secreted by Sphingomonas sp. WG in the submerged fermentation. The data presented in this study will provide evidences for exploring the role of cyclic dipeptides in regulating the production of welan gum., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This work presents the selection and characterization of erythropoietin (EPO)-binding cyclic peptide ligands. The sequences were selected by screening a focused library of cyclic depsipeptides cyclo[(N α -Ac)Dap(A)-X 1 -X 6 -AE], whose structure and amino acid compositions were tailored to mimic the EPO receptor. The sequences identified through library screening were synthesized on chromatographic resin and characterized via binding-and-elution studies against EPO to select a pool of candidate ligands. Sequences with higher hydrophobicity consistently showed stronger binding to EPO, with the exception of FSLLSH, which was noted for its lower hydrophobicity and high EPO binding. Mutagenesis studies performed on FSLLSH with natural and non-natural amino acid substitutions led to the identification of critical EPO-binding determinants, and the discovery of new peptide ligands. In particular, histidine-scanning mutagenesis performed on three lead sequences yielded the discovery of variants whose EPO-binding is more pH-sensitive, which facilitates EPO recovery. Selected ligands were studied to correlate the elution yield to the salinity of the binding buffer and the elution pH. Elution yields were consistently higher when EPO binding was performed at low ionic strength. The crystal structures of lead cyclic peptides were docked in silico against EPO to estimate the binding affinity in solution. Isotherm adsorption studies performed on FSLLSH indicated that the cyclic version of the ligand (K D =0.46μM) has a higher affinity for EPO than its corresponding linear variant (K D =1.44μM). Collectively, these studies set the stage for use of the cyclic peptide ligands as EPO purification and detection tools., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Seven cyclopeptide alkaloids were isolated from the stem bark of Ziziphus nummularia and Ziziphus spina-christi. Three previously undescribed compounds were identified: nummularine-U, spinanine-B and spinanine-C, together with the known compounds mauritine-F, nummularine-D, nummularine-E and amphibine-D. For their purification either semi-preparative HPLC with DAD and ESIMS detection or HPLC-PDA-(HRMS)-SPE-NMR was applied, together with conventional separation methods. Their structures were elucidated by spectroscopic means., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

