Supercritical CO2 Plant Extracts Show Antifungal Activities against Crop-Borne Fungi

Fungal infections of cultivated food crops result in extensive losses of crops at the global level, while resistance to antifungal agents continues to grow. Supercritical fluid extraction using CO₂ (SFE-CO₂) has gained attention as an environmentally well-accepted extraction method, as CO₂ is a non-toxic, inert and available solvent, and the extracts obtained are, chemically, of greater or different complexities compared to those of conventional extracts. The SFE-CO₂ extracts of Achillea millefolium, Calendula officinalis, Chamomilla recutita, Helichrysum arenarium, Humulus lupulus, Taraxacum officinale, Juniperus communis, Hypericum perforatum, Nepeta cataria, Crataegus sp. and Sambucus nigra were studied in terms of their compositions and antifungal activities against the wheat- and buckwheat-borne fungi Alternaria alternata, Epicoccum nigrum, Botrytis cinerea, Fusarium oxysporum and Fusarium poae. The C. recutita and H. arenarium extracts were the most efficacious, and these inhibited the growth of most of the fungi by 80% to 100%. Among the fungal species, B. cinerea was the most susceptible to the treatments with the SFE-CO₂ extracts, while Fusarium spp. were the least. This study shows that some of these SFE-CO₂ extracts have promising potential for use as antifungal agents for selected crop-borne fungi.     

Direct determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight mass spectrometry

O-Fucosylation is an unusual posttranslational modification present in several proteins that play important roles in physiological processes such as coagulation, cell signaling and metastasis. Although the exact function of the modification is still unclear, the number of proteins found to be modified is increasing, and there is a need for further structural and functional analyses. Here we report on a rapid and straightforward approach in the analysis of glycosylation status and determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight (nano-ESI Q-TOF) mass spectrometry. In a single measurement of previously chemically untreated O-fucosylated peptides originating from the thrombospondin-1 repeats, we were able to determine the glycosylation status of the analyzed peptide, the glycosylation site, and the glycan structure. The abundance of glycosylated peptide fragment ions in MS(2) spectra suggests that nano-ESI Q-TOF mass spectrometry can be used as a general approach in structural studies of O-fucosylation in proteins.     

Gangliosides from human granulocytes: a nano-ESI QTOF mass spectrometry fucosylation study of low abundance species in complex mixtures

Nano-ESI QTOF MS was used for sensitive mapping and sequencing of single molecular species in complex ganglioside mixtures obtained from human granulocytes, where the fucosylated carbohydrate chains of granulocyte gangliosides carry sLex and VIM-2 epitopes postulated to interact with E-selectin of the blood vessel wall in the early phase of the inflammation process. Functionally relevant components are expressed only at a low level, but using the negative ion detection it is possible to trace and identify such species, which were not detectable even by TLC. Advantage of the low-energy CID fragmentation for low abundance components of the complex ganglioside mixtures in the negative ion mode is to produce clear-cut series of fragment ions for sequencing. Fucosylation analysis carried out for each molecular species by MS/MS permits the clear distinction between sLex and VIM-2 epitope. VIM-2 epitope was expressed in all species with a longer sugar core, while in the short oligosaccharide chain with a sLex only, using biological material at a mid-femtomol level detection.     

Effects of counterion size on the attraction between similarly charged surfaces

Interaction between similarly charged surfaces can be attractive at high electrostatic coupling constants Ξ = l(B)Z(2)/μ(GC), where l(B) is the Bjerrum length, μ(GC) the Gouy-Chapman length, and Z the valency of counterions. While this effect has been studied previously in detail, as a function of surface charge density and valency of the pointlike counterions, much less is known about the effect of counterion size. We apply the Wang-Landau sampling Monte Carlo (MC) simulation method to compute the free energy F as a function of the scaled distance between the plates D?=D/μ(GC) for a range of Ξ and scaled counterion radii R?=R/μ(GC). We find that for large Ξ and small ion radius, there is a global equilibrium distance D?=D?(eq)=2(1+R?), correctly giving the expected value at the point counterion limit. With increasing R? the global minimum in F(D?) changes to a metastable state and finally this minimum vanishes when R? reaches a critical value, which depends on Ξ. We present a state diagram indicating approximate boundaries between these three regimes. The Wang-Landau MC method, as it is applied here, offers a possibility to study a wide spectrum of extended problems, which cannot be treated by the use of contact value theorem.     

Domoic acid--a new toxin in the Croatian Adriatic shellfish toxin profile

This is the first study that presents concentrations of domoic acid detected in the whole shellfish tissue from breeding and harvesting areas along the Croatian coast of the Adriatic Sea during the period 2006 to 2008. Shellfish sample analyses after SAX cleaning procedures, using a UV-DAD-HPLC system, showed the presence of domoic acid in four species. The most prevalent of those species were the blue mussel (Mytilus galloprovincialis), followed by European flat oyster (Ostrea edulis), Mediterranean scallop (Pecten jacobaeus) and proteus scallop (Flexopecten proteus). Domoic acid, a potentially lethal phycotoxin that causes amnesic shellfish poisoning (ASP), was detected for the first time in January 2006 with the highest value of 6.5486 μg g?1 in whole shellfish tissue. Pseudo-nitzschia spp. bloom events preceded these high domoic acid concentrations. According to this study, retention of domoic acid in the blue mussel M. galloprovincialis is more than 42 days. This investigation indicates the first presence of domoic acid in Croatian shellfish, but in concentrations under the regulatory limit (20 μg g?1), therefore shellfish consumption was not found to endanger human health.     

