Lapachol biotransformation by filamentous fungi yields bioactive quinone derivatives and lapachol-stimulated secondary metabolites

Abstract

Lapachol is a natural naphthoquinone with a range of biological effects, including anticancer activity. Microbial transformations of lapachol can lead to the formation of new biologically active compounds. In addition, fungi can produce secondary metabolites that are also important for drug discovery. The goal of this study was to evaluate the ability of filamentous fungi to biotransform lapachol into biologically active compounds and identify secondary metabolites produced in the presence of lapachol. Seven out of nine strains of filamentous fungi tested exhibited the ability to biotransform or biodegrade lapachol. The bioactive derivatives norlapachol and isolapachol were identified among biotransformation products. Moreover, lapachol stimulated the production of pyrrolo-[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) and phenol-2,4-bis-(1,1-dimethylethyl), secondary metabolites already known to have antimicrobial and antioxidant activities. These results open the perspective of using these strains of filamentous fungi for lapachol biotransformation and efficient production of several biologically active compounds.     

Volatile compounds from the symbiotic system Azolla filiculoides‐Anabaena azollae bacteria

Abstract

Azolla filiculoides is an aquatic pteridophyte that may be used as animal food, biofertilizer and phytoremediation. Its volatile composition was never studied although several phytochemical analyses were performed. The volatile composition of A. filiculoides grown outdoors in a pond at the Botanical Garden of Lisbon University (BGLU) or in culture conditions as well as the effect of different harvesting times and the storage type were evaluated. The volatiles isolated by hydrodistillation and distillation‐extraction were analysed by gas chromatography and gas chromatography‐mass spectrometry. The oil of all the A. filiculoides samples studied affords a yellowish colour and an unpleasant odour in a yield of 0.01% (v/fw). Alcohols, aldehydes, alkanes and ketones dominated the culture samples, while aldehydes, alcohols, terpenoids and alkanes represented the main volatiles of the BGLU samples. Some quantitative differences were detected in seasonal and type of storage (fresh, dry or frozen at ?20°C) studies of A. filiculoides from the BGLU. The BGLU and culture volatiles showed qualitative differences: 2‐ethyl‐1‐hexanol was only identified in the fern culture, whereas acetophenone, pentylfuran, acetylpyridine and 2‐octanone were only detected in BGLU samples. The dendrogram showed two distinct clusters (culture and BGLU samples). The possible biological origin and bioactivity of some of the volatile compounds is discussed.

Abbreviations: FID, flame ionization detector; i.d., internal diameter; v/fw, volume by fresh weight; GC, gas chromatography; GC‐MS, gas chromatography‐mass spectrometry; u, unified atomic mass unit     

Micropropagation of Santolina canescens Lagasca and in vitro volatiles production by shoot explants

Shoot-tips of Santolina canescens, an aromatic species producing a novel diacetylene derivative, were in vitromultiplied on Murashige-Skoog basal medium (MS) containing different concentrations of 6-benzyladenine (BA) and kinetin (KIN). Rooting phase was performed on media supplemented with different auxins: 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA) and 1-naphthaleneacetic acid (NAA). The best axillary-bud proliferation was recorded on MS medium containing 1.33 M BA plus 0.32 M NAA. Plantlets rooted easily on the different tested media, but more abundant and stronger roots occurred on media containing 2.68 M NAA. The composition of oil obtained through the cultured explants was analyzed by headspace gas chromatography (HS-GC), and santolindiacetylene (SDA) in vitro production was tested. In order to stimulate the production of this product, different concentrations of linoleic acid were supplemented to the media. The best santolindiacetylene production was obtained when the rooting media was supplemented with 0.36 M linoleic acid.     