[1-9-NαC]-crourorb A1 is a cyclic peptide isolated from Croton urucurana Baillon latex, found in midwestern Brazil, that has been shown to exert cytotoxic effects against a panel of cancer cell lines. However, the underlying mechanisms responsible for the crourorb A1-induced cytotoxicity in cancer cells remain unknown. In this study, the effects of crourorb A1 on the viability, apoptosis, cell cycle and migration of Huh-7 (human hepatocarcinoma) cells were investigated. We evaluated the viability of Huh-7 cells treated with crourorb A1 in 2D and 3D collagen cultures and found that cells in 3D culture exhibited increased resistance to crourorb A1 compared to cells in 2D culture (IC 50 : 62μg/ml versus 35.75μg/ml). Crourorb A1 treatment decreases the viability of Huh-7 cells in a dose- and time-dependent manner and is associated with the induction of apoptosis, in the absence of necrotic cells, through the activation of caspase-3/7 and increased expression of the pro-apoptotic proteins Bak, Bid, Bax, Puma, Bim, and Bad. The effects of crourorb A1 are also associated with G2/M phase cell cycle arrest and increases in cyclin-dependent kinase (CDK1) and cyclin B1 expression. A significant reduction in Huh-7 cell migration induced by crourorb A1 was also observed in the presence of mitomycin C. Finally, we showed that the JNK/MAP pathway, but not ERK signaling, is involved in crourorb A1-induced hepatocarcinoma cell mortality., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently it has been demonstrated that catanionic mixtures of oppositely charged surfactants have improved physicochemical-biological properties compared to the individual components. Isotherms of mixtures of an anionic biosurfactant (lichenysin) and a cationic aminoacid surfactant (C 3 (LA) 2 ) indicate a strong interaction suggesting the formation of a new "pseudo-surfactant". The antimicrobial properties of the mixture lichenysin and C 3 (LA) 2 M80:20, indicate a synergistic effect of the components. The mechanism of action on the bacterial envelope was assessed by flow cytometry and Transmission Electron Microscopy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ustiloxins are cyclopeptide mycotoxins produced by the pathogenic fungus Ustilaginoidea virens of rice false smut. Quantification of ustiloxins is essential to assess the food safety of rice infected by rice false smut disease. This paper describes a sensitive method for the simultaneous quantification of ustiloxins A, B, C, D and F in rice grains using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since notable matrix enhancement effects (21%-78%) occurred for all of the target analytes (except for ustiloxin A), several solid phase extraction materials were tested for their ability to retain ustiloxins from aqueous solutions prior to the LC-MS/MS analysis, including C18 sorbents, polymer anion exchange sorbents resin (PAX), and polymer cation exchange resin (PCX). The PCX resin was adopted due to its higher extraction capability and selectivity for all targets compared to others, and in this case, almost no matrix effects (-5% to 8%) were observed for all of the ustiloxins monitored. The developed method reached limits of quantification of 0.2-2ngg -1 , and linearity was statistically verified over two orders of magnitude with regression coefficients (R 2 )>0.991. The mean recoveries were from 85% to 109%, and the inter-day precisions (n=11) were less than 16%, with intra-day precisions (n=6) within 12%. Analysis of samples showed that ustiloxin A was the dominant species, with the content ranging from 5.5 to 273.8ngg -1 , followed by ustiloxin B (≤88.7ngg -1 ), while concentrations of ustiloxins C, D and F were slightly lower (≤43.2ngg -1 ). To our knowledge, this is the first report on the determination and analysis of five ustiloxins simultaneously in a single analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Five Ib-type cyclopeptide alkaloids, jubanines F-J (1-5), and three known compounds, nummularine B (6), daechuine-S3 (7), and mucronine K (8) were isolated from the roots of Ziziphus jujuba. Their structures were fully characterized by spectroscopic analyses in combination with chemical derivatization. Compounds 1-3, and 6 were evaluated for their antiviral activity against the porcine epidemic diarrhea virus (PEDV). Compounds 2, 3, and 6 showed potent inhibitory effects on PEDV replication., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Naturally occurring macrocyclic peptides represent a unique class of compounds that exhibit various biological activities ranging from antibiotics to immunosuppressant. Although the discovery of such macrocyclic peptides had relied on their isolation from living organisms, recent advances in ribosomal peptide synthesis and in display techniques made it possible to use artificially generated macrocyclic peptide libraries for selection of ligands for biologically relevant proteins. In this review, we discuss the technologies and their applications for the discovery of peptide ligands., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Roles of lipids in the cell membrane are poorly understood. This is partially due to the lack of methodologies, for example, tool chemicals that bind to specific membrane lipids and modulate membrane function. Theonellamides (TNMs), marine sponge-derived peptides, recognize 3β-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells through as yet unknown mechanisms. Here, we show that TNMs recognize cholesterol-containing liquid-disordered domains and induce phase separation in model lipid membranes. Modulation of membrane order was also observed in living cells following treatment with TNM-A, in which cells shrank considerably in a cholesterol-, cytoskeleton-, and energy-dependent manner. These findings present a previously unrecognized mode of action of membrane-targeting natural products. Meanwhile, we demonstrated the importance of membrane order, which is maintained by cholesterol, for proper cell morphogenesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Surfactin-type lipopeptides are suspected of being implicated in the rare food poisonings caused by Bacillus species outside the Bacillus cereus cluster. In order to get information on surfactin levels in actual human foods, bacilli from three commercial samples of a Japanese traditional bean product, natto, were isolated in order to clarify their potential to produce the suspect lipopeptides. The isolated bacilli were characterized as Bacillus subtilis. They were β-hemolytic and gave a positive signal in the PCR screen for genes associated with surfactin production, and their culture extracts were cytotoxic to boar sperm cells. Organic extracts of both Bacillus cultures and the natto samples were analyzed for their surfactin content using ultrahigh-performance liquid chromatography with high-resolution mass spectrometry. All the strains proved to be surfactin producers (15 to 39 μg/ml culture medium); the natto samples contained as much as 2.2 mg g(-1) of surfactins. This means that consumers can ingest at least approximately 80 to 100 mg of surfactins per single 50-g natto serving apparently without suffering any ill effects, indicating a very low human toxicity.

