Secondary metabolites produced by fungi derived from a microbial mat encountered in an iron-rich natural spring

A collection of fungal isolates was obtained from a complex microbial mat, which occupied an iron-rich freshwater spring that feeds into Clear Creek, Golden, Colorado, USA. Two of the fungal isolates, a Glomeromycete (possibly Entrophospora sp.) and a Dothideomycete (possibly Phaeosphaeria sp.), were investigated for bioactive secondary metabolites. In total, six new compounds consisting of clearanols A-E (5, 6, 10-12) and disulochrin (7) were purified and their structures were determined. Disulochrin exhibited modest antibacterial activity against methicillin-resistant Staphylococcus aureus, whereas clearanol C showed weak inhibitory activity against Candida albicans biofilm formation.     

Probing the Intracellular Glutathione Redox Potential by In‐Cell NMR Spectroscopy

Non‐invasive and real‐time analysis of cellular redox processes has been greatly hampered by lack of suitable measurement techniques. Here we describe an in‐cell nuclear magnetic resonance (NMR) based method for measuring the intracellular glutathione redox potential by direct and quantitative measurement of isotopically labeled glutathione introduced exogenously into living yeast. By using this approach, perturbations in the cellular glutathione redox homeostasis were also monitored as yeast cells were subjected to oxidative stress.     

Alteration of foliar flavonoid chemistry induced by enhanced UV-B radiation in field-grown Pinus ponderosa, Quercus rubra and Pseudotsuga menziesii

Chromatographic analyses of foliage from several tree species illustrate the species-specific effects of UV-B radiation on both quantity and composition of foliar flavonoids. Pinus ponderosa, Quercus rubra and Pseudotsuga menziesii were field-grown under modulated ambient (1x) and enhanced (2x) biologically effective UV-B radiation. Foliage was harvested seasonally over a 3-year period, extracted, purified and the flavonoid fraction applied to a mu Bondapak/C(18) column HPLC system sampling at 254 nm. Total flavonoid concentrations in Quercus rubra foliage were more than twice (leaf area basis) that of the other species; Pseudotsuga menziesii foliage had intermediate levels and P. ponderosa had the lowest concentrations of total flavonoids. No statistically significant UV-B radiation-induced effects were found in total foliar flavonoid concentrations for any species; however, concentrations of specific compounds within each species exhibited significant treatment effects. Higher (but statistically insignificant) levels of flavonoids were induced by UV-B irradiation in 1- and 2-year-old P. ponderosa foliage. Total flavonoid concentrations in 2-year-old needles increased by 50% (1x ambient UV-B radiation) or 70% (2x ambient UV-B radiation) from that of 1-year-old tissue. Foliar flavonoids of Q. rubra under enhanced UV-B radiation tended to shift from early-eluting compounds to less polar flavonoids eluting later. There were no clear patterns of UV-B radiation effects on 1-year-old P. menziesii foliage. However, 2-year-old tissue had slightly higher foliar flavonoids under the 2x UV-B radiation treatment compared to ambient levels. Results suggest that enhanced UV-B radiation will alter foliar flavonoid composition and concentrations in forest tree species, which could impact tissue protection, and ultimately, competition, herbivory or litter decomposition.     

Multi-scale magnetic resonance measurements and validation of Discrete Element Model simulations

This short review describes the capabilities of magnetic resonance (MR) to image opaque single- and two-phase granular systems, such as rotating cylinders and gas-fluidized beds operated in different fluidization regimes. The unique capability of MR to not only image the solids’ distribution (voidage) but also the velocity of the particulate phase is clearly shown. It is demonstrated that MR can provide measurements over different length and time scales. With the MR equipment used for the studies summarized here, temporal and spatial scales range from sub-millisecond to hours and from a few hundred micrometres to a few centimetres, respectively. Besides providing crucial data required for an improved understanding of the underlying physics of granular flows, multi-scale MR measurements were also used to validate numerical simulations of granular systems. It is shown that predictions of time-averaged properties, such as voidage and velocity of the particulate phase, made using the Discrete Element Model agree very well with MR measurements.     

Mass and charge state assignment for proteins and peptide mixtures via noncovalent adduction in electrospray mass spectrometry

A method has been developed that takes advantage of the formation of noncovalent compounds in electrospray mass spectrometry. Mixtures of proteins and peptides are shown to produce an intense ion that corresponds to a 1:1 complex with a crown ether (18-crown-6). Although the crown ether may be added directly to the solution, for the current experiments it is introduced via the methanol liquid sheath. The spacing of these complexed species in the mass spectrum allows unambiguous determination of the charge state of the ions and their actual mass. Through constant neutral loss scans, charge state may be determined, mass assigned, spectra simplified, and chemical noise may be reduced for the analysis of complex peptide samples without chromatographic separation. Finally, the prevalence of single complexation permits mass assignments based on the mass difference of a single protein ion and its complexed form at any charge state. In essence, the method performs a separation based on charge state. It can be used to complement chromatographic separation and deconvolution algorithms for the electrospray mass spectrometry analysis of peptide-protein mixtures.     