Health monitoring of plants by their emitted volatiles: trichome damage and cell membrane damage are detectable at greenhouse scale

Pathogen attack and herbivore infestation have a major impact on plant health. In a model study, these two plant health issues were simulated to study whether plant health can be monitored at greenhouse scale through the analysis of volatile organic compounds (VOCs) in greenhouse atmosphere. To simulate pathogen attack and herbivore infestation, we repeatedly stroked the stems of tomato plants ( Lycopersicon esculentum ) and repeatedly removed their side shoots. In addition, we studied the effect of fruit picking on the concentration of plant-emitted VOCs in greenhouse atmosphere. Analysis of air samples obtained before these treatments revealed up to 17 VOCs that are known to be released from tomato plants, of which the most dominant one was the monoterpene β-phellandrene. When plants were 7 weeks old, the concentration of this VOC was approximately 0.06 ppbv before treatment. When plants were 12 weeks old, this concentration was raised to approximately 0.14 ppbv. Stroking of the stems, removing the side shoots and fruit picking resulted in an increase in the concentrations of all mono- and most sesquiterpenes up to 60-fold, which was expected because these VOCs are well-known constituents of trichomes. The treatments did not result in substantially increased concentrations of the stress-related compounds α-copaene, methyl salicylate and ( E,E )-4,8,12-trimethyl-1,3,7,11-tridecatetraene. In contrast to stroking and fruit picking, shoot removal resulted in the emission of the lipoxygenase-derived product ( Z )-3-hexenol in greenhouse atmosphere expressing cell membrane degradation. The findings presented in this paper focus on the feasibility of monitoring plant health through the analysis of VOCs in greenhouse air, but findings might also be relevant for atmospheric chemistry.     

Biosurfactant Facilitated Biodegradation of Quinalphos at High Concentrations by Pseudomonas aeruginosa Q10

Previous studies indicate that high concentration of pesticides and their associated toxic effects are high at their point source of application. Use of pesticide-degrading bacteria at point sources could augment degradation and thereby reduce toxic effects associated with pesticide persistence in soil. Quinalphos, an organophosphorus insecticide, though ranked “moderately hazardous” in the WHO's acute hazard ranking, still continues to be used extensively in developing countries. The presence of a chloride radical usually makes this pesticide sparingly soluble in water and hence difficult to degrade. The present study aimed to isolate autochthonous bacterial strains capable of utilizing quinalphos as a carbon source. Primary screening of pesticide-contaminated soil by enrichment culture and degradation analysis by UV-VIS spectrophotometry led to the isolation of 12 different bacterial isolates, of which three efficient isolates of Pseudomonas sp, Serratia sp, and Pseudomonas aeruginosa with degradation rate 86%, 82%, 94%, respectively, were selected. GC-MS studies with P.aeruginosa confirmed the formation of 2-hydroxy quinoxaline and phosphorothioic acid as a result of biodegradation. The present study succeeded in isolating autochthonous bacterial strains capable of utilizing high concentrations of quinalphos as a carbon source in a shorter incubation period. This strain also possessed biosurfactant-production ability, which makes quinalphos available to cells at higher concentrations.     

Correlation between biofilm production,antibiotic susceptibility and exopolysaccharide composition in Burkholderia pseudomallei bpsI,ppk, and rpoS mutant strains

Burkholderia pseudomallei is the cause of melioidosis, a fatal tropical infectious disease, which has been reported to have a high rate of recurrence, even when an intensive dose of antibiotics is used. Biofilm formation is believed to be one of the possible causes of relapse because of its ability to increase drug resistance. EPS in biofilms have been reported to be related to the limitation of antibiotic penetration in B. pseudomallei. However, the mechanisms by which biofilms restrict the diffusion of antibiotics remain unclear. The present study presents a correlation between exopolysaccharide production in biofilm matrix and antibiotic resistance in B. pseudomallei using bpsI, ppk, and rpoS mutant strains. CLSM revealed a reduction in exopolysaccharide production and disabled micro‐colony formation in B. pseudomallei mutants, which paralleled the antibiotic resistance. Different ratios of carbohydrate contents in the exopolysaccharides of the mutants were detected, although they have the same components, including glucose, galactose, mannose, and rhamnose, with the exception being that no detectable rhamnose peak was observed in the bpsI mutant. These results indicate that the correlation between these phenomena in the B. pseudomallei biofilm at least results from the exopolysaccharide, which may be under the regulation of bpsI, ppk, or rpoS genes.     