A cyclic peptide, jatrophidin I, was isolated from the latex of Jatropha curcas L. Its structure was elucidated by extensive 2D NMR spectroscopic analysis, with additional conformational studies performed using Molecular Dynamics/Simulated Annealing (MD/SA). Jatrophidin I had moderate protease inhibition activity when compared with pepstatin A; however, the peptide was inactive in antimalarial, cytotoxic and antioxidant assays., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The purification and enrichment of most natural products with potential pharmaceutical applications has been performed mainly employing conventional batch-mode chromatographic processes. There is a growing interest in use of simulated moving bed (SMB) chromatography for natural product enrichment as this method enables conservation of mobile phase, while increasing productivity of chromatography medium. SMB increases yield while decreasing cost. Cyclolinopeptides C ([1-9-NaC],[1-MetO]-CLB, 3) and E ([1-8-NaC],[1-MetO]-CLE, 8) were extracted as a mixture from flaxseed oil and then enriched using a three-zone simulated moving bed. The current research extends the SMB technology to enrichment of cyclolinopeptides (CLs), a group of biologically active hydrophobic cyclic peptides that occur in flaxseed oil. Of interest are [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) that provide synthetic scaffolds for modified CLs. The influence of flow rate (feed, desorbent, and extract) on the separation of [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) was investigated., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although >100 thiopeptides have been discovered, the number of validated gene clusters involved in their biosynthesis is lagging. We use genome mining to identify a silent thiopeptide biosynthetic gene cluster responsible for biosynthesis of lactazoles. Lactazoles are structurally unique thiopeptides with a 32-membered macrocycle and a 2-oxazolyl-6-thiazolyl pyridine core. We demonstrate that lactazoles originate from the simplest cluster, containing only six unidirectional genes (lazA to lazF). We show that lazC is involved in the macrocyclization process, leading to central pyridine moiety formation. Substitution of the endogenous promoter with a strong promoter results in an approximately 30-fold increase in lactazole A production and mutagenesis of lazC precursor gene in production of two analogs. Lactazoles do not exhibit antimicrobial activity but may modulate signaling cascades triggered by bone morphogenetic protein. Our approach facilitates the production of a more diverse set of thiopeptide structures, increasing the semisynthetic repertoire for use in drug development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Languishing antibiotic discovery and flourishing antibiotic resistance have prompted the development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against Mycobacterium tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2-catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for the bactericidal activity of lassomycin., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Myriad applications of surfactin in environmental and biomedical field prompt understanding the self-assembly behaviour of surfactin in aqueous solution as well as its interaction with counterions. Effect of four divalent counterions namely, Ni(2+), Zn(2+), Cd(2+), and Ca(2+) on the self-assembly of the surfactin, a biosurfactant isolated from Bacillus subtilis YB7 is studied by fluorescence spectroscopy, dynamic light scattering, optical and electron microscopic studies. The critical micelle concentration (CMC) and aggregation number (Nagg) of surfactin are 96.76 ± 15.49 μM and 101.12 ± 2.53, respectively. The degree of counterion association increases as its ionic radius decreases. Ni(2+) exhibits the highest and Ca(2+) the least degree of counterion association. Addition of counterion reduces the size of the microstructures, aggregation number (Nagg) and zeta potential. The reduction in the zeta potential indicates the neutralization of the negative charges on the electrical double layer of the microstructures. Differential interference contrast (DIC) and transmission electron microscopic (TEM) images of surfactin show the presence of vesicles and large aggregates including giant vesicles. On the addition of Ca(2+), fusion of vesicles into large aggregates is predominantly observed. Ni(2+) induces the transition of large spherical vesicles into small spherical, worm-like vesicles and multicompartment-like structures (vesosome). Such structures are the evidences for metal ion coordinated intervesicular interactions. This study reveals that the self-assembly process of surfactin can be controlled by the addition of metal ions according to the requirements., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Two biosurfactants, surfactin and fatty acyl-glutamate, were produced from genetically-modified strains of Bacillus subtilis on 2% glucose and mineral salts media in shake-flasks and bioreactors. Biosurfactant synthesis ceased when the main carbohydrate source was completely depleted. Surfactin titers were ∼30-fold higher than fatty acyl-glutamate in the same medium. When bacteria were grown in large aerated bioreactors, biosurfactants mostly partitioned to the foam fraction, which was recovered. Dispersion effectiveness of surfactin and fatty acyl-glutamate was evaluated by measuring the critical micelle concentration (CMC) and dispersant-to-oil ratio (DOR). The CMC values for surfactin and fatty acyl-glutamate in double deionized distilled water were 0.015 and 0.10 g/L, respectively. However, CMC values were higher, 0.02 and 0.4 g/L for surfactin and fatty acyl-glutamate, respectively, in 12 parts per thousand Instant Ocean®[corrected].sea salt, which has been partly attributed to saline-induced conformational changes in the solvated ionic species of the biosurfactants. The DORs for surfactin and fatty acyl-glutamate were 1:96 and 1:12, respectively, in water. In Instant Ocean® solutions containing 12 ppt sea salt, these decreased to 1:30 and 1:4, respectively, suggesting reduction in oil dispersing efficiency of both surfactants in saline. Surfactant toxicities were assessed using the Gulf killifish, Fundulus grandis, which is common in estuarine habitats of the Gulf of Mexico. Surfactin was 10-fold more toxic than fatty acyl-glutamate. A commercial surfactant, sodium laurel sulfate, had intermediate toxicity. Raising the salinity from 5 to 25 ppt increased the toxicity of all three surfactants; however, the increase was the lowest for fatty acyl-glutamate., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The current study was designed to evaluate the antinociceptive profile of five cyclopeptide alkaloids isolated from Ziziphus oxyphylla, including Oxyphylline-B 1, Oxyphylline C 2 Oxyphylline-D 3, Nummularin-C 4, and Nummularin-R 5. The effect was studied in acetic acid induced writhing and formalin induced flinching behavior tests, at 2.5 and 5mg/kg i.p. In the post-acetic acid induced writhing test, the compounds significantly ameliorated abdominal constrictions in a dose dependent manner, with compounds 1 and 5 showing 80.98% and 77.87% protection, respectively. When challenged in the formalin induced test, pretreatment of compounds significantly attenuated painful sensation in both phases. Moreover, compounds 1 and 5 were more effective with 45.32% and 75.32% for 1 and 36.77% and 71.10% protection for 5, in the 1st and 2nd phases respectively. The peripheral analgesia was strongly augmented by the central effects of these compounds. The current finding strongly supports the ethnomedicinal use of this valuable medicinal plant in various painful conditions., (© 2013.)