Laser desorption ion trap mass spectrometry of self-assembled monolayers

It is demonstrated that laser desorption ion trap mass spectrometry (LD-ITMS) can be successfully applied to the chemical analysis of a monolayer of adsorbates on a solid surface. Negative ion spectra obtained from LD-ITMS of self-assembled monolayers adsorbed from solutions of alkanethiols (CH₃(CH₂)_nSH with N = 5, 9, and 15) onto polycrystalline gold surfaces displayed clear ion peaks corresponding to the sulfonate adsorbate species. Sulfonate ions with the general formula CH₃(CH₂)_nSO⁻₃ were detected at m/z 165, 221, and 305, respectively, and were derived from the partial oxidation of the corresponding alkanethiol self-assembled monolayers. Little fragmentation and no clustering was observed in these mass spectra. These results indicate that the sensitivity of LD-ITMS is sufficient to allow its application to a wide array of problems in surface science.     

Ion formation from charged droplets: Roles of geometry,energy, and time

The formation of ions from the charged droplets produced in the several spray ionization techniques is viewed as an activated rate process involving field-assisted desorption, in accord with the ideas first set forth by Iribame and Thomson. The novel features of the present treatment are particularly relevant to the unique ability of electrospray ionization to transform large molecules in solution to free ions in the gas phase, with extensive multiple charging. These new features stem mainly from the realization that the spacing of charges on a desorbed ion must relate to the spacing of charges on the surface of the droplet whence it came. The consequences of this “rule” can account for the existence of maxima and minima in the number of charges on the ions of a particular species as well as the nature of the distribution of ions among the intervening charge states. They also explain the dependence of charge state on the configuration in solution of the parent molecule of the desorbed ion. In addition, they provide insight into the sequence in time at which ions in the various charge states leave an evaporating droplet.     

Metastable ion study of substituted cyclopentadienylmanganese cations in the gas phase

The behavior of some substituted cyclopentadienylmanganese ions has been studied by tandem mass spectrometry. This metastable ion study showed that only C₅H₅Mn⁺ and (C₅H₄CN)Mn⁺ ions retain their nido-cluster structure (1), which is characterized by a simple metal-ligand bond cleavage. Other substituted ions, RXC₅H₄Mn⁺, rearrange to a different extent, depending on the nature of the substituent. The first rearrangement step is R radical migration to the central metal atom, leading to RMnC₅H₄X⁺-type ions (2). These ions decompose by elimination of X (for X=CO) or with formation of RMnX⁺, but further rearrangements can also occur. These are the reverse migration of R from the metal atom to the π-ligand (for R=H, Ph) and cyclopentadienyl ring expansion (for X=CH₂). Collisional activation mass spectra contained an Mn⁺ ion peak, which can indicate the existence of stable type 1 structures for most cyclopentadienylmanganese ions. Carboxyl and hydroxymethyl derivatives exist, presumably as ions of type 2. The neutralization-reionization mass spectra of RXC₅H₄Mn⁺ ions are also discussed.     

Search of sequence databases with uninterpreted high-energy collision-induced dissociation spectra of peptides

We have broadened the utility of the SEQUEST computer algorithms to permit correlation of uninterpreted high-energy collision-induced dissociation spectra of peptides with all sequences in a database. SEQUEST now allows for the additional fragment ion types observed under high-energy conditions. We analyzed spectra from peptides isolated following trypsin digestion of 13 proteins. SEQUEST ranked the correct sequence first for 90% (18/20) of the spectra in searches of the OWL database, without constraint by enzyme cleavage specificity or species of origin. All false-positives were flagged by the scoring system. SEQUEST searches databases for sequences that correspond to the precursor ion mass ±0.5 u. Preliminary ranking of the top 500 candidates is done by calculation of fragment ion masses for each sequence, and comparison to the measured ion masses on the basis of ion series continuity, summed ion intensity, and immonium ion presence. Final ranking is done by construction of model spectra for the 500 candidates and constructing/performing of a cross-correlation analysis with the actual spectrum. Given the need to relate mounting genome sequence information with corresponding suites of proteins that comprise the cellular molecular machinery, tandem mass spectrometry appears destined to play the leading role in accelerating protein identification on the large scale required.