Chemical composition of scent marks in the ringtailed lemur (Lemur catta): glandular differences, seasonal variation, and individual signatures

The apocrine and sebaceous scent glands of ringtailed lemurs (Lemur catta) appear to serve different social functions. In behavioral experiments, lemurs modulate their responses to scent marks based on the type of odorant, their own physiological state, the signaler's physiological state, and prior social experience. To examine variation in odorant chemistry relative to olfactory behavior, we used gas chromatography and mass spectrometry to analyze over 86 samples of glandular secretion collected over 2 years from 15 adult lemurs. Labial and scrotal secretions contained organic acids and esters, whereas male brachial secretions were composed almost entirely of squalene and cholesterol derivatives. Principal component and linear discriminant analyses revealed glandular, individual-specific, and seasonal variation in chemical profiles but no relationship to the signaler's social status. The chemical composition of the various secretions provides further clues about the function of the different glands: the higher molecular weight compounds in genital and brachial secretions may increase signal longevity and provide lasting information to conspecifics, consistent with a role in advertising resource ownership or reproductive state. Conversely, the lower molecular weight compounds of antebrachial secretions produce ephemeral signals used primarily in social dominance displays and require integration of multiple sensory modalities for effective signal transmission.     

Microbial Eukaryotes that Lack Sterols

It is widely held that sterols are key cyclic triterpenoid lipids in eukaryotic cell membranes and are synthesized through oxygen‐dependent multienzyme pathways. However, there are known exceptions―ciliated protozoans, such as Tetrahymena, along with diverse low‐oxygen‐adapted eukaryotes produce, instead of sterols, the cyclic triterpenoid lipid tetrahymanol that does not require molecular oxygen for its biosynthesis. Here, we report that a number of anaerobic microbial eukaryotes (protists) utilize neither sterols nor tetrahymanol in their membranes. The lack of detectable sterol‐like compounds in their membranes may provide an opportunity to reconsider the physiological function of sterols and sterol‐like lipids in eukaryotes.     

细叶韭花化学成分的研究

细叶韭的花用乙醇浸提后制成膏,用乙醚萃取,蒸发除去大部分乙醚,残留物通过气相色谱－质谱仪分析,鉴定了46种化合物,占峰面积的83 .30%,其中含硫化合物4种;烃基芳香化合物11种,醛、酮类5种,长链烷烃6种,烯烃2种,醇、酚类10种,酯类5种,有机酸3种。细叶韭花叶的呈香物质主要为含硫化合物、醛、酮类、烃基芳香化合物、长链烯烃等,Vita-min E、β－,γ－生育酚、3β－羟基－5－烯－麦角甾烷、3β－羟基－5,16－二烯－妊酮是具有强生理活性物质。     

Estimation of N-amino-N,N-dimethylaminoethanol, choline and their acetate esters by gas chromatography mass spectrometry

A method is described for measurement of choline, N-aminodeanol, and their acetyl esters by gas chromatography mass spectrometry. The preparation of N-aminodeanol and its isotopic variants is also described. This method allows a thorough quantitative analysis of the replacement of true with false neurotransmitter in biological preparations.     

An optimized protocol for large‐scale in situ sampling and analysis of volatile organic compounds

Chemical ecology is an ever‐expanding field with a growing interest in population‐ and community‐level studies. Many such studies are hindered due to lack of an efficient and accelerated protocol for large‐scale sampling and analysis of chemical compounds. Here, we present an optimized protocol for such large‐scale study of volatiles. A large‐scale in situ study to understand role of semiochemicals in variation in mating success of lekking blackbuck was conducted. Suitable methods for sampling and statistical analysis were identified by testing and comparing the efficiencies of available techniques to reduce analysis time while retaining sensitivity and comprehensiveness. Solid‐phase extraction using polydimethylsiloxane, analysis using a semiautomated detection of retention time and base peak, and statistical analysis using random forest algorithm were identified as the most efficient methods for large‐scale in situ sampling and analysis of volatiles. The protocol for large‐scale volatile analysis can facilitate evolutionary and metaecological studies of volatiles in situ from all types of biological samples. The protocol has potential for wider application with the analysis and interpretation methods being suitable for all kinds of semiochemicals, including nonvolatile chemicals.     