Backbone-cyclized proteins, with their characteristic stability toward denaturants such as heat and chemicals, are becoming increasingly significant in many applications. Intein-mediated protein cyclization is the most efficient and frequently used method of choice and has been successfully applied to various targets, achieving stable proteins. However, the detection and isolation of the cyclic protein from the linear one after cyclization is very difficult because the backbone-cyclized protein and the linear one (a by-product formed during the cyclization reaction), which originated from the same molecule, are almost identical in terms of their size. Thus, we first developed a split c-myc tag system; the active c-myc tag was formed only in the backbone-cyclized protein and not in the linear by-product from the inactive precursor, and this helps both the detection and purification of the backbone-cyclized proteins. This tag system, which we called a cyclization tag, was further engineered in its sequence to develop an engineered c-myc (e-myc) tag with enhanced efficiency in the backbone cyclization reaction while keeping its specificity toward the commercial antibody intact. Using two different proteins as models, we show that the cyclization tag developed here can be used as a specific tag for the backbone-cyclized protein, thereby facilitating detection and purification., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Microcystins and nodularin are cyclic peptides hepatotoxins produced by cyanobacterial genera (blue-green algae). Toxic cyanobacterial blooms are a worldwide problem, as reported in several countries, like China, Australia, or the United States. Therefore, it is necessary to develop sensitive and reliable analytical methodology to determine this type of toxins in water at parts per billion levels, or even lower. In this work, the potential of solid-phase extraction coupled on-line to ultra-high-pressure liquid chromatography/electrospray tandem mass spectrometry (SPE-UHPLC-MS/MS) has been investigated for the efficient quantification and confirmation of microcystins LR, RR, YR, LY, LW, LF and nodularin in surface and drinking water samples, at sub-ppb levels. The method developed involves the injection of only 1 mL of water sample into the on-line SPE-UHPLC-MS/MS system and allows the rapid determination of the compounds selected (8 min of chromatographic run), avoiding laborious sample treatment. The method was validated in surface and drinking water by means of recovery experiments at 0.25 and 1 μg L(-1). Average recoveries (n=5) ranged from 71 to 116%, with relative standard deviations (RSDs) lower than 15%. For microcystins LR, RR, YR and nodularin, a third level was also assayed (0.1 μg L(-1)) obtaining satisfactory data too. Limits of detection between 0.002 and 0.0405 μg L(-1) were estimated (0.0005 μg L(-1) for nodularin). The developed method was applied to the analysis of water samples collected in the province of Castellón (Spain). The acquisition of three MS/MS transitions for each compound allowed the unequivocal confirmation of positive samples, which was supported by the accomplishment of ion intensity ratios and retention time when compared with reference standards., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Three monolithic C(18)-bonded silica gel columns i.e. Chromolith SpeedROD (CSR), Chromolith Performance (CP), and Chromolith High Resolution (CHR), MerckKGaA Darmstadt, Germany and two particle-based columns i.e. ZORBAX Eclipse XDB-C(18) (ZEX), Agilent and POROS R1/20 (POR), Applied Biosystems were compared for their performance in separating a mixture of flaxseed cyclolinopeptides (CLs). Gradient mobile phases of acetonitrile and water were optimized for each column. The performance of CHR column in profiling CL standards, measured as the resolution of individual CL, selectivity, and peak asymmetry exceeded the performance of traditional particle-packed columns and the other monolithic columns. The profiling of CLs in aqueous methanolic flaxseed extract was optimized for high-throughput analysis. A total analysis time of 1.5 min at a flow rate of 3.0mLmin(-1) was achieved on a CSR column. Injection of over 2000 methanol extracts of flaxseed on a CSR column had no impact on backpressure or resolution of a standard CL mixture., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A mini-Tn10 transposon mutagenesis library of strain Bacillus amyloliquefaciens BAhja NK10 was constructed and one mutant, L4, exhibiting markedly enhanced antagonistic activity against Micrococcus luteus, was screened and possessed an inactivated purL gene. A similar antibacterial spectrum was observed for both L4 and wild-type strains, but L4 displayed a markedly strengthened antimicrobial effect. Extracts of the corresponding culture media were separated by means of liquid chromatography and subjected to antimicrobial assay wherein the active molecule was further purified. Mass spectrometry and genetic analysis data revealed that the bacteriocin subtilosin A was produced and was responsible for the antibacterial activity of B. amyloliquefaciens. Expression of the subtilosin-A-encoding gene sboA was determined to occur earlier in the purL mutant, while the transcription level of the negative regulator AbrB declined by 3.3-fold. Correspondingly, an increase was observed in the mRNA levels of two early sporulation genes spo0A and sigH, which are implicated in repressing abrB expression and regulating subtilosin A production. Moreover, RT-PCR showed that the expression of three Kin family kinases was coordinately upregulated. We speculated that purL mutation could result in induced expression of Kin kinases and accelerated accumulation of phosphorylated Spo0A, with the latter repressing transcription of abrB which ultimately led to earlier and increased production of the bacteriocin subtilosin A., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Contamination of water, foods and food supplements by various genera of cyanobacteria is a serious health problem worldwide for humans and animals, largely due to the toxic effects of microcystins (MCs) and nodularin (NOD), a group of hepatotoxic cyclic peptides. The toxins occur in variable structures resulting in more than 90 different MCs and 8 different NODs, many of them not having been investigated for their toxic potency. Potent MCs such as MC-LR have been shown to elicit their hepatotoxic potency via inhibition of hepatic protein phosphatases (PP) 1 and 2A leading to over-phosphorylation of vital cellular proteins. This mechanism of action is also thought to be responsible for the long term tumor promoting action of certain MCs and NOD in the liver. Here, we report on the isolation of certain MCs and NOD as well as a number of their desmethylated derivatives from algae bloom. Subsequently, we determined the cytotoxicity of these compounds in isolated primary human and rat hepatocytes in culture. In parallel experiments, we analyzed the inhibitory potency of these congeners on PP1 and 2A using commercially available enzymes. We found in primary rat hepatocytes that MC-LR, -YR and NOD were cytotoxic, namely in the 10 to >50 nM range, while MC-RR was not. The desmethylated congeners of MC-LR, -YR, and NOD were equally or more-toxic as/than their fully methylated counterparts. In primary human hepatocytes we could show that MC-LR, NOD and the desmethylated variants [³Asp]MC-LR, [⁷Dha]MC-LR and [¹Asp]NOD were cytotoxic in the 20 to >600 nM range. Inhibition data with human, bovine and rabbit protein phosphatases 1 and 2A were roughly in accordance with the cytotoxicity findings in human and rat hepatocytes, i.e. desmethylation had no pronounced effects on the inhibitory potencies. Thus, a variety of naturally occurring desmethylated MC and NOD congeners have to be considered as being at least as toxic as the corresponding fully methylated derivatives., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Two anabaenopeptin-type peptides, lyngbyaureidamides A and B, together with two previously reported peptides lyngbyazothrins C and D, were isolated from the cultured freshwater cyanobacterium Lyngbya sp. (SAG 36.91). Their structures were determined by spectroscopic and chemical methods. Lyngbyazothrins C and D were also able to inhibit the 20S proteasome with IC(50) values of 7.1 μM and 19.2 μM, respectively, while lyngbyaureidamides A and B were not active at 50 μM., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

