Alkylation of DNA by melphalan: investigation of capillary liquid chromatography-electrospray ionization tandem mass spectrometry in the study of the adducts at the nucleoside level

Nitrogen mustards are among the oldest cancer chemotherapeutic agents and remain the drugs of choice for treatment of many human cancers. A serious complication of treatment with nitrogen mustards is the increased risk of a secondary leukaemia in long-term survivors because not all alkylating agent interactions with DNA result in cell death. In an earlier study 2'-deoxy-5'-mononucleotide/melphalan adducts have been analysed by us by LC-ES MSMS. In this work we want to present the first results of the analysis of the corresponding 2'-deoxynucleoside/melphalan adducts from DNA hydrolysates by column switching/capillary LC-ES tandem mass spectrometry. Nucleosides, compared to nucleotides, give better chromatographic results and show a good sensitivity under electrospray (+) [ES(+)] ionisation. Several adducts were identified under ES(+) conditions. Mono-alkylated nucleoside adducts alkylated at the base moiety were identified for dGuo, dCyd and dAdo. Structures were identified by recording the low-energy CAD product ion scans. Also a mono-alkylated nucleotide pdA with alkylation position at the phosphate moiety could be detected. This proves that in the case of phosphate alkylation the enzymatic dephosphorylation reaction was inhibited. A Jurkat cell suspension was treated with melphalan (1 mM) and incubated at 37 degrees C (5% CO(2)). After 6 and 48 h, the DNA was isolated and enzymatically hydrolysed. The corresponding nucleoside pool was evaluated with the developed LC-MS method. In the 48-h experiment, one adduct could be identified as a N-7 alkylated dGuo. In the 6-h experiment, no adducts could be found. Additional experiments were done wherein Jurkat-DNA, isolated from a non-treated cell culture, was treated with melphalan. These results were analogous with the data found in melphalan-treated calf thymus DNA. Additionally, we tried to determine the exact alkylation position by interpreting high-resolution fragmentation spectra.     

Ion-pair reversed-phase high-performance liquid chromatography of adenine nucleotides and nucleoside using triethylamine as a counterion

An isocratic HPLC method for the simple and selective determination of adenine nucleoside and nucleotides has been developed. The separation is achieved at room temperature by reversed-phase chromatography (Shiseido, Capcell Pak C18). A mixture of 0.1 M triethylamine (TEA) phosphate buffer and methanol (95:5, v/v) is used as a standard eluent. Influence of pH and concentrations of organic modifiers and TEA ion on capacity factors of adenine compounds has been investigated. It has been also found that the TEA ion in the eluent is adsorbed onto the reversed-phase surface. The results clearly demonstrate that ion-pair formation with TEA ion occurs probably both in the mobile phase and on the stationary phase and governs the retention of adenine and nucleotides in the present system. The HPLC system is applied to the analysis of adenine nucleotides formed as intermediates in the synthesis of 3′-phosphoadenosine 5′-phosphosulphate (PAPS) and to the assays of ATPases and 5′-nucleotidase activities in rat liver plasma membrane. This method is a new type of ion-pair reversed-phase HPLC system and is suitable for the separation of highly polar organic anions, especially for adenine nucleotides.     

Functional analysis of site-directed glycosylation mutants of the human equilibrative nucleoside transporter-2

Protein glycosylation is important for nucleoside transport, and this has been demonstrated for the human equilibrative nucleoside transporter-1 (hENT1). It is not known whether glycosylation affects the functions of hENT2 or where hENT2 is glycosylated. We address these questions using N-glycosylation mutants (N48D, N57D, and N48/57D) and demonstrate that hENT2 is glycosylated at Asn(48) and Asn(57). Our results show that although the apparent affinities for [3H]uridine and [3H]cytidine of the mutants were indistinguishable from those of the wild-type protein, N-glycosylation was required for efficient targeting of hENT2 to the plasma membrane. All mutants had a two- to threefold increase in IC(50) for dipyridamole. N57D and N48/57D, but not N48D, also had a twofold increase in IC(50) for NBMPR. We conclude that the relative insensitivity of hENT2 to inhibitors is primarily due to its primary structure and not to glycosylation. Glycosylation modulates hENT1 function, but is not required for hENT2.     

