Combined cysteine and homocysteine quantitation in plasma by trap and release membrane introduction mass spectrometry

Recently, a new and efficient method for total homocysteine (tHcy) quantitation in plasma using trap and release membrane introduction mass spectrometry (T&R-MIMS) with a versatile removable direct introduction membrane probe (DIMP) was described [R. Haddad, M. A. Mendes, N. F. Hoehr and M. N. Eberlin, Analyst, 2001, 126, 1212]. Herein we report on the use of the DIMP-T&R-MIMS technique for total cysteine (tCys) quantitation; hence combined tCys and tHcy quantitation in plasma or serum can be achieved. The method employs Cys and Hcy derivatization with ethyl chloroformate (after disulfide bond reduction with dithiothreitol and protein precipitation with trichloroacetic acid), preconcentration in a capillary silicone membrane, and their thermal desorption to the gas phase inside the ion source region of a mass spectrometer, at a point exactly between the two ionization filaments. Thermal desorption uses the uniform heat radiation provided by the two ionization filaments. The analytes are then ionized by electron ionization and both Cys and Hcy are quantitated by mass spectrometry using selected ion monitoring. For tCys quantitation, good linearity and reproducibility was observed for concentrations ranging from 5 to 350 microM, recovery was near 95%, and the limit of detection (LOD) was of 2 microM. This LOD is well below the mean Cys concentration in plasma, and serum samples from a large group of healthy people showed a mean tCys concentration of 132 +/- 45 microM.     

Identification of the function of gene lndM2 encoding a bifunctional oxygenase-reductase involved in the biosynthesis of the antitumor antibiotic landomycin E by Streptomyces globisporus 1912 supports the originally assigned structure for landomycinone

The angucycline antibiotic family of the landomycins displays potent antitumor activity. To elucidate early post polyketide synthase (PKS) tailoring steps of the landomycin E biosynthetic pathway in Streptomyces globisporus 1912, the mutant S. globisporus M12 was prepared through gene replacement experiment of lndM2. It encodes an enzyme with putative oxygenase and reductase domains, according to sequencing of the gene and its counterpart lanM2 from S. cyanogenus S136 landomycin A biosynthetic gene cluster. The isolation of the novel shunt products 11-hydroxytetrangomycin and 4-hydroxytetrangomycin along with the well-known angucyclines tetrangomycin and tetrangulol from the culture of S. globisporus M12 provides evidence for the involvement of lndM2 in the early biosynthetic pathway of the landomycins, in particular in the formation of the alicyclic 6-hydroxy function of the landomycin aglycon. We therefore propose LndM2 to be responsible for both hydroxylation of the 6-position and its subsequent reduction. These reactions are necessary before the glycosylation reactions can occur. The results are in agreement with the originally published structure of landomycin but do not support the recently suggested revised structure.     

Arsenic Speciation in Urine and Blood Reference Materials

Acute and chronic exposure to arsenic is a growing problem in the industrialized world. Arsenic is a potent carcinogen and toxin in humans. In the body, arsenic is metabolized to produce several species, including inorganic forms, such as trivalent (As^III) and pentavalent (As^V), and the methylated metabolites such as monomethylarsonic acid, (MMA^V), and dimethylarsinic acid (DMA^V), in addition to arsenobetaine (AsB) which is ingested and excreted from the body in the same form. Each of these species has been reported to possess a specific but different degree of toxicity. Thus, not only is the measurement of total As required, but also quantification of the individual metabolites is necessary to evaluate the toxicity and risk assessment of this element. There are a large number of reference materials that are used to validate methodology for the analysis of As in blood and urine, but they are limited to total As concentrations. In this study, the speciation of five arsenic metabolites is reported in blood and urine from commercial available control materials certified for total arsenic levels. The separation was performed with an anion exchange column using inductively coupled plasma mass spectrometry as a detector. Baseline separation was achieved for As^III, As^V, MMA^V, DMA^V, and AsB, allowing us to quantify all five species. Excellent agreement between the total arsenic levels and the sum of the speciated As levels was obtained.     

Sequence-dependent conformational energy of DNA derived from molecular dynamics simulations: toward understanding the indirect readout mechanism in protein-DNA recognition

Sequence dependence of DNA conformation plays a crucial role in its recognition by proteins and ligands. To clarify the relationship between sequence and conformation, it is necessary to quantify the conformational energy and specificity of DNA. Here, we make a systematic analysis of dodecamer DNA structures including all the 136 unique tetranucleotide sequences at the center by molecular dynamics simulations. Using a simplified conformational model with six parameters to describe the geometry of adjacent base pairs and harmonic potentials along these coordinates, we estimated the equilibrium conformational parameters and the harmonic potentials of mean force for the central base-pair steps from many trajectories of the simulations. This enabled us to estimate the conformational energy and the specificity for any given DNA sequence and structure. We tested our method by using sequence-structure threading to estimate the conformational energy and the Z-score as a measure of specificity for many B-DNA and A-DNA crystal structures. The average Z-scores were negative for both kinds of structures, indicating that the potential of mean force from the simulation is capable of predicting sequence specificity for the crystal structures and that it may be used to study the sequence specificity of both types of DNA. We also estimated the positional distribution of conformational energy and Z-score within DNA and showed that they are strongly position dependent. This analysis enabled us to identify particular conformations responsible for the specificity. The presented results will provide an insight into the mechanisms of DNA sequence recognition by proteins and ligands.     

