The production of peptaibols by a marine-related Trichoderma longibrachiatum strain was studied using electrospray ionisation multiple-stage ion trap mass spectrometry (ESI-MSn-IT) and gas chromatography/electron impact mass spectrometry (GC/EI-MS). Two major groups of peptaibols were identified, those with long sequences (20 amino acids) and others with short sequences (11 amino acids). This paper describes the methodology used to establish the sequences of short peptaibols in a mixture without previous individual separation. Nine peptaibols were identified. Among them, eight are new, namely as trichobrachin A I-IV (Aib9-Pro10 sequence) and as trichobrachin B I-IV (Val9-Pro10 sequence). Original Pro6-Val7 and Val9-Pro10 sequences have to be noted.

Peptaibols are known membrane-modifying peptides that were recently detected in marine sediments and mussels collected from a shellfish farming area (Fier d'Ars, Atlantic coast, France). In this investigation, embryotoxicity bioassays with oysters (Crassostrea gigas) were performed to assess acute toxicity of alamethicin and different groups of peptaibols produced by a Trichoderma longibrachiatum strain isolated from marine environment. C. gigas embryos appeared very sensitive to all the metabolites examined with higher toxic effects for long-sequence peptides (EC50 ranging from 10 to 64 nM). D-shaped larvae with mantle abnormality were particularly noticed when peptaibol concentrations increased. Disturbances of embryogenesis were also observed following exposure to organic and aqueous extract of sediments from Fier d'Ars (EC50=42.4 and 6.6 g L(-1) dry weight, respectively). Although peptaibol concentrations measured in these sediments could explain only a part of the toxic effects observed, this study suggests that these mycotoxins can induce larval abnormalities in a population of exposed animals at environmentally realistic concentrations. Their detection in coastal areas devoted to bivalve culture should be taken into account.

Studying electronic absorption and fluorescence spectra of 1,6-diphenyl-1,3,5-hexatriene (DPH) in twenty-two solvents, several factors were found to affect the spectral parameters. Two groups of DPH spectral behaviour were defined, related to the refractive index and the dispersion interactions parameters of the solvents. DPH molar absorptivity is hardly affected by the nature of the solvent or the increase of temperature (20-50°C). By contrast, DPH fluorescence intensity increases along with the viscosity and polarizability of solvents and is decreased by elevating the temperature. Investigations on the combined effects of solvent nature and temperature on DPH spectral parameters allowed the solvents to be classed into four groups (referred to as 1 to 4). When the temperature was increased from 20 to 50°C, DPH fluorescence intensity was only slightly modified in solvents from group 1 (as tetrahydrofuran, I = 100 at 20°C, I = 80 ± 5 at 50°C), but was severely decreased in solvents from groups 2 (as ethanol, I = 55 at 50°C) and 3 (as acetic acid, I = 45 at 50°C), and still more in solvents from group 4 (tetrachloromethane, I = 10 at 50°C). Data on the reversibility of the phenomenon in these solvents after returning the temperature from 50 to 20°C show a concomitant decrease in DPH stability in these solvents. When a water-miscible solvent was used, the decrease in fluorescence intensity was found to be higher after adding water to this solvent. These data may help to assess the spectral behaviour of DPH in the more complex biological media.Fluorescent probes are extensively used in physical, chemical and biological sciences for investigating the structure and dynamics of living systems. 1 One of the most commonly used probes for the assessment of membrane microviscosity by fluorescence polarization is the polyene 1,6-diphenyl-1,3,5-hexatriene (DPH). Due to its long rod-like structure and its non-polar properties, DPH aligns parallel to the phospholipid fatty acyl chains of biological membranes. These properties explain its wide use for measuring the fluidity of membrane bilayers. 2 Yet, difficulties have been reported in using this probe, due to its poorly known location in the membranes, 3,4 its partition in cellular compartments, 5-7 and its fluorescence quenching caused by DPH-water interactions. 8 Unusual fluorescence properties of DPH have been listed 8 but only a few solvents, namely methylcyclohexane, heptane, benzene, ethyl iodide, perfluorohexane, acetonitrile, 1,4-dioxane and ethanol, have been used for studying the physicochemical properties of the probe. 9-11 Although excited states of DPH have been well studied because of its structural analogy with retinol, 12-15 the influence of the surrounding medium on the spectral properties of DPH is not yet clear. In the present study, the interactions between DPH and various solvents were examined; it is shown that the stability of the probe largely depends on the temperature and the nature or hydration of solvents. ExperimentalTwenty-two solvents of spectrop...

No abstract

Extraction followed by reverse phase liquid chromatography (LC)/electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS) analysis has been successfully developed for the determination of peptaibols, fungal toxic metabolites, in marine sediments. Spiking experiments showed that the mean recovery of target compounds exceeded 85% at a spiking level of 10 ng/g of sediment (wet weight). Detection and quantification limits were 250 and 830 pg/g of sediment, respectively. The method developed constituted the first sensitive assay for quantification of peptaibol trace amounts in a natural environment. A concentration of 5 ng/g in sediment samples collected from Fier d'Ars was found.