One method of mobilization of iron by mycorrhizal organisms is through the secretion of small organic chelators called siderophores. Hydroxamate donor chelators are a common type of siderophore that is frequently used by fungal organisms. The primary siderophore that is produced by fungi from the genera Ceratobasidium and Rhizoctonia is the tripodal trishydroxamate siderophore basidiochrome. To gain some insight into the iron uptake mechanisms of these symbiotic fungi, the iron binding characteristics of basidiochrome were determined. It was found that basidiochrome exhibits a log β(110) of 27.8±0.1 and a pFe value of 25.0. These values are similar to those of another fungal trishydroxamate siderophore, ferrichrome. The similarity in iron affinity between the two siderophores suggests that the structure of the backbone has little influence in complex formation due to the length of the pendant arms, although the identity of the terminating groups of the pendant arms is likely related to complex stability. The role of basidiochrome in the biogeochemical cycling of iron is also discussed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Iturin A, a powerful antifungal surfactant, is a kind of bacterial lipopeptide produced by Bacillus strains. This study addresses the use of an aqueous two-phase system (ATPS) using ethanol/ammonium sulfate to extract iturin A from Bacillus amyloliquefaciens NJN-6 fermentation broth and the quantification of iturin A by HPLC. Baseline separation of iturin A homologues was performed using an RP-C(18) column with a mixture of water and acetonitrile. The results showed that the correlation coefficient between integral area and concentration was 0.9961 within the range of 20-140 mg/l. The RSD of the retention time and the peak area were 1.29% and 1.45%, respectively. The effects of some operating parameters in ATPS, e.g., pH, temperature and centrifugation time, were also studied. This method can be successfully used for the rapid quantification of iturin A., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Investigation of the MeOH extract obtained from the root of the Ziziphus mauritiana grown in Thailand resulted in the isolation of two 14- and 13-membered cyclic alkaloids, mauritine L (1) and mauritine M (2), and three known cyclopeptide alkaloids, nummularines H (3), B (4) and hemsine A (5). Their structures were elucidated on the basis of extensive NMR spectroscopic analysis. The first single crystal X-ray diffraction study of the 13-membered ring cyclopeptide, nummularine B methiodide (4'), revealed all S configurations on the amino acid residues. The isolated alkaloids exhibited potent antiplasmodial activity against the parasite Plasmodium falciparum with the inhibitory concentration (IC50) ranging from 3.7 to 10.3 μM. Compounds 2 and 3 also demonstrated antimycobacterial activity against Mycobacterium tuberculosis with the MIC of 72.8 and 4.5 μM, respectively., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Scutianene E (1), 3,4,28-tris-epi-scutiaene E (2), 28-epi-scutianene E (3) and scutianene L (4), four neutral cyclopeptide alkaloids, were isolated from Scutia buxifolia Reiss, together with four known cyclopeptide alkaloids, scutianines B, C, D and E. Scutianenes 1-3 are diastereoisomeric compounds, with 3-hydroxyleucine as a β-hydroxy amino acid unit, which is connected to the styryl fragment via an ether bridge, β-phenylserine, as a common ring-bonded amino acid residue. Attached to the amino group of β-hydroxyamino acid is a side chain [trans-CH=CH-Ph]. The structures of the peptides were elucidated by means of spectroscopic analysis, including extensive 2D NMR studies. The stereochemistry for the diastereomeric 3,4,28-tris-epi-scutiaene E and 28-epi-scutianene E was confirmed by X-ray diffraction analysis of their O-acetyl derivatives., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Following an initial clean-up step on silica gel, high-speed counter-current chromatography (HSCCC) was used to separate cyclic peptides from an extract of the seeds of Vaccaria segetalis. The two-phase solvent system used for HSCCC separation was composed of petroleum ether-ethyl acetate-methanol-water at an optimized volume ratio of 0.5:3.5:1:5. From 190 mg of crude extract, 38.0 mg of segetalin B and 28.5 mg of segetalin A were obtained with purities of 98.1% and 95.6% as determined by HPLC, respectively. The chemical structures of the target compounds were confirmed by high resolution electrospray ionization time of flight MS (HRESI-TOF-MS) and (1)H NMR analyses., (Copyright © 2011. Published by Elsevier B.V.)

