Biosynthesis of 13-desmethyl spirolide C by the dinoflagellate Alexandrium ostenfeldii

Biosynthetic origins of the cyclic imine toxin 13-desmethyl spirolide C were determined by supplementing cultures of the toxigenic dinoflagellate Alexandrium ostenfeldii with stable isotope-labeled precursors [1,2-13C2]acetate, [1-13C]acetate, [2-13CD3]acetate, and [1,2-13C2,15N]glycine and measuring the incorporation patterns by 13C NMR spectroscopy. Despite partial scrambling of the acetate labels, the results show that most carbons of the macrocycle are polyketide-derived and that glycine is incorporated as an intact unit into the cyclic imine moiety. This work represents the first conclusive evidence that such cyclic imine toxins are polyketides and provides support for biosynthetic pathways previously defined for other polyether dinoflagellate toxins.     

Confinement and controlled release of bisphosphonates on ordered mesoporous silica-based materials

Bisphosphonates have been confined in siliceous ordered mesoporous materials, and the drug release rate of these systems has been investigated. The bisphosphonate adsorption rate has been increased from 1% in oral administration to around 40% locally delivered. Drug dosage can be modulated through amine modification of the material surface, leading to a bisphosphonate adsorption in the ordered mesoporous matrices 3 times larger than that for unmodified materials. The use of these drug delivery systems opens new fields with new possibilities for tissue engineering.     

Breaking the N2 triple bond: insights into the nitrogenase mechanism

Nitrogenase is the metalloenzyme that performs biological nitrogen fixation by catalyzing the reduction of N2 to ammonia. Understanding how the nitrogenase active site metal cofactor (FeMo-cofactor) catalyzes the cleavage of the N2 triple bond has been the focus of intense study for more than 50 years. Goals have included the determination of where and how substrates interact with the FeMo-cofactor, and the nature of reaction intermediates along the reduction pathway. Progress has included the trapping of intermediates formed during turnover of non-physiological substrates (e.g., alkynes, CS2) providing insights into how these molecules interact with the nitrogenase FeMo-cofactor active site. More recently, substrate-derived species have been trapped at high concentrations during the reduction of N2, a diazene, and hydrazine, providing the first insights into binding modes and possible mechanisms for N2 reduction. A comparison of the current state of knowledge of the trapped species arising from non-physiological substrates and nitrogenous substrates is beginning to reveal some of the intricacies of how nitrogenase breaks the N2 triple bond.     

Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

Analysis of acrylamide in coffee and cocoa by isotope dilution liquid chromatography–tandem mass spectrometry

An accurate and precise method for the quantification of acrylamide using stable isotope dilution liquid chromatography–tandem mass spectrometry was developed and used to measure acrylamide in coffee and cocoa samples. The sample preparation involved extraction of the analyte and its internal standard, ¹³C₃-acrylamide, into water and subsequent defatting of the aqueous extract with dichloromethane. An aliquot of the resulting aqueous extract was then azeotropically dried under reduced pressure and subsequently purified using an aminopropyl-bonded silica cartridge. The purified extracts were then chromatographed on a 5-μm 2.1×150 mm Hypercarb column, the effluent of which was monitored for the analyte and its internal standard using positive-ion APCI-selected reaction monitoring. The intra-laboratory reproducibility of the method, expressed as a relative coefficient of variation (%, n=5), was determined at four levels of concentration (12.3, 42.3, 139.3 and 464.8 μg kg⁻¹) and was found to vary between 0.6–2.5%. The accuracy of the method was assessed using a reference sample of coffee. The average result obtained using our method differed from the assigned value of the reference material by less than 1%. An analysis of a cocoa sample revealed that the method is capable of precisely estimating acrylamide in challenging matrices down to a level of at least 12.3 μg kg⁻¹.     

Occurrence of photoprotective compounds in yeasts from freshwater ecosystems of northwestern Patagonia (Argentina)

In this paper we present the results of research on the occurrence, induction and role of photoprotective compounds (PPCs) present in native aquatic yeasts from freshwater Patagonian ecosystems. We focus on the effect of UV radiation (UVR) as a factor that controls the level of photoprotection of yeasts, and explore its potential significance in shaping yeast distributional patterns. The research presented here combines field surveys and laboratory work, including the isolation and culture of native yeasts strains, and laboratory assays under different radiation conditions. The results obtained suggest that yeasts are common dwellers of oligotrophic Patagonian water bodies, and provide the first evidence of the distribution of PPC (carotenoid and mycosporine)-producing yeasts in temperate freshwaters. A greater proportion of carotenogenic yeasts were observed in high-elevation lakes. The yeast strains isolated from these environments were found to produce higher amounts of mycosporines (MYCs), and to present higher tolerance to UVB exposure than those from piedmont lakes. Patagonian yeasts have only one type of MYC, mycosporine-glutaminol-glucoside (myc-glu-glu), which seems common to all other yeasts. By analyzing the production of myc-glu-glu in a large number of yeasts belonging to different taxonomic groups, we propose that this compound may have potential use as a chemotaxonomic marker in yeast systematics. Collectively, our work reveals that in Patagonian freshwater yeasts there is an apparent relationship between the ability to produce PPCs, their tolerance to UV exposure and their success in colonizing habitats highly exposed to UVR.     