Bis­(ethyl­enedi­amine‐N,N′)(pyrimidine‐2‐carboxyl­ato‐N1,O)­cobalt(III) bis­(tri­fluoro­methane­sulfonate) dihydrate

In the title compound, the coordination geometry of the Co^III atom is only slightly distorted from regular octahedral, and the racemic nature of the material was confirmed by X‐ray structure analysis.     

Software utilities for the interpretation of mass spectrometric data of glycoconjugates: application to glycosphingolipids of human serum

Glycosphingolipids (GSLs) are major components of the outer leaflet of the cell membrane. These lipids are involved in many cell surface events and show disease‐related expression changes. GSLs could thus serve as useful targets for biomarker discovery. The GSL structure is characterized by two entities: a hydrophilic glycan and a hydrophobic ceramide moiety. Both components exhibit numerous structural variations, the combination of which results in a large diversity of GSL structures that can potentially exist. Mass spectrometry (MS) is a powerful tool for high‐throughput analysis of GSL expression analysis and structural elucidation. Yet, the assignment of GSL structures using MS data is tedious and demands highly specialized expertise. SysBioWare, a software platform developed for MS data evaluation in glycomics, was here applied for the MS analysis of human serum GSLs. The program was tuned to provide automated compositional assignment, supporting a variety of glycan and ceramide structures. Upon in silico fragmentation, the masses of predicted ions arising from cleavages in the glycan as well as the ceramide moiety were calculated, thus enabling structural characterization of both entities. Validation of proposed structures was achieved by matching in silico calculated fragment ions with those of experimental MS/MS data. These results indicate that SysBioWare can facilitate data interpretation and, furthermore, help the user to deal with large sets of data by supporting management of MS and non‐MS data. SysBioWare has the potential to be a powerful tool for high‐throughput glycosphingolipidomics in clinical applications. Copyright © 2010 John Wiley & Sons, Ltd.     

Combining size-exclusion chromatography and fully automated chip-based nanoelectrospray quadrupole time-of-flight tandem mass spectrometry for structural analysis of chondroitin/dermatan sulfate in human decorin

Chondroitin/dermatan sulfate (CS/DS) chain of decorin (DCN) from human skin fibroblasts (HSk) was released by reductive β-elimination reaction and digested with chondroitin AC I lyase. Enzymatic hydrolysis mixture of CS/DS chains was separated by size-exclusion chromatography (SEC). Collected octasaccharide fraction was subjected to fully automated chip-based nanoelectrospray (nanoESI) quadrupole time-of-flight (QTOF) MS and tandem MS (MS/MS). MS of human skin fibroblasts DCN CS/DS displayed a high complexity due to the large variety of glycoforms, which under chip-nanoESI MS readily ionized to form multiply charged ions. Except for the regularly tetrasulfated octasaccharide, the investigated fraction contained four additional octasaccharides of atypical sulfation status. Two new oversulfated glycoforms and two undersulfated species were identified. Remarkably, the series of decasaccharides discovered in the same SEC pool was found to encompass a trisulfated and a novel hexasulfated [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?] species. MS/MS by collision-induced dissociation (CID) on the [M-4H]? ion corresponding to the previously not reported [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?](5S) corroborated for a novel motif in which three N-acetylgalactosamine (GalNAc) moieties are monosulfated, 4,5-Δ-GlcA and the first IdoA from the non-reducing end bear one sulfate group each, while the second N-acetylgalactosamine from the reducing end is unsulfated.     

Lipophilic toxin profile in Mytilus galloprovincialis during episodes of diarrhetic shellfish poisoning (DSP) in the N.E. Adriatic Sea in 2006

Dinophysis spp. blooms and related shellfish toxicity events of diarrhetic shellfish poisoning (DSP) have been the most reported toxicity event through the Croatian National monitoring program. With the aim to characterize the DSP toxin profile in shellfish farmed in Croatia, for the first time a complete analysis of the toxin profile of Croatian mussels has been carried out using the LC-MS/MS technique. The obtained results showed okadaic acid (OA) as the main toxin contaminating Croatian mussels at that time. The maximum concentration of OA in shellfish tissue was recorded 12 days after the Dinophysis fortii bloom, thus suggesting that rapid growth of the toxin level in the shellfish occurred in the first week after the bloom while it was slower in the second week. Furthermore, the presence of only OA at concentrations which could endanger human health suggests D. fortii as the main organism responsible for the toxic event that occurred in Lim Bay. The presence of gymnodimine and spirolides in Croatian mussel has been detected for the first time, while the presence of yessotoxin and pectenotoxin-2 is confirmed.