The complete separation of structurally similar compounds has been a challenge due mainly to their similarity on physical and chemical properties. In the present study, a simple and effective chromatographic method to separate and purify nocathiacin acid from its structural analogue nocathiacin I was developed. After evaluating mobile phase compositions on the retention characteristics by reversed phase high-performance liquid chromatography (HPLC), the elution order of nocathiacin I and nocathiacin acid was completely reversed, and the resolution value between the two analogues was improved, by varying pH value and ionic strength, to greater than 10 from merged peaks under initial conditions. In addition, a preparative isolation of nocathiacin acid was performed by reversed phase column chromatography under the guidance of the HPLC study. This chromatographic method resulted in an efficient process to obtain pure nocathiacin acid with a recovery rate of 83%. The present approach offers a new methodology for the separation of structurally closely related secondary metabolites., (2010 Elsevier B.V. All rights reserved.)

Hantupeptins B (2) and C (3) were isolated, along with the previously reported hantupeptin A (1), from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu Besar, Singapore. Their structures were elucidated by interpretation of extensive 1D and 2D NMR spectroscopic data. Compounds 2 and 3 are cyclic depsipeptides consisting of five alpha-amino/hydroxy acid residues, including phenyllactic acid, proline, N-methyl-valine, valine, N-methyl-isoleucine, and a beta-hydroxy acid unit with different degrees of unsaturation at the terminal end of each molecule. The absolute configurations of the common amino acids and phenyllactic acid were determined by the advanced Marfey's and chiral HPLC analyses, respectively. The complete stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid moiety in hantupeptin A was elucidated by a combination of homonuclear J-resolved 2D NMR experiments and by Mosher's method. Hantupeptins B and C showed moderate in vitro cytotoxicity when tested against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines., (2009 Elsevier Ltd. All rights reserved.)

Four ultra performance liquid chromatography (UPLC) columns with different reversed-phase characteristics were tested in the chromatographic separation of 10 microcystins and three nodularins, cyanobacterial peptide toxins. The columns had been designed by the manufacturer to withstand the ultra-high pressure generated by sub-2microm stationary phase particles and the Waters ACQUITY UPLC system in ultra-fast separations. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, with three gradient rise times: 1, 1.5 and 2min. The UV detection of the toxins was performed by a photodiode array detector. The chromatographic performance was evaluated both visually and by calculating chromatographic parameters such as capacity factor, resolution, peak width at half height, selectivity and peak asymmetry. The best chromatographic performance as judged by visual inspection was given by the ACQUITY BEH Shield RP18 and ACQUITY BEH Phenyl columns. The BEH Shield RP18 column showed excellent selectivity and resolution of chosen peak pairs considered as critical. A further advantage of the UPLC system was the high sample throughput with a total analysis time of 3.12min (injection-to-injection) equalling to 461 separations per 24h.