New strategies of photoprotection

Adequate photoprotection is essential to control UV-related disorders, including sunburn, photoaging and photocarcinogenisis. Sun avoidance, protection of skin with clothing, and sunscreens are presently the best way of photoprotection, assuming that they are used properly. However, new strategies, which are based on or make use of the endogenous protective response to UV light, may further improve currently used photoprotective means. The addition of repair enzymes and/or antioxidants has a positive effect on skin's recovery from UV-induced DNA-damage. Several botanical agents, mainly vitamins and polyphenols, have shown to influence signal transduction pathways leading to photoprotective effects. Also stimulation of endogenous UV-response pathways via irradiation with a low UV dose or via simulation of UV-induced DNA-damage results in photoprotective effects. Future research in this field and combination of different photoprotective strategies will hopefully lead to improved photoprotection.     

Determination of polycyclic aromatic hydrocarbons in olive oil by a completely automated headspace technique coupled to gas chromatography-mass spectrometry

A new and completely automated method for the determination of ten relevant polycyclic aromatic hydrocarbons (PAHs) in olive oil is proposed using an extraction by the headspace (HS) technique. Quantification and confirmation steps are carried out by gas chromatography-mass spectrometry (GC-MS) combining simultaneously selected-ion monitoring (SIM) and tandem mass spectrometry (MS/MS). This combination offers on one hand an increased sensitivity and on the other hand, selective and reliable qualitative information. Sample pretreatment or clean-up are not necessary because the olive oil sample is put directly into an HS vial, automatically processed by HS and introduced into the GC-MS instrument for analysis. Because of its high selectivity and sensitivity, a triple-quadrupole (QqQ) detector coupled with the gas chromatograph allows us to limit handling. Each sample is completely processed in approximately 63 min (45 min for HS isolation and 18 min for GC-MS determination), a reduced time compared with previously published methods. The chemical and instrumental variables were preliminarily optimized using uncontaminated olive oil samples spiked with 25 microg kg(-1) of each target compound. The final method was validated to ensure the quality of the results. The precision was satisfactory, with relative standard deviation (RSD) values in the range 3-9%. Recovery rates ranged from 96 to 99%. Limits of detection (LOD) were calculated as 0.02-0.06 microg kg(-1) and the limits of quantification (LOQ) were obtained as 0.07-0.26 microg kg(-1). It must be mentioned that the LOD and LOQ are much lower than the maximum levels established by the European Union (EU) in oils and fats intended for direct human consumption or for use as an ingredient in foods, which are set at 2 microg kg(-1). All the figures of merit are completely in accordance with the latest EU legislation. This fact makes it possible to consider the proposed method as a useful tool for the control of PAHs in olive oils.     

Chemisorption of isocyanate (NCO) on the Pd(1 0 0) surface at different coverages

The chemisorption of isocyanate (NCO) species on Pd(1 0 0) was studied within the density functional formalism (DFT) using a periodic slab model. The NCO was adsorbed on top, bridge and hollow sites of the metal surface at different coverages. At low coverages, the adsorption energies are in the range of ?2.5/?3.0 eV, indicating an important adsorbate–substrate interaction for the three sites studied. The lateral repulsive interaction between neighboring NCO species is almost negligible or weak at lower and intermediate coverages, and very strong at complete monolayer. At low coverages, both bridge and hollow sites are energetically preferred; yet the bridge site becomes the only favoured site at intermediate and complete coverages. Work function and dipole moment calculations can be interpreted by an important charge transfer from the metal surface to NCO. Interestingly, while on hollow site the charge taken by NCO is essentially the same over all the range of coverage, an increasing depolarization is observed on bridge and top sites as the coverage increases. Symmetric and asymmetric NCO stretching modes were also calculated and compared with recent infrared spectroscopy measurements.     

Constant-sign solutions of systems of higher order boundary value problems with integrable singularities

We consider some systems of boundary value problems where the nonlinearities may be singular in the independent variable and may also be singular in the dependent arguments. Using the Schauder fixed point theorem, we establish criteria such that the systems of boundary value problems have at least one constant-sign solution.