Fraction Of Adducts Research Articles

Branched chain fatty acids concentrate prepared from butter oil via urea adduction

Saturated branched chain fatty acids (BCFA) intake in the US is greater than that of other bioactive fatty acids (FA), yet little information is available on methodologies to concentrate them. We report here the effect of urea‐to‐FA (urea:FA) ratio, adduction time, and temperature on the enrichment of branched chain fatty acids (BCFA) from butter oil. Urea adducts precipitate both saturated and monounsaturated hydrocarbon chains as urea complexes, leaving solubilized polyunsaturated FA and BCFA in the non‐urea adduct fraction (NUA). The optimum urea:FA ratio was found to be 4:1 and the optimum temperature to be 4°C. Adduction time had negligible effect on BCFA enrichment. Anteiso‐15:0 was most enriched across major BCFA under all conditions of temperature, time, and urea:FA ratio studied. In our preferred embodiment, a two‐stage urea adduction procedure applied to hydrolyzed butter oil resulted in an enrichment from <2% BCFA in the starting oil to >11% BCFA, indicating an enrichment factor of >6. The best method has a first stage performed at 4°C and urea:FA ratio of 4:1, and a second stage at 30°C and lower urea:FA ratio (2:1). Overall yield of BCFA in enriched fraction was about 10% of starting BCFA for two stages.Practical applications: BCFA are constituents of the GI tract of healthy newborns and are known to prevent necrotizing enterocolitis. The average per capita BCFA intake of Americans is estimated to be about 220 mg/day from dairy whereas based on the current dietary recommendations of USDA it should be about 400 mg/day from dairy alone with total amounting to 575 mg/day including beef. Using the results of the current study, enriched BCFA concentrates can be prepared to meet the demand.Enrichment in major branched chain fatty acids (BCFA) of butter oil after two stage urea adduction. First stage (NUA‐1) and second stage (NUA‐2) were conducted using 4:1; 4°C 2 h and 2:1, 30°C 2 h of urea‐to‐fatty acids ratio (wt/wt), temperature and time of adduction, respectively.

Alkylating potential of styrene oxide: reactions and factors involved in the alkylation process.

The chemical reactivity of styrene-7,8-oxide (SO), an alkylating agent with high affinity for the guanine–N7 position and a probable carcinogen for humans, with 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases, was investigated kinetically in water/dioxane media. UV–vis spectrophotometry and ultrafast liquid chromatography were used to monitor the reactions involved. It was found that in the alkylation process four reactions occur simultaneously: (a) the formation of a β-NBP–SO adduct through an SN2 mechanism; (b) the acid-catalyzed formation of the stable α-NBP–SO adduct through an SN2′ mechanism; (c) the base-catalyzed hydrolysis of the β-adduct, and (d) the acid-catalyzed hydrolysis of SO. At 37.5 °C and pH = 7.0 (in 7:3 water/dioxane medium), the values of the respective reaction rate constants were as follows: kalkβ = (2.1 ± 0.3) × 10–4 M–1 s–1, kalkα = (1.0 ± 0.1) × 10–4 M–1 s–1, khydAD = (3.06 ± 0.09) × 10–6 s–1, and khyd = (4.2 ± 0.9) × 10–6 s–1. These values show that, in order to determine the alkylating potential of SO, none of the four reactions involved can be neglected. Temperature and pH were found to exert a strong influence on the values of some parameters that may be useful to investigate possible chemicobiological correlations (e.g., in the pH 5.81–7.69 range, the fraction of total adducts formed increased from 24% to 90% of the initial SO, whereas the adduct lifetime of the unstable β-adduct, which gives an idea of the permanence of the adduct over time, decreased from 32358 to 13313 min). A consequence of these results is that the conclusions drawn in studies addressing alkylation reactions at temperatures and/or pH far from those of biological conditions should be considered with some reserve.

