Mercury(II) Site-Selective Binding to a DNA Hairpin. Relationship of Sequence-Dependent Intra- and Interstrand Cross-Linking to the Hairpin-Duplex Conformational Transition

Hg(II) interacted site selectively with only one of three deoxyribooligonucleotides examined; these "oligos" each had a different number of unmatched T residues. Thus, Hg(II) formed an intrastrand T-Hg-T cross-link between the first and fourth T residues of the hairpin, d(GCGCTTTTGCGC) (T4). The DNA strand formed a loop around the Hg, as if the Hg atom had been lassoed. The interactions of Hg(II) with two other oligos, d(ATGGGTTCCCAT) (T2) and d(GCGCTTTGCGC) (T3), were less specific. Previously, we found that at high DNA and salt concentrations, T2 was a mixture of hairpin and duplex forms while T3 and T4 had the hairpin form; modeling studies showed that in the free T4 hairpin the two T's at the ends of the (T)(4) loop form a T.T wobble base pair. Only in T4 are the T residues positioned to form an intrastrand cross-link readily. The Hg(II)-oligo adducts formed as a function of added Hg(II) were investigated by titrations monitored by UV, CD, and (1)H NMR spectroscopy. The appearance of a new set of (1)H signals with the concomitant decay of the free oligo (1)H signals indicated that 1:1 Hg(II):T2, 1.5:1 Hg(II):T3, and 1:1 Hg(II):T4 adducts were formed with Hg(NO(3))(2). In H(2)O, these adducts all had spectra with very downfield signals for the exchangeable TN(3)H and GN(1)H groups, a characteristic of base-paired regions. All upfield N(3)H signals from the (T)(2) and (T)(3) sequences of the free oligo disappeared in the spectra of the 1:1 Hg(II):T2 and 1.5:1 Hg(II):T3 adducts. The disappearance of the NH signals, the UV spectral changes, and the stoichiometries (1:1 Hg(II):T2 and 1.5:1 Hg(II):T3) indicate that these adducts are duplexes containing two and three T-Hg-T interstrand cross-links for T2 and T3, respectively. The (1)H and (13)C signals of the 1:1 Hg(II):T4 adduct in D(2)O were nearly completely assigned by 2D NMR spectroscopy. The spectrum of the adduct in H(2)O had only two of the four original TN(3)H signals from the (T)(4) sequence present in the spectrum of T4; this result is consistent with the presence of a TN3-Hg-TN3 cross-link. The (13)C chemical shift changes upon Hg(II) binding indicated that the TN3-Hg-TN3 cross-link was between the T's at each end of the (T)(4) loop. The NOESY, CD, and UV spectra were all consistent with a hairpin conformation for the 1:1 Hg(II):T4 adduct. A hairpin conformation also appeared reasonable from molecular modeling calculations. In conclusion, the length of the central (T)(n)() sequence influenced the type of T-Hg-T cross-link formed and, in turn, the conformation of the adducts. For (T)(2) and (T)(3), interstrand T-Hg-T cross-linking favored the duplex form. In contrast, for (T)(4), intrastrand T-Hg-T cross-linking stabilized the hairpin form.     

N,N-dimethyl-trimethylsilyl-carbamate as a derivatizing agent in gas chromatography of trichothecene mycotoxins

N,N-dimethyl-trimethylsilyl-carbamate, a commercially available silylating agent, has been tested in the derivatization of trichothecenes for the first time. Its reaction with alcohols is an autocatalytic, non-equilibrium process with volatile by-products. As a consequence of these advantageous characteristics N,N-dimethyl-trimethylsilyl-carbamate proved to be easily utilizable and effective for the derivatization of all studied trichothecenes, namely deoxynivalenol (DON), nivalenol (NIV) and 4,15-diacetoxyscirpenol (DAS). During testing also, optimum conditions (temperature: 15 °C, reaction time: 35 min) were established for the trimethylsilylation and for the gas chromatographic determination of the above-mentioned three trichothecenes. The linearity of the detector response was found to be proper in the 5-1100 ng range. (Given in the grams of trichothecene equivalent to the derivatives injected). The limit of detection is less than 1 ng for each of the studied trichothecenes. Relative standard deviations of the peak heights ranged from 0.75 to 4.32%.     

Resolution of 8-aminonaphthalene-1,3,6-trisulfonic acid-labeled glucose oligomers in polyacrylamide gel electrophoresis at low gel concentration

A discontinuous Tris-Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled malto-oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris-OAc behind a moving boundary formed between the leading chloride ion of Tris-HCl gel buffer and the trailing OAc ion provided by a catholyte of NH(4)OAc. In contrast with the results obtained with Tris-CI/glycinate buffer commonly used in electrophoresis, or with Tris-CI/borate, the best resolution of the glucose oligomers containing 1-4 glucose units in Tris-OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH(4)OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS-glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, R(f), vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, K(R), of the plots. The molecular weight of the oligomers is directly but nonlinearly related to K(R). The novel electrophoretic system illustrated here for separation of short ANTS-saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.     

