Mass spectrometric methods for the analysis of chlorinated and nitrated isoflavonoids: a novel class of biological metabolites

Electrospray ionization combined with tandem mass spectrometry was applied to a study of some representative chlorinated and nitrated isoflavones-potential metabolites of isoflavones in inflammatory cells. Upon collision-induced dissociation of deprotonated [M - H](-) ions of these compounds, a number of structurally characteristic product ions were produced. The product ion analysis of 3'- and 8-chlorodaidzein in the tandom mass spectra led to ready differentiation of these isomers. 3-Nitro derivatives of both genistein and daidzein have product ions due to the losses of HNO(2) and two OH groups. Chlorinated derivatives of isoflavones were detected in cell-based experiments and their structures were proposed by comparing the tandem mass spectra of their product ions with those of standards. This work provides a suitable analytical basis to aid the characterization of chlorinated and nitrated metabolites in studies in vivo and in vitro.     

Electrochemical templating of metal nanoparticles and nanowires on single-walled carbon nanotube networks

The use of single-walled carbon nanotube (SWNT) networks as templates for the electrodeposition of metal (Ag and Pt) nanostructures is described. Pristine SWNTs, grown on insulating SiO2 surfaces using catalyzed chemical vapor deposition, served as the working electrode. In the simplest case, electrical contact was made by depositing a gold strip on the SWNT substrate (device 1). Deposition of Ag and Pt over extensive periods (30 s) resulted in a high density of particles on the SWNTs, with almost contiguous nanowire formation from the Au/SWNT boundary moving to isolated nanoparticles at further distances from the contact. For direct electrochemical studies of Ag and Pt nucleation, the assembly was coated in a resist layer and a small window opened up to expose only the electrically connected SWNTs to solution (device 2). In this case, the electrochemical signature in voltammetric and amperometric studies of metal deposition was due solely to processes at the SWNTs. Coupled with high-resolution microscopy measurements (atomic force microscopy and field emission scanning electron microscopy), this approach provided detail on the nucleation and growth mechanisms of Ag and Pt on SWNTs under electrochemical control. In particular, Ag growth was found to be rapid and progressive with an increasing nanoparticle density with time, whereas Pt deposition was characterized by lower nucleation densities and slower growth rates with a tendency for larger particles to be produced over long times.     

CCR5 antagonists: 3-(pyrrolidin-1-yl)propionic acid analogues with potent anti-HIV activity

[reaction: see text] A novel approach to alpha,alpha-disubstituted-beta-amino acids (beta(2,2)-amino acids) was employed in the synthesis of a series of 3-(pyrrolidin-1-yl)propionic acids possessing high affinity for the CCR5 receptor and potent anti-HIV activity. The rat pharmacokinetics for these new analogues featured higher bioavailabilities and lower rates of clearance as compared to cyclopentane 1.     

Scanning electrochemical microscopy as a probe of Ag+ binding kinetics at Langmuir phospholipid monolayers

A new method has been developed for measuring local adsorption rates of metal ions at interfaces based on scanning electrochemical microscopy (SECM). The technique is illustrated with the example of Ag+ binding at Langmuir phospholipid monolayers formed at the water/air interface. Specifically, an inverted 25 microm diameter silver disc ultramicroelectrode (UME) was positioned in the subphase of a Langmuir trough, close to a dipalmitoyl phosphatidic acid (DPPA) monolayer, and used to generate Ag+ via Ag electro-oxidation. The method involved measuring the transient current-time response at the UME when the electrode was switched to a potential to electrogenerate Ag+. Since the Ag+/Ag couple is reversible, the response is highly sensitive to local mass transfer of Ag+ away from the electrode, which, in turn, is governed by the interaction of Ag+ with the monolayer. The methodology has been used to determine the influence of surface pressure on the adsorption of Ag+ ions at a phospholipid (dipalmitoyl phosphatidic acid) Langmuir monolayer. It is shown that the capacity for metal ion adsorption at the monolayer increased as the density of surface adsorption sites increased (by increasing the surface pressure). A model for mass transport and adsorption in this geometry has been developed to explain and characterise the adsorption process.     