In vitro studies on cisplatin focusing on kinetic aspects of intracellular chemistry by LC-ICP-MS

To date, preclinical studies have addressed drug accumulation and intracellular distribution of cisplatin by determination of the total Pt content. In this work, the use of liquid chromatography in combination with inductively coupled plasma mass spectrometry (LC-ICP-MS) enabled accurate intact cisplatin quantification in cell model experiments. Hence, for the first time, intracellular drug degradation, drug accumulation and drug efflux were studied by actually quantifying the intact drug, along with the total Pt content of the cell nucleus, the cytosol and the low molecular weight fraction of the cytosol. The latter fraction was obtained by centrifugal filtration (cut-off filter of 10 kDa). Flow injection (FI)-ICP-MS was implemented for platinum quantification. In a first step, kinetics of intracellular cisplatin degradation was addressed by incubating cell extracts with sub-μM drug concentration levels. A half-life of 2 hours was observed in cell extracts of two different cancer cell lines (colon carcinoma and human mesothelioma), which was significantly shorter than that observed in sodium chloride. Hence, it was suggested that intact and nonaquated cisplatin was reacting with cellular components. Due to the large excess of potential binding partners pseudo first order kinetics were observed. The drug accumulation experiments revealed rapid uptake of the drug into the cytosol and the nucleus. Moreover, a significant fraction of Pt was bound to intracellular high molecular weight biomolecules after one hour of exposure. With ongoing time, the intracellular Pt concentration was increasing. However, the cisplatin concentration remained constant during 5 hours of continuous exposure. Assuming a cell volume of 10(-12) L, an intracellular concentration corresponding to the cisplatin concentration in the cell culture medium (5 μM) was estimated. At any time of investigation, intact cisplatin was the predominant species in the low molecular weight fraction of the cytosol. These findings support the hypothesis of passive diffusion as an uptake mechanism. Finally, a model experiment was designed resembling the situation of limited drug exposure time. Human mesothelioma cells were incubated with 5 μM cisplatin for 3 hours. Then the culture medium was replaced and the drug efflux was studied. The observed efflux was biphasic, with the intact cisplatin being removed within the first hour of investigation, while the Pt-protein adduct fraction was removed only partially (30% were still found in the cytosol after 24 hours). No net transfer of Pt from the cytosol to the nucleus fraction was observed after medium replacement.

Taurine–nitrite interaction as a precursor of alkylation mechanisms

Taurine (2-aminoethanesulphonic acid) is an amino acid-like-compound widely used as an ingredient in some nutraceuticals and energy drinks. Here the interaction of taurine (Tau) with nitrite was investigated. The reactions were carried out mimicking the conditions of the stomach lumen. The conclusions drawn are as follows: (i) Nitrite showed nitrosating capacity on Tau. The rate equation was ν(N)=k(obs)[Tau](o)[nitrite](o)(2), this result suggesting that the yield of nitrosation products in the human stomach would increase sharply with higher nitrate/nitrite intakes; (ii) the experimental results suggest a mechanism for the nitrosation, whose rate-limiting step is bimolecular attack by N(2)O(3); (iii) the nitrosation of taurine affords ethanesultone (ES), which displays alkylating capacity on the nucleophile 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases. Although the NBP alkylation rate for ethanesultone is much higher than those for carcinogenic four-membered ring lactones, resulting in the nitrosation of amino carboxylic acids, the fraction of ES-forming adduct with NBP is much smaller; (iv) in spite of the low risk to human health, since the stomach lumen conditions could be a favourable medium for Tau nitrosation, attention should be paid to potential situations of the concurrence of high contents of taurine and nitrite/nitrate in the diet.

Comparative LC–MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC–MS/MS using a stable isotope dilution assay. The glycation profiles for N ɛ -fructoselysine (FL), N ɛ -carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 ± 3.81 nmol CML per μmol of free Lys (Lys free) and 81.5 ± 87.8 nmol Pyr μmol −1 Lys free −1 vs. 3.72 ± 1.29 nmol FL μmol −1 Lys free −1. In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 ± 0.08 nmol FL μmol −1 of protein-bound Lys (Lys p-b), 0.04 ± 0.03 nmol CML μmol −1 Lys p-b −1 and 0.06 ± 0.02 nmol Pyr μmol −1 Lys p-b −1. It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

The 32P-postlabeling assay for DNA adducts

32P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen-DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3'-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5'-labeling of the adducts by transfer of 32P-orthophosphate from [gamma-32P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 10(10) nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d.