Sample degradation leads to false-positive copy number variation calls in multiplex real-time polymerase chain reaction assays

The recent implication of genomic copy number variations (CNVs) in multiple human genetic disorders has led to increased interest in CNV discovery technologies. There is a growing consensus that, in addition to the method used for detection, at least one additional technology should be employed for validation. Real-time quantitative polymerase chain reaction (qPCR) analysis, incorporating a normal (2N) copy number standard, is commonly used as a means of validating CNVs. Whereas it has previously been reported that formalin-fixed paraffin-embedded (FFPE) DNA samples can yield spurious CNV calls in real-time qPCR assays, here we report that sample degradation under standard laboratory storage conditions generates a significant increase in false-positive CNV results. Results suggest the possibility of biased degradation among genomic regions and emphasize the need to assess sample integrity immediately prior to real-time qPCR experiments.     

C-terminal sequencing method for proteins in polyacrylamide gel by the reaction of acetic anhydride

A successive C-terminal amino acid truncation reaction with acetic anhydride was applied on proteins in polyacrylamide gel. Protein bands separated by conventional SDS-PAGE were excised, partially fixed in the gel with glutaraldehyde ethanol solution, dehydrated with ACN and subjected to the truncation reaction with acetic anhydride formamide solution. Pre-treatment of the gel with pyridine aqueous solution was found to enhance the truncation reaction yields. After the truncation reaction, the products were treated with an aqueous solution of dimethylaminoethanol to hydrolyze oxazolone rings at the C termini of the truncated products and O-acetylated products of serine, threonine and/or tyrosine. Several commercially available proteins of 10-40 kDa, as determined by SDS-PAGE, such as myoglobin, trypsin inhibitor, alpha-hemolysin, cytochrome c, chymotrypsin C chain, elastase, acylase and histone H4, were subjected to the C-terminal analysis. The truncated proteins were in-gel digested with trypsin and the extracted peptides were analyzed by MALDI-TOF MS, giving rise to a series of molecular mass ions of the C-terminal truncated fragments corresponding to the C-terminal amino acid sequence of the relevant protein.     

Abiogenic nucleoside synthesis in the presence of phosphorus salts

By means of UV-spectroscopy, gel-filtration, ion-exchange and thin layer chromatography it has been shown that the action of ionizing radiation on the mixture of dry preparations of adenine and deoxyribose in the presence of the K, Na and Ca phosphates results in the formation of nucleoside-like substances. The phosphates catalyze or inhibit the nucleoside synthesis but they are not phosphorylating agents. The data obtained indicate the reality of abiogenic synthesis of nucleoside-like substances from the mixture of dry preparations of adenine and deoxyribose in the lithosphere during chemical evolution.     

Pinocytic uptake of divinyl ether-maleic anhydride (pyran copolymer) and its failure to stimulate pinocytosis

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of ¹²⁵I-labelled PVP and colloidal ¹⁹⁸Au by the rat visceral yolk sac and by rat peritoneal macrophages was studied in vitro. Contrary to expectations from some earlier data, there was no enhancement of pinocytosis and in some cases inhibition was seen. [¹⁴C]DIVEMA and ¹²⁵I-labelled DIVEMA were accumulated rapidly by rat peritoneal macrophages, the results indicating that this is by an adsorptive pinocytic mechanism.     