Nature of the oxomolybdenum-thiolate pi-bond: implications for Mo-S bonding in sulfite oxidase and xanthine oxidase

The electronic structure of cis,trans-(L-N(2)S(2))MoO(X) (where L-N(2)S(2) = N,N'-dimethyl-N,N'-bis(2-mercaptophenyl)ethylenediamine and X = Cl, SCH(2)C(6)H(5), SC(6)H(4)-OCH(3), or SC(6)H(4)CF(3)) has been probed by electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies to determine the nature of oxomolybdenum-thiolate bonding in complexes possessing three equatorial sulfur ligands. One of the phenyl mercaptide sulfur donors of the tetradentate L-N(2)S(2) chelating ligand, denoted S(180), coordinates to molybdenum in the equatorial plane such that the OMo-S(180)-C(phenyl) dihedral angle is approximately 180 degrees, resulting in a highly covalent pi-bonding interaction between an S(180) p orbital and the molybdenum d(xy) orbital. This highly covalent bonding scheme is the origin of an intense low-energy S --> Mo d(xy) bonding-to-antibonding LMCT transition (E(max) approximately 16000 cm(-)(1), epsilon approximately 4000 M(-)(1) cm(-)(1)). Spectroscopically calibrated bonding calculations performed at the DFT level of theory reveal that S(180) contributes approximately 22% to the HOMO, which is predominantly a pi antibonding molecular orbital between Mo d(xy) and the S(180) p orbital oriented in the same plane. The second sulfur donor of the L-N(2)S(2) ligand is essentially nonbonding with Mo d(xy) due to an OMo-S-C(phenyl) dihedral angle of approximately 90 degrees. Because the formal Mo d(xy) orbital is the electroactive or redox orbital, these Mo d(xy)-S 3p interactions are important with respect to defining key covalency contributions to the reduction potential in monooxomolybdenum thiolates, including the one- and two-electron reduced forms of sulfite oxidase. Interestingly, the highly covalent Mo-S(180) pi bonding interaction observed in these complexes is analogous to the well-known Cu-S(Cys) pi bond in type 1 blue copper proteins, which display electronic absorption and resonance Raman spectra that are remarkably similar to these monooxomolybdenum thiolate complexes. Finally, the presence of a covalent Mo-S pi interaction oriented orthogonal to the MOO bond is discussed with respect to electron-transfer regeneration in sulfite oxidase and Mo=S(sulfido) bonding in xanthine oxidase.     

Resistant starch as related to companion animal nutrition

Companion animal diets may contain up to 50% starch, derived from cereal grains. The amount of resistant starch (RS) in an ingredient depends on the origin and form of the ingredient and on the processing conditions to which the ingredient has been exposed. Extrusion has proven to be a means of optimizing utilization of starch by companion animals. Although the RS fraction of starch typically decreases by extrusion, retrogradation can result in increased concentrations of this fraction. Limited research exists regarding the effects of RS in companion animal nutrition and gastrointestinal health. Existing in vitro and in vivo research indicates that certain RS sources are readily fermented in the large bowel, producing short-chain fatty acids, whereas others are less fermentable, resulting in excellent laxation properties. Feeding dogs a diet high in RS may result in an increase in fecal bulk due to an increased excretion of microbial matter in those cases where RS is highly fermentable, or to indigestibility of the RS source in other cases. RS has a role to play as a potential proxy for dietary fiber, especially for those companion animals fed diets high in protein and fat and devoid of traditional dietary fiber.     