Enzymatic purification of dihomo-γ-linolenic acid from Mortierella single-cell oil

Purification of dihomo-γ-linolenic acid (20:3n−6; DGLA) from a single-cell oil containing 39 wt.% DGLA was attempted. The process comprised: (i) non-selective hydrolysis of the oil to prepare a mixture of free fatty acids (FFAs); (ii) urea adduct fractionation of the FFA mixture to remove saturated fatty acids; and (iii) repeated selective esterification of the resulting mixture with two kinds of lipases. In the first step, Candida rugosa lipase (Lipase-OF from Meito Sangyo Co. Ltd., Aichi, Japan) was the most effective for preparation of the FFAs from the oil; 99% hydrolysis was achieved by the reaction at 40 °C for 72 h. Urea adduct fractionation of the FFA mixture removed almost completely behenic and lignoceric acids, and the content of DGLA increased from 39 to 55 wt.%. The FFAs were esterified with 2 mol equivalent of lauryl alcohol (LauOH) using C. rugosa lipase (Lipase-AY from Amano Enzyme Inc., Aichi, Japan). In consequent, DGLA was enriched to 86 wt.% in the unesterified FFA fraction. To further increase the content of DGLA, the esterification was repeated using the same lipase. Accordingly, the content of DGLA increased to 91 wt.%, but the preparation was contaminated with 3.3 wt.% γ-linolenic acid. This contaminant was removed finally by selective esterification of the FFAs with 2 mol equivalent of LauOH using Pseudomonas aeruginosa lipase. A series of procedures purified DGLA to 95 wt.% in a yield of 51% of the initial content in the single-cell oil.

4‘-Substituted-4-biphenylyloxenium Ions: Reactivity and Selectivity in Aqueous Solution

Azide trapping shows that the 4'-substituted-4-biphenylyloxenium ions 1b-d are generated during hydrolysis of 4-aryl-4-acetoxy-2,5-cyclohexadienones, 2c and 2d, and O-(4-aryl)phenyl-N-methanesulfonylhydroxylamines, 3b and 3c. In addition, the 4'-bromo-substituted ester, 2d, undergoes a kinetically second-order reaction with N3- that accounts for a fraction of the azide adduct, 5d. Since both first-order and second-order azide trapping occurs simultaneously in 2d, the second-order reaction is not enforced by the short lifetime of 1d, which has similar azide/solvent selectivity to the unsubstituted ion, 1a. In contrast the 4'-CN and 4'-NO2 ions 1e and 1f cannot be detected by azide trapping during the hydrolysis of the dichloroacetic acid esters 2e' and 2f' even though 18O labeling experiments show that a fraction of the hydrolysis of both esters occurs through C(alkyl)-O bond cleavage. These esters exhibit only second-order trapping by azide. Correlations of the azide/solvent selectivities of 1a-d with the calculated relative driving force for hydration of the ions (DeltaE of eq 4) determined at the pBP/DN//HF/6-31G and BP/6-31G//HF/6-31G levels of theory suggest that 1e and 1f have lifetimes in the 1-100 ps range. Ions with these short lifetimes are not in diffusional equilibrium with nonsolvent nucleophiles, and must be trapped by such nucleophiles via a preassociation mechanism. The second-order trapping that is observed in these two cases is enforced by the short lifetime of the cations, and may occur by a concerted S(N)2' mechanism or by internal azide trapping of an ion sandwich produced by azide-assisted ionization. Comparison of azide/solvent selectivities of the oxenium ions 1a-c with the corresponding biphenylylnitrenium ions 8a-c shows that 4'-substituent effects on reactivity in both sets of ions are similar in magnitude, although the nitrenium ions are ca. 30-fold more stable in an aqueous environment than the corresponding oxenium ions. The magnitude of the 4'-substituent effects for electron-donating substituents suggest that both sets of ions are more accurately described as 4-aryl-1-imino-2,5-cyclohexadienyl or 4-aryl-1-oxo-2,5-cyclohexadienyl carbocations. Calculated structures of the oxenium ions are also consistent with this interpretation.