Synthesis of vasoactive intestinal peptide (VIP) via the mixed anhydride method

Porcine VIP was synthesized from three segments. The segments, VIP(1-6), VIP(7-13), and VIP(14-28), were synthesized via the Repetitive Excess Mixed Anhydride (REMA) method. The low solubility of the C-terminal segment was greatly improved by a temporary substitution of Asn28 by a beta-t-butyl aspartic acid ester. The segments VIP(1-6) and VIP(7-13) were purified by HPLC and coupled via the mixed anhydride method. The product was purified by gel filtration. VIP was synthesized from VIP(1-13) and VIP(14-28) by the same procedure. After deprotection, Met17-sulfoxide reduction, and purification by ion-exchange chromatography, the product was found to have the expected amino acid composition and biological potency. A HPLC purified sample was compared with several commercial preparations of varying purity.     

A mild and quantitative procedure for the removal of nucleoside alkoxycarbonyl groups using pig liver esterase or Candida antarctica B lipase

A set of eight mono-, di-, tri- and tetraalkoxycarbonylated nucleosides was tested in order to assess their enzymatic hydrolysis. All the alkoxycarbonyl groups of the assayed substrates, from both carbonate and carbamate functions, were quantitatively hydrolysed using pig liver esterase (PLE) at pH 7 and 60 °C, regardless of the nucleoside base. Quantitative full alkoxycarbonyl groups removal was also reached by Candida antarctica B lipase (CAL B) under mild conditions, but in this case, longer reaction times were required. Thus, PLE appears as an useful catalyst for the mild and quantitative deprotection of nucleoside carbonates and carbamates in the synthesis of modified nucleosides.     

Immobilization of levan fructotransferase for the production of di-fructose anhydride from levan

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was immobilized on various carriers of which Chitopearl BCW2501 beads showed the higher activity of 320 U g^–1 for the formation of di-fructose anhydride compounds. The immobilized enzyme retained about 60% of its initial activity after being used for 20 cycles.     

Development of a dinoflagellate-oriented PCR primer set leads to detection of picoplanktonic dinoflagellates from Long Island Sound

We developed dinoflagellate-specific 18S rRNA gene primers. PCR amplification using these oligonucleotides for a picoplanktonic DNA sample from Long Island Sound yielded 24 clones, and all but one of these clones were dinoflagellates primarily belonging to undescribed and Amoebophrya-like lineages. These results highlight the need for a systematic investigation of picodinoflagellate diversity in both coastal and oceanic ecosystems.     

Homogeneous modification of sugarcane bagasse cellulose with succinic anhydride using a ionic liquid as reaction medium

The homogeneous chemical modification of sugarcane bagasse cellulose with succinic anhydride using 1-allyl-3-methylimidazolium chloride (AmimCl) ionic liquid as a reaction medium was studied. Parameters investigated included the molar ratio of succinic anhydride/anhydroglucose units in cellulose in a range from 2:1 to 14:1, reaction time (from 30 to 160min), and reaction temperature (between 60 and 110 degrees C). The succinylated cellulosic derivatives were prepared with a low degree of substitution (DS) ranging from 0.071 to 0.22. The results showed that the increase of reaction temperature, molar ratio of SA/AGU in cellulose, and reaction time led to an increase in DS of cellulose samples. The products were characterized by FT-IR and solid-state CP/MAS (13)C NMR spectroscopy, and thermal analysis. It was found that the crystallinity of the cellulose was completely disrupted in the ionic liquid system under the conditions given. The data also demonstrated that homogeneous modification of cellulose with succinic anhydride in AmimCl resulted in the production of cellulosic monoester. The thermal stability of the succinylated cellulose decreased upon chemical modification.     

Synthesis and antiviral activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT

Phospholipid conjugates of 3′-azido-3′-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112–6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with ³¹P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC₅₀ values of AZTTP-DSG were 0.33 and 0.79 μM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 μM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.     

Environmental degradation of streams leads to the loss of ecomorphologically similar fish species

Hydrobiologia - Human activities change the environmental conditions of streams and alter their assemblages. However, the environmental factors associated with the change in the ecomorphological...