Synthetic models for the cysteinate-ligated non-heme iron enzyme superoxide reductase: observation and structural characterization by XAS of an Fe(III)-OOH intermediate

Superoxide reductases (SORs) belong to a new class of metalloenzymes that degrade superoxide by reducing it to hydrogen peroxide. These enzymes contain a catalytic iron site that cycles between the Fe(II) and Fe(III) states during catalysis. A key step in the reduction of superoxide has been suggested to involve HO(2) binding to Fe(II), followed by innersphere electron transfer to afford an Fe(III)-OO(H) intermediate. In this paper, the mechanism of the superoxide-induced oxidation of a synthetic ferrous SOR model ([Fe(II)(S(Me2)N(4)(tren))](+) (1)) to afford [Fe(III)(S(Me2)N(4)(tren)(solv))](2+) (2-solv) is reported. The XANES spectrum shows that 1 remains five-coordinate in methanolic solution. Upon reaction of 1 with KO(2) in MeOH at -90 degrees C, an intermediate (3) is formed, which is characterized by a LMCT band centered at 452(2780) nm, and a low-spin state (S = 1/2), based on its axial EPR spectrum (g(perpendicular) = 2.14; g(parallel) = 1.97). Hydrogen peroxide is detected in this reaction, using both (1)H NMR spectroscopy and a catalase assay. Intermediate 3 is photolabile, so, in lieu of a Raman spectrum, IR was used to obtain vibrational data for 3. At low temperatures, a nu(O-O) Fermi doublet is observed in the IR at 788(2) and 781(2) cm(-)(1), which collapses into a single peak at 784 cm(-1) upon the addition of D(2)O. This vibrational peak diminishes in intensity over time and essentially disappears after 140 s. When 3 is generated using an (18)O-labeled isotopic mixture of K(18)O(2)/K(16)O(2) (23.28%), the vibration centered at 784 cm(-1) shifts to 753 cm(-1). This new vibrational peak is close to that predicted (740 cm(-1)) for a diatomic (18)O-(18)O stretch. In addition, a nu(O-O) vibrational peak assigned to free hydrogen peroxide is also observed (nu(O-O) = 854 cm(-1)) throughout the course of the reaction between Fe(II)-1 and superoxide and is strongest after 100 s. XAS studies indicate that 3 possesses one sulfur scatterer at 2.33(2) A and four nitrogen scatterers at 2.01(1) A. Addition of two Fe-O shells, each containing one oxygen, one at 1.86(3) A and one at 2.78(3) A, improved the EXAFS fits, suggesting that 3 is an end-on peroxo or hydroperoxo complex, [Fe(III)(S(Me2)N(4)(tren))(OO(H))](+). Upon warming above -50 degrees C, 3 is converted to 2-MeOH. In methanol and methanol:THF (THF = tetrahydrofuran) solvent mixtures, 2-MeOH is characterized by a LMCT band at lambda(max) = 511(1765) nm, an intermediate spin-state (S = 3/2), and, on the basis of EXAFS, a relatively short Fe-O bond (assigned to a coordinated methanol or methoxide) at 1.94(10) A. Kinetic measurements in 9:1 THF:MeOH at 25 degrees C indicate that 3 is formed near the diffusion limit upon addition of HO(2) to 1 and converts to 2-MeOH at a rate of 65(1) s(-1), which is consistent with kinetic studies involving superoxide oxidation of the SOR iron site.     

Boiling Temperature and Reversed Deliquescence Relative Humidity Measurements for Mineral Assemblages in the NaCl + NaNO3 + KNO3 + Ca(NO3)2 + H2O System