This experiment was performed to investigate the anxiolytic-like effects of sanjoinine A, one of the major alkaloid compounds in Zizyphi Spinosi Semen (ZSS), by using experimental paradigms of anxiety in comparison with a known anxiolytic, diazepam. Sanjoinine A (2.0 mg/kg) increased the percentage of time spent on the open arms and the number of open arms entries in the elevated plus-maze test, increased the number of head dips in the hole-board test, and increased the percentage of time spent in the center zone and the center zone locomotor distance in the open field box experiment. However, sanjoinine A (0.5, 1.0, 2.0 mg/kg) had no effect on locomotor activity, while diazepam (2.0 mg/kg) significantly reduced locomotor activity. Sanjoinine A (0.5, 1.0, 2.0 mg/kg) did not influence the grip force in the grip strength meter test either. Molecular experiments showed that sanjoinine A (2.0, 5.0 microM) increased chloride influx in cultured cerebellar granule cells. In addition, sanjoinine A (5.0 microM) treatment resulted in over-expression of alpha- and gamma-subunits of GABA(A) receptors and glutamic acid decarboxylase (GAD65/67) in cultured cerebellar granule cells. It is concluded that sanjoinine A may have anxiolytic-like effects in the elevated plus-maze, hole-board test and open field test, and these effects may be mediated by GABAergic transmission.

Two new minor cyclopeptides, named japonicin A (1), japonicin B (2), were isolated from the whole plants of Sagina japonica (Caryophyllaceae). Their structures were determined as cyclo-(Pro(1)-Pro(2)-Leu(2)-Leu(1)-Phe(2)-Pro(3)-Gly-Ser-Phe(1)) (1) and cyclo-(Pro(1)-Ile-Tyr-Asp-Pro(2)-Phe(2)-Pro(3)-Phe(1)) (2) on the basis of spectroscopic data, especially by two-dimension NMR technologies.

Three Bacillus strains isolated from honey samples and bee gut were pre-selected for their in vitro antimicrobial activity against Paenibacillus larvae and Ascosphaera apis, important honeybee pathogens. The analysis of their 16S rRNA sequences revealed that C4, M1 and G2III strains belong to the subtilis species. Surfactin synthesis was verified by IR spectroscopy and HPLC studies. Surfactin inhibited P. larvae but it failed to affect A. apis. Vegetative cells of P. larvae were affected as soon as they came in contact with the surfactin sample; two orders of magnitude less in log scale were recorded. Optimal surfactin production was observed in MEL medium, a broth with molasses as the only carbon source. Bacillus subtilis G2III strain exhibited the highest levels of surfactin synthesis in BHI and MEL broths: 1391AU/ml and 2782AU/ml, respectively. Since only A. apis inhibition was observed when cell suspensions were assayed, we suspect that there may be an antimycotic compound within cells. The co-production of surfactin and a fungicide by these strains might biologically control bee pathogens in apiculture.

The problem of toxicity of cyanobacterial toxins is of increasing concern, as the incidence of such blooms grows. Among the toxins, the most abundant in the environment are hepatotoxins known as nodularins and microcystins. These toxins are responsible for almost all known cases of fresh and brackish water intoxication and are responsible for recurrent episodes of human and animal illness and death. Moreover, they are believed to be potent tumor promoters and initiators. However, the mechanisms by which these toxins induce liver cancer are not well understood. The aim of the present study was to determine the effect of nodularin on the kinetics of nucleotide excision repair (NER) in Chinese hamster ovary (CHO) cells exposed to UV radiation. The first set of experiments was performed to define the optimal treatment conditions for nodularin to avoid the possibility of encountering false positive signals in the comet assay due to the apoptogenic activity of nodularin. Based on the analysis of apoptosis, the 6-h treatment time of cells with nodularin (1mug/ml, 10mug/ml and 20mug/ml) was chosen for the alkaline comet assay. The kinetics of NER was determined in CHO cell lines: AA8 (wild-type) and mutant cell lines: UV135 (XPG(-)), UV41 (XPF(-)) and UV20 (ERCC1(-)) exposed to 20J/m(2) UV radiation. The micronucleus assay was performed to determine a residual DNA damage in four cell lines treated with nodularin (10mug/ml) and exposed to equitoxic doses UV radiation. Radiation doses of UV producing 50% of survival for AA8, UV135, UV20 and UV41 cell lines were calculated from UV survival curves. The results show that nodularin impairs the incision/excision step of NER in CHO cells by the ERCC1/XPF inactivation and leads to an increased level of UV-induced cytogenetic DNA damage.