Mutagenic Activity of 4-Hydroxyestradiol, but Not 2-Hydroxyestradiol, in BB Rat2 Embryonic Cells, and the Mutational Spectrum of 4-Hydroxyestradiol

Estrogens are hypothesized to contribute to breast cancer via estrogen receptor-mediated increases in cell proliferation and via genotoxic processes leading to mutations. In this latter process, estradiol (E(2)) is thought to be oxidized to 4-hydroxyestradiol and then to E(2)-3,4-quinone, which reacts with DNA leading to apurinic sites. These sites represent premutagenic lesions. Additionally, E(2)-3,4-quinone can undergo redox cycling with E(2)-3,4-hydroquinone, leading to the release of reactive oxygen species. Although there is evidence that estradiol and E(2)-3,4-quinone are carcinogenic or mutagenic in several systems, 4-hydroxyestradiol, a key intermediate in the proposed genotoxic pathway, has thus far been negative in mutagenesis assays. Another major metabolite of estradiol, 2-hydroxyestradiol, is essentially inactive in carcinogenicity or mutagenicity assays. Here, we report that when using multiple low-dose exposures 4-hydroxyestradiol is mutagenic in the cII assay in BB rat2 cells. Under similar conditions, 2-hydroxyestradiol is inactive. Furthermore, the mutational spectrum of 4-hydroxyestradiol contains a considerable proportion of mutations at A:T base pairs, consistent with the known ability of E(2)-3,4-quinone to form a significant fraction of DNA adducts at adenines. Thus, the results of this study support the proposal that estradiol can contribute to carcinogenesis via a genotoxic pathway.

Purification of Arachidonic acid from Mortierella single‐cell oil by selective esterification with Burkholderia cepacia lipase

AbstractPurification of arachidonic acid (AA) from Mortierella alpina single‐cell oil was attempted. The process comprised three steps: (i) preparation of FFA by nonselective hydrolysis of the oil with Alcaligenes sp. lipase; (ii) elimination of long‐chain saturated FA from the resulting FFA by urea adduct fractionation; and (iii) enrichment of AA through lipase‐catalyzed selective esterification with lauryl alcohol (LauOH). In the third step, screening of industrially available lipases indicated that Burkholderia cepacia lipase (Lipase‐PS, Amano Enzyme Inc., Aichi, Japan) acted on AA more weakly than on other FA and was the most effective for enrichment of AA in the FFA fraction. When the FFA obtained by urea adduct fractionation were esterified with 2 molar equivalents of LauOH at 30°C for 16 h in a mixture with 20% water and 20 units (U)/g‐mixture of Lipase‐PS, the esterification reached 39% and the content of AA in the FFA fraction was raised from 61 to 86 wt%. To further increase the content of AA, unesterified FFA were allowed to react again under the same conditions as those in the first selective esterification except for the use of 50 U/g Lipase‐PS. A series of procedures raised the content of AA to 97 wt% with a 49% recovery based on the initial content in the single‐cell oil. These results indicated that the three‐step process for selective esterification with Lipase‐PS was effective for purifying AA from the single‐cell oil.

Supercritical CO2 extraction and concentration of n-3 polyunsaturated fatty acid ethyl esters from tuna cooking juice

Cooking is an important step in the tuna canning process; it usually produces functional solubilized proteins and lipids. The objective of this study was to recover and increase the concentration of functional n−3 fatty acids from the lipids in cooking juice. The lipids contained 12.98% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) after ethyl esterification and increased to 37.4% with subsequent urea adduct fractionation. Supercritical carbon dioxide extraction was further used to increase the concentration of EPA and DHA ethyl esters. The ratio of EPA+DHA ethyl esters (high-molecular-weight components) to that of C16 and C18∶1 fatty acid ethyl esters (low-molecular-weight components) was used as a separation index for evaluating the process variables. Experimental results indicated that a high CO2 density caused a low separation factor. At 1500 psig and 328.2 K, the extraction collected over 600 L CO2, displaying an accepted concentration factor of EPA+DHA ethyl esters herein. About 80% yield of EPA and DHA was obtained and the ethyl esters increased from 37.4% to 54.3%.

