Covalent binding of human serum albumin and ovalbumin by chloramine-T and chemical modification of the proteins

The covalent binding of ³⁵S-chloramine-T to human resum albumin (HSA) and ovalbumin is described. At pH 6.5, up to 24 chloramine-T molecules were found to be covalently bound per molecule of HSA; with ovalbumin the binding was only 5–7 molecule per protein molecule. Binding was accompanied by extensive modification of methionine, cysteine, histidine, tyrosine and lysine. Three new peaks appeared in the amino acid profiles of the modified proteins; two were identified as 1-aminoadipic acid (oxidation of lysine) and 3-chlorotyrosine. The most sites for covalent binding are lysine residues.     

Capillary liquid chromatography with MS3 for the determination of enkephalins in microdialysis samples from the striatum of anesthetized and freely-moving rats

In vivo microdialysis sampling was coupled to capillary liquid chromatography (LC)/electrospray ionization quadrupole ion trap mass spectrometry (MS) to monitor [Met]enkephalin and [Leu]enkephalin in the striatum of anesthetized and freely-moving rats. The LC system utilized a high-pressure pump to load 2.5 microl samples and desalt the 25 microm i.d. by 2 cm long column in 12 min. Samples were eluted with a separate pump at approximately 100 nl min(-1). A rapid gradient effectively separated the endogenous neuropeptides in 4 min. A comparison was made for operating the mass spectrometer in the MS2 and MS3 modes for detection of the peptides. In standard solutions, the detection limits were similar at 1-2 pM (2-4 amol injected); however, the reproducibility was improved with MS3 as the relative standard deviation was <5% compared with 20% for MS2 for 60 pM samples. For dialysate solutions, reconstructed ion chromatograms and tandem mass spectra had much higher signal-to-noise ratios in the MS3 mode, resulting in more confident detection at in vivo concentrations. The method was successfully used to monitor the peptides under basal conditions and with stimulation of peptide secretion by infusion of elevated K+ concentration.     

Carboxylation and Mitsunobu reaction of amines to give carbamates: retention vs inversion of configuration is substituent-dependent

A mild method for the synthesis of carbamates from amino alcohols involves sequential carboxylation with carbon dioxide, followed by a Mitsunobu reaction. Unexpectedly, the stereochemical course of the Mitsunobu reaction is dependent on whether the carbamic acid intermediate is N-substituted with hydrogen (retention) or carbon (inversion).     

High-throughput ribozyme-based assays for detection of viral nucleic acids

Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.     

Liquid crystal effects on bacterial viability

The primary objective of this research was to test the hypothesis that lyotropic chromonic liquid crystals (neutral grey, red 14, blue 27, cromolyn) are not toxic to bacteria as compared with surfactant-based lyotropic (CPCl and CsPFO) or thermotropic (5CB and E7) liquid crystals. Biocompatibility of most liquid crystals is currently unknown and is required for the development of systems interfacing liquid crystals and biological systems. Potential liquid crystal toxicity was evaluated by two methods. The first examined bacterial survival measured by bacterial growth over 24 hours, after exposure to various liquid crystals. The second toxicity method evaluated liquid crystal effects on bacterial membrane permeability using two fluorescent dyes. Three different types of bacteria were evaluated to assess bacterial structure differences with respect to liquid crystal toxicity. The results of this study indicate that lyotropic chromonic liquid crystals are not toxic to bacteria, whereas thermotropic and surfactant-based lyotropic liquid crystals are toxic to one or more forms of bacteria. We conclude that lyotropic chromonic liquid crystals may be the preferred material in designing liquid crystal-based systems that interact with biological systems, especially in the use of liquid crystal-based biosensors.     

Quantification of monohydroxy-PAH metabolites in urine by solid-phase extraction with isotope dilution-GC–MS

For measurement of biomarkers from polycyclic aromatic hydrocarbon (PAH) exposure, an analytical method is described quantifying hydroxylated PAH (OH-PAH) in urine samples. This method determined monohydroxy metabolites of naphthalene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, benzo[c]phenanthrene, and benz[a]anthracene. The sample preparation consisted of enzymatic hydrolysis, solid-phase extraction and derivatization with a silylating reagent. Five carbon-13 labeled standards were used for isotope dilution. Analytes were separated by gas chromatography (GC) and quantified with high-resolution mass spectrometry (HRMS). This method produced good recoveries (41-70%), linearity, and specificity. Data were corrected for blank levels from the naphthalene, fluorene, and phenanthrene metabolites. Method detection limits ranged from 2 ng L(-1) for 1-hydroxypyrene to 43.5 ng L(-1) for 1-hydroxynaphthalene. Using quality control charts from two urine pools, the method can be readily applied to biomonitoring PAH exposure.     

Crystal Structure and Magnetic Properties of SrCaMnGaO5+δ

The room-temperature crystal structure of the brownmillerite SrCaMnGaO_5+δ (δ=0.035) has been refined from neutron powder diffraction data; space group Ima2, a=15.7817(6), b=5.4925(2), c=5.3196(2)> Å. Mn and Ga occupy 99.0(2)% of the 6- and 4-coordinate sites, respectively. A combination of magnetometry, neutron diffraction and μSR spectroscopy has shown that the compound orders magnetically at 180 K, and that the low-temperature phase has a G-type antiferromagnetic structure, with an ordered magnetic moment of 3.30(2) μ_B per Mn at 2 K. Displaced hysteresis loops provide evidence that the atomic moment has an additional, glassy component. Magnetometry shows that significant short-range magnetic interactions persist above 180 K, and μSR that the spin fluctuations are thermally activated in this temperature region. The compound is an electrical insulator which at 159 K shows an unusually large magnetoresistance of 85% in 6 T, increasing to 90% in 13 T.     

Mass spectrometric interrogation of thioester-bound intermediates in the initial stages of epothilone biosynthesis

Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS). Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC. Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.0 Da mass shift consistent with the formation of the methylthiazolyl-methacrylyl product by incorporation of acetyl-CoA, cysteine, and methylmalonyl-CoA. Thioester-templated reaction intermediates and products are typically characterized by quantifying radioactive substrates, either enzyme bound or chemically hydrolyzed. In contrast, the MS-based methodology described here provides semiquantifiable ratios of free enzyme, intermediate, and product occupancy and reveals that certain substrates result in a >50% formation of nonproductive intermediates.     

Avalanches and self-organized criticality in simulations of particle piles

Simulations are carried out for an athermal pile of particles in the presence of a gravity-like force. As the force increases, the pile structure rearranges through discrete avalanche events. The sizes of the avalanche events are characterized by the change in energy, and it is found that the avalanche size follows a power-law probability distribution with a power-law exponent of −0.96. These results suggest that the particle pile is in a self-organized critical state.