In the Baltic Sea regular, intensive cyanobacterial blooms rich in the cyanobacterium Nodularia spumigena occur during the summer season. N. spumigena is known to produce the cyclic pentapeptide nodularin (NOD) in high concentrations. Marine macroalgae, together with sea-grass meadows, are an extremely important habitat for life in the sea. In addition to this, the decaying macroalgae substantially contribute to the substrate for the microbial loop in coastal food webs. Uptake of nodularin into the brown macroalga Fucus vesiculosus was assessed using an ELISA technique resulting in an uptake of up to 45.1 microg kg(-1) fresh weight (fw). Nodularin was also detected in the reproductive part of the algae (receptacle) at 14.1 microg kg(-1) fw. The induction of oxidative stress in F. vesiculosus, after exposure to NOD, was also shown by monitoring cellular damage as changes in lipid peroxidation and the activation of antioxidative defence systems (antioxidative capacity, superoxide dismutase and soluble glutathione S-transferase).

The cyclic peptide alkaloid, chamaedrine, was isolated from the roots of Melochia chamaedris (Sterculiaceae), along with four known cyclic peptide alkaloids (adouetine X, frangulaline, scutianine B and scutianine C), and waltherione A, parasorbic acid, propacine, and (-)-epicatequine. Their structures were elucidated on the basis of spectroscopic analysis, especially by 2D NMR ((1)H-(1)H-COSY, NOESY, HMQC, HMBC).

In this issue of Chemistry & Biology, Gross et al. report development of a novel genome mining method for isolating products of orphan biosynthetic gene clusters, and the application of this method to the isolation of orfamide A, a novel cyclic lipopeptide.

With the increasing number of genomes sequenced and available in the public domain, a large number of orphan gene clusters, for which the encoded natural product is unknown, have been identified. These orphan gene clusters represent a tremendous source of novel and possibly bioactive compounds. Here, we describe a "genomisotopic approach," which employs a combination of genomic sequence analysis and isotope-guided fractionation to identify unknown compounds synthesized from orphan gene clusters containing nonribosomal peptide synthetases. Analysis of the Pseudomonas fluorescens Pf-5 genome led to the identification of an orphan gene cluster predicted to code for the biosynthesis of a lipopeptide natural product. Application of the genomisotopic approach to isolate the product of this gene cluster resulted in the discovery of orfamide A, founder of a group of bioactive cyclic lipopeptides.

Contribution on microcystin variant analysis by capillary electrochromatography (CEC) with easily affordable spectrophotometric detection is presented. Two types of reversed-phase capillary columns formed by inorganic or organic polymer monoliths were prepared for this purpose. The analyses were performed isocratically by means of tris(hydroxymethyl) aminomethane (TRIS) buffers of mildly alkaline pH containing 30% (v/v) acetonitrile as the mobile phases. The samples were injected electrokinetically and the analyses were done at the same separation field strength of 500 V/cm. Microcystins were detected at 238 nm. Although both column types differ not only in monolith quality (inorganic versus organic) but also in the length of the aliphatic moiety (C8 versus C12) similar results were achieved. The on-column preconcentration as the encouraging prospect of electrochromatographic technique was also tested. Consequently 5% of column volume was injected in contrast with 0.5% at standard injection scheme resulting in the six times enrichment of the low concentrated cyanobacterial extract at the top of the separation column. From these preliminary results can be seen that the CEC method is fully applicable for rapid microcystin screening.

The development of a simplified assay for detection of congeners of the microcystin (MC) hepatotoxin is described that combines the extreme sensitivity of surface-enhanced laser desorption/ionization time-of-flight MS (SELDI TOF-MS) with the superior selectivity of immunoaffinity interactions. Using methods similar to those of conventional immunoassays, MC standards were captured and enriched on immunoreactive ProteinChips coated with an MC-antibody and analyzed by TOF-MS. Unlike with conventional immunoassays, individual congeners were resolved from mixed pools. Assay conditions were optimized for the quantification of MC from untreated raw pond water at concentrations as low as 0.025 microg L(-1), well below the public health relevant guideline of 1 microg L(-1).

Preparation and evaluation of a number of stationary phases for improved chromatographic purification of pneumocandin B0, a key intermediate in the synthesis of the antifungal agent, Cancidas, has led to the identification of several materials with potential for improved performance.

The present study reports a cyclopeptide alkaloid, scutianine M, isolated from the methanolic root bark extract of Scutia buxifolia Reiss (Rhamnaceae) along with six known compounds, scutianines-B, -C, -D, -E, -F, and scutianene D. Its structure was established on the basis of spectroscopic analyses, including application of 2D NMR spectroscopic techniques. As part of a study of the bioactive compounds of medicinal plants from southern Brazil, we also compared the antimicrobial activity of the isolated compounds towards Gram (+), Gram (-) bacteria, and yeasts.