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by (13)C-labeling experiments

Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.     

Characterisation of different types of hay by solid-phase micro-extraction-gas chromatography mass spectrometry and multivariate data analysis

The volatile organic compounds of different ground hay samples from Austria, Italy and Switzerland were collected at 50 degrees C on a Supelco Carbowax Divinylbenzene headspace solid-phase microextraction fibre, separated by capillary gas chromatography on an HP5-ms column running a temperature programme and using helium as carrier gas, detected with a mass sensitive detector and studied with principal component analyses after autoscaling selected variables. The analytes, mainly mono- and sesquiterpenes, were able to cluster differences resulting from the site of production. Coumarin can be used to differentiate hay grown north and south of the main chain of the Alps. Acetic acid is appropriate for distinguishing between hay from Kastelruth and Passeier Valley, two South Tyrolean regions. The average linalool content in aftermath is higher than in hay.     

Role of odour compounds in the attraction of gamete vectors in endophytic Epichloë fungi

Grass-infecting Epichlo? endophytes (Ascomycota, Calvicipitaceae) depend on Botanophila flies for gamete transfer, while fly larvae feed and develop on the fertilized fungal fruiting structures. Flies are known to be attracted by volatile signals, but the exact mechanisms of chemical communication and the degree of specialization are unknown. Headspace samples collected from five different Epichlo? species were analysed with respect to physiologically active substances using Botanophila flies. In field bioassays using synthetic compounds, their attractiveness and the specificity of the Epichlo?-Botanophila attraction were investigated. The identification of a new natural product, methyl (Z)-3-methyldodec-2-enoate, attracting Botanophila flies is reported here, and chokol K is confirmed as an attractive compound. Different blends of the two compounds attracted Botanophila flies under field conditions, but the three fly taxa present at the study site showed no preference for specific blends of volatiles. Chemical communication in the Epichlo?-Botanophila system relies on a few specific compounds, known as a communication system with 'private channels'. Although ratios of emitted compounds vary in different Epichlo? species, this seems not to lead to specialized attraction of Botanophila flies. Low selective pressure for specialization may have maintained a more generalist interaction between fungi and flies.     

Floral volatiles fromClarkia breweri andC. concinna (Onagraceae): Recent evolution of floral scent and moth pollination

Clarkia breweri (Onagraceae) is the only species known in its genus to produce strong floral fragrance and to be pollinated by moths. We used gas chromatography-mass spectrometry (GC-MS) to identify 12 abundant compounds in the floral headspace from two inbred lines ofC. breweri. These volatiles are derived from two biochemical pathways, one producing acyclic monoterpenes and their oxides, the other leading from phenylalanine to benzoate and its derivatives. Linalool and linalool oxide (pyran form) were the most abundant monoterpenoids, while linalool oxide (furan form) was present at lower concentrations. Of the aromatic compounds detected, benzyl acetate was most abundant, whereas benzyl benzoate, eugenol, methyl salicylate, and vanillin were present as minor constituents in all floral samples. The two inbredC. breweri lines differed for the presence of the additional benzenoid compounds isoeugenol, methyleugenol, methylisoeugenol, and veratraldehyde. We also analyzed floral headspace fromC. concinna, the likely progenitor ofC. breweri, whose flowers are odorless to the human nose. Ten volatiles (mostly terpenoids) were detected at low concentrations, but only when headspace was collected from 20 or more flowers at a time. Trans--ocimene was the most abundant floral compound identified from this species. Our data are consistent with the hypothesized recent evolution of floral scent production and moth pollination inC. breweri.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.