Microwave Digestion of Thermoluminescent Aluminium-Oxide Powders and Determination of Trace Impurities by Inductively Coupled Plasma Optical Emission Spectroscopy

 Five commercial and three laboratory prepared thermoluminescent aluminium-oxide powders as well as three reference samples were digested by a microwave digestion system and the impurities were determined by ICP-OES. The physico-chemical properties of the different samples were found to influence highly the decomposition efficiency. Optimized decompositi on parameters were determined for the samples with different physico-chemical properties (phase composition, grain size). The detection limits of the impurities of interest are presented, the analysis results for the aluminium- oxide dosimetric samples and the standards are given as well for Be, Ca, Ce, Cu, Fe, Cd, Mg, Mo, Na, Ni, Ti and Zn in the < 2–400 μg/g range. Received August 24, 1999. Revision December 1, 1999.     

Statistical evalution of aroma and metal content in Tokay wines

Wine qualities are predominantly determined by the qualitative and quantitative composition of organic and inorganic components present, consequently elucidating them is one of the tasks of greatest significance facing wine analysis. It is, on the other hand, of unique importance from the economic viewpoint to make sure and keep a continuous check on whether wine qualities are satisfactory enough, taking the trend into consideration that nowadays among products for human consumption it is solely those that show a high likelihood to succeed on the market in the long run that manage to satisfy quality requirements of gradually increasing demands. Research into food analytic on the other hand, plays a considerably significant role in origin protection which is an issue of particular importance in the case of a wine-growing area of such long tradition. During the course of the investigation the answer was sought to the question of how qualitative and quantitative relations of volatile organic and metal components present in traditional wines produced in the wine-growing area depend on the vintage, the location on which it is grown, as well as the type of wine grape and to what extent these are characteristic of wines of given type and vintage.     

A steroidal phenyldi­hydro‐1,3‐oxazine derivative

The structure of methyl (6R)‐6‐(3′β‐acetoxy‐5′‐androsten‐17′β‐yl)‐2‐phenyl‐5,6‐dihydro‐4H‐[1,3]oxazine, C₃₁H₄₁NO₃, synthesized from an azidopregnene derivative, is reported. The di­hydro‐1,3‐oxazine ring is connected in the β position to the sterane skeleton at C‐17′. An R configuration was found at C‐6.     

Thermal Plasma Decomposition of Tetrachloroethylene

Tetrachloroethylene (C₂Cl₄) has been used widely as a solvent and dry cleaning agent, but was later specified as possible human carcinogen. As a result, its safe treatment became a priority. In this paper, we report on its decomposition in an atmospheric radiofrequency thermal plasma reactor. Main components of the exhaust gases were determined by Fourier transform infrared spectroscopy. We found that complete decomposition can be achieved in either oxidative or reductive conditions but not in neutral one. The solid soot product was characterised by transmission electron microscopy and specific surface area measurement. Organic compounds adsorbed on the surface of the soot were extracted by toluene and comprised, based on gas chromatography mass spectrometry, of various perchlorinated aliphatic (for example hexachlorocyclopentadiene) and aromatic compounds (like hexachlorobenzene, octachloronaphthalene or octachloroacenaphthylene). Several nitrogen containing molecules were also identified whose presence are rare during thermal plasma treatments. Further investigation of the extract by mass spectrometry revealed various higher molar mass chlorinated carbon clusters and two types of fullerenes (C₆₀ and C₇₀).     

Shotgun electroelution: a proteomic tool for simultaneous sample elution from whole SDS-polyacrylamide gel slabs

A high-throughput device has been constructed which allows parallel electroelution of separated SDS-protein bands directly from intact unsectioned polyacrylamide gel slabs as well as single electroelution of certain protein spots into a 384-well standard flat-bottom multiwell plate. The prototype provides complete, quick elution for proteomics from 1-D or from 2-D gels without gel sectioning. Since the elution chamber matrix requires no assembly, sample handling can be easily carried out by existing robotic workstations. The current design is a good candidate for automation of spot elution since there are no moving liquid containing components in the apparatus. Eight SDS-proteins were eluted in test runs and an average 70% sample recovery was achieved by re-electrophoresis of the electro-eluates.     

High-yielding synthesis of 1-isoindolinone derivatives via palladium-catalysed cycloaminocarbonylation

1-Isoindolinone derivatives were synthesised in high yields (up to 89%) by using 2-iodobenzyl bromide and 2-iodobenzylamine as bifunctional substrates in palladium-catalysed carbonylation. Depending on the N-nucleophiles, two types of compounds were synthesised with 2-iodobenzyl bromide: the use of primary amines, including amino acid methylesters, resulted in the formation of N-substituted 1-isoindolinones, while secondary amines react both with the benzyl bromide and iodoarene moieties resulting in the corresponding ortho-(N-piperidino/morpholinomethyl)-benzamides. The parent 1-isoindolinone was obtained in a facile, highly chemoselective intramolecular aminocarbonylation of 2-iodobenzylamine. The mechanistic details of the ring-closure reaction and the conditions leading to side-products are discussed as well.     

Rearrangement of aryl- and benzylthiopyridinium imides with participation of a methyl substituent

6-Methyl substituted 2-aryl- and 2-benzylthiopyridinium N-imides reacted with an excess of isocyanates to give N,N-disubstituted exocyclic1H-imidazo[4,5-b]pyridin-2(3H)-ones. The products easily underwent spontaneous [1,5] hydrogen shift to provide the heteroaromatic imidazopyridinone isomers. The transformation implied the initial formation of [1,2,4]triazolo[2,3-a]pyridinium salt, followed by deprotonation and carbamoylation of the methylene moiety, and, finally, a rearrangement following a [1,3] sigmatropic pattern. Mechanistic considerations suggest and some experimental findings reveal the nonconcerted two-step mechanism of the ring transformation step.