Boiling temperature measurements have been made at ambient pressure for saturated ternary solutions of NaCl + KNO₃ + H₂O, NaNO₃ + KNO₃ + H₂O, and NaCl + Ca(NO₃)₂ + H₂O over the full composition range, along with those of the single salt systems. Boiling temperatures were also measured for the four component NaCl + NaNO₃ + KNO₃ + H₂O and five component NaCl + NaNO₃ + KNO₃ + Ca(NO₃)₂ + H₂O mixtures, where the solute mole fraction of Ca(NO₃)₂, x{Ca(NO₃)₂}, was varied between 0 and 0.25. The maximum boiling temperature found for the NaCl + KNO₃ + H₂O system is ≈134.9 ^∘C; for the NaNO₃ + KNO₃ + H₂O system is ≈165.1 ^∘C at x(NaNO₃) ≈ 0.46 and x(KNO₃) ≈ 0.54; and for the NaCl + Ca(NO₃)₂ + H₂O system is 164.7 ± 0.6 ^∘C at x{NaCl} ≈ 0.25 and x{Ca(NO₃)₂} ≈ 0.75. The NaCl + NaNO₃ + KNO₃ + Ca(NO₃)₂ + H₂O system forms molten salts below their maximum boiling temperatures and the temperatures corresponding to the cessation of boiling (dry-out temperatures) of these liquid mixtures were determined. These dry-out temperatures range from ≈300 ^∘C when x{Ca(NO₃)₂} = 0 to ≥ 400 ^∘C when x{Ca(NO₃)₂} = 0.20 and 0.25. Mutual deliquescence/efflorescence relative humidity (MDRH/MERH) measurements were also made for the NaNO₃ + KNO₃ and NaCl + NaNO₃ + KNO₃ salt mixture from 120 to 180 ^∘C at ambient pressure. The NaNO₃ + KNO₃ salt mixture has a MDRH of 26.4% at 120 ^∘C and 20.0% at 150 ^∘C. This salt mixture also absorbs water at 180 ^∘C, which is higher than expected from the boiling temperature experiments. The NaCl + NaNO₃ + KNO₃ salt mixture was found to have a MDRH of 25.9% at 120 ^∘C and 10.5% at 180 ^∘C. The investigated mixture compositions correspond to some of the major mineral assemblages that are predicted to control brine composition due to the deliquescence of salts formed in dust deposited on waste canisters in the proposed nuclear repository at Yucca Mountain, Nevada.     

Further study of theE/f 1 (1420) meson in central production

We have studied the reactions \(({{\pi ^ + } \mathord{\left/ {\vphantom {{\pi ^ + } p}} \right. \kern-0em} p})p \to ({{\pi ^ + } \mathord{\left/ {\vphantom {{\pi ^ + } p}} \right. \kern-0em} p})(K\bar K\pi )p\) where the \(K\bar K\pi \) system is centrally produced, at 85 GeV/c and 300 GeV/c using the CERN Omega spectrometer. A spin-parity analysis of theK _S ⁰ K ^± π ^? system shows the presence of a strongJ ^PC=1⁺⁺ signal which we identify as theE/f ₁ (1420) meson. We also find evidence for the decayE/f ₁(1420)→K _S ⁰ K _S ⁰ π ⁰ which determines theC-parity of this state to be positive. Alternative explanations of the data have been tested and ruled out. Hence we obtain the quantum numbers of theE/f ₁ (1420) to beI ^G(J^PC)=0⁺(1⁺).     

LC-mass spectrometry analysis of N- and C-terminal boundary sequences of polypeptide fragments by limited proteolysis

Limited proteolysis is an important and widely used method for analyzing the tertiary structure and determining the domain boundaries of proteins. Here we describe a novel method for determining the N- and C-terminal boundary amino acid sequences of products derived from limited proteolysis using semi-specific and/or non-specific enzymes, with mass spectrometry as the only analytical tool. The core of this method is founded on the recognition that cleavage of proteins with non-specific proteases is not random, but patterned. Based on this recognition, we have the ability to determine the sequence of each proteolytic fragment by extracting a common association between data sets containing multiple potential sequences derived from two or more different mass spectral molecular weight measurements. Proteolytic product sequences derived from specific and non-specific enzymes can be accurately determined without resorting to the conventional time-consuming and laborious methods of SDS-PAGE and N-terminal sequencing analysis. Because of the sensitivity of mass spectrometry, multiple transient proteolysis intermediates can also be identified and analyzed by this method, which allows the ability to monitor the progression of proteolysis and thereby gain insight into protein structures.