Thermal and FTIR spectroscopic analysis of the interactions of aniline adsorbed on to MCM-41 mesoporous material

The adsorption of aniline on Na-AlMCM-41 synthesized by us has been characterized by infrared spectroscopy, temperature programmed desorption (TPD), and differential thermal analysis methods. Aniline would be mostly bound to the mesostructure through weak pi interactions. On the mesostructure containing adsorbed water, the co-adsorption of aniline could occur by weak hydrogen bonding through surface water molecules. For water, two possible modes of adsorption have been identified. Different associations between aniline and hydrated and nonhydrated mesostructures have been evaluated in order to favor the posterior in situ polymerization of adsorbed aniline.     

Effects of trivalent phosphorus compounds on vinyl polymerizations—VII. A nonstationary state radical polymerization of methylmethacrylate in the presence of phenyldichlorophosphine

In the radical polymerization of methylmethacrylate in the presence of p-phenyldichlorophosphine, the initial rate and degree of polymerization increased with polymerization time. They first decreased with increase in the phosphine concentration but increased with further increase in the phosphine concentration. Termination was first order with respect to the initiator. The degree of polymerization was independent of the initiator concentration, but dependent on the conversion. The polymer contained no phosphorus units regardless of the phosphine concentration. The ESR spectra of system showed existence of a phosphorus radical. In order to explain these characteristics of the polymerization, it is proposed that there may be a nonstationary state in which the polymer radicals are regenerated during the polymerization from the phosphoranyl radicals.     

An NMR study of merocyanine-type dyes derived from barbituric acid

The 13C NMR of two solvatochromic dyes derived from a barbituric acid acceptor and dimethylaminophenyl donor fragments, compound 1 and the related merocyanine 2, were recorded in various solvents. The observed chemical-shift variations were used to interpret their structural differences and solvatochromic behavior in solution.     

Chemistry of the phenoxathiins and isosterically related heterocycles. XXIII . 1-azathianthrene: First reported synthesis of a monoazathianthrene and the investigation of the 13C-NMR spectrum using two-dimensional NMR techniques

The reaction of the dianion of 3-mercaptopyridin-2(1H)-thione with 2-chloronitrobenzene in N,N-dimethylformamide leads to the formation of 1-azathianthrene, the first reported mono-aza analog of the thianthrene ring system. A partial assignment of the ¹³C-nmr spectrum of the title compound is reported, the assignment based on chemical shift arguments, spin-lattice (T₁) relaxation times and ¹H-¹³C spin coupling constants. Amplitude modulated two-dimensional Fourier transform (AM2DFT) techniques were employed for the acquisition of the heteronuclear spin-coupling constants.     

Investigating the possibility of simultaneously finding an electron-hole and an electron-pair in a molecule: Delocalization,competition of ionic vs. covalent character,and related effects in push–pull ethylenes

This work examines the possibility of finding an electron-hole and an electron-pair simultaneously in a π-system substituted by an electron-donating (NH₂) and/or electron-withdrawing (NO₂) group. The contributions of various ionic [(+), ( ↑ ↓ )] structures are calculated from ab initio SCF –CI wave functions, using a recently developed general multielectron population analysis. The molecules studied are two monosubstituted ethylenes, aminoethylene and nitroethylene, and a disubstituted ethylene, the 2-nitroethenamine (push–pull ethylene) in its two configurational forms. The influence of the NH₂ and/or NO₂ group in delocalization and ionic (vs. covalent) character of the C?C double bond are investigated, along with examining the experimental chemist formalism of electron-pair “displacements” in several resonance structures. Analysis of mutual dependence of an electron-hole and an electron-pair, at short and long distances, leads to the conclusion that a push–pull π-system can stimulate the simultaneous existence of an electronhole and an electron-pair even for nonvicinal positions. The relationship between the electronpair distributions and contributions of the corresponding structures are also examined; the effects of electronic correlation are analyzed as well. © 1992 John Wiley & Sons, Inc.     

Synthesis and biological evaluation of spongistatin/altohyrtin analogues: E-ring dehydration and C46 side-chain truncation

Simplified analogues of the potent antimitotic marine macrolide spongistatin 1/altohyrtin A were synthesised and evaluated as growth inhibitory agents against a range of human tumour cell lines, including Taxol-resistant strains, revealing that E-ring dehydration leads to enhanced cytotoxicity at the low picomolar level while truncation of the side-chain at C46 results in a drastic decrease in activity.