Duplex-Promoted Platination of Adenine-N3 in the Minor Groove of DNA: Challenging a Longstanding Bioinorganic Paradigm

The interactions of [Pt(en)Cl(ACRAMTU-S)](NO3)2 (PT-ACRAMTU, en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) with adenine in DNA have been studied using a combination of analytical and high-resolution structural methods. For the first time, a cytotoxic platinum(II) complex has been demonstrated to form adducts in the minor groove of DNA through platination of the adenine-N3 endocyclic nitrogen. An acidic depurination assay was developed that allowed the controlled and selective (pH 2, 60 degrees C, 12 h) release of platinum-modified adenine from drug-treated nucleic acid samples. From the digested mixtures, three adducts were isolated by semipreparative reverse phase high-performance liquid chromatography and studied by electrospray ionization mass spectrometry (in-line LC-MS), variable-pH 1H NMR spectroscopy, and, where applicable, X-ray crystallography. The three species were identified as the N7 (A-I), N3 (A-II), and N1 (A-III) linkage isomers of [Pt(en)(ACRAMTU-S)(adenine)]3+ (A). Incubations carried out with the single- and double-stranded model sequences, d(TA)5 and d(TA)15, as well as native DNA indicate that the adduct profiles (A-I:A-II:A-IIIratios) are sensitive to the nature of the nucleic acid template. A-II was found to be a double-strand specific adduct. The crystal structure of this adduct has been determined, providing ultimate evidence for the N3 connectivity of platinum. A-II crystallizes in the triclinic space group P in the form of centrosymmetric dimers, {[Pt(en)(ACRAMTU-S)(adenine-N3)]2}6+. The cations are stabilized by a combination of adenine-adenine base pairing (N6...N1 2.945(5) A) and mutual acridine-adenine base stacking. Tandem mass spectra and 1H chemical shift anomalies indicate that this type of self-association is not merely a crystal packing effect but persists in solution. The monofunctional platination of adenine at its N7, N3, and N1 positions in a significant fraction of adducts breaks a longstanding paradigm in platinum-DNA chemistry, the requirement for nucleophilic attack of guanine-N7 as the principal step in cross-link formation. The biological consequences and potential therapeutic applications of the unique base and groove recognition of PT-ACRAMTU are discussed.

Fluorescence Lifetimes of Benzofurazan Adducts with Thiol Groups in Biomole- cules

Fluorescence lifetimes have been measured for the adducts of ammonium 4-chloro-2,1,3-benzoxadiazole-7-sulfonate, which is known as a fluorescence probe specific to thiol groups Although the adduct with simple molecule such as mercaptoethanol showed a single exponential fluorescence decay, most adducts with amino acid, peptide, coenzyme and protein showed the bi exponential decay. The fluorescence lifetimes and ratio of the short lifetime fraction to long lifetime fraction of the SBD adducts with biomolecules vary with the molecular size, solvent polarity and micro environment around the fluorescence probe.

Purification of conjugated linoleic acid isomers through a process including lipase-catalyzed selective esterification.

A mixture of conjugated linoleic acids (CLAs) was prepared by alkali conjugation of high purity linoleic acid. The preparation contained 45.1 wt% cis-9, trans-11 (c9,t11)-CLA, 46.8 wt% trans-10, cis-12 (t10,c12)-CLA, and 5.3 wt% other CLAs. A process comprising Candida rugosa lipase-catalyzed selective esterification with lauryl alcohol, molecular distillation, and urea adduct fractionation under strict conditions in ethanol was very effective for purification of c9,t11- and t10,c12-CLAs. In particular, the urea adduct fractionation efficiently eliminated CLAs except c9,t11- and t10,c12-isomers. Purification of c9,t11- and t10,c12-CLAs from 1.0 kg of the CLA mixture increased the c9,t11-CLA purity to 93.1% with 34% recovery of the initial content, and increased the t10,c12-CLA purity to 95.3% with 31% recovery.

Enzymatic fractionation and enrichment of n−9 PUFA

AbstractA single‐cell oil from a Mortierella alpina mutant (TGM17 oil) contains n−9 PUFA: 14.3 wt% 6,9‐octadecadienoic acid (18∶2n−9; n−9 LnA) and 17.1 wt% Mead acid (20∶3n−9; MA). Lipase screening indicated that Pseudomonas aeruginosa lipase acted strongly on n−9 LnA and weakly on MA, and Candida rugosa lipase acted weakly on the two PUFA. Hence, fractionation and enrichment of the two FA were conducted with the lipases. The first step was selective hydrolysis of IGM17 oil with P. aeruginosa lipase. The hydrolysis fractionated the oil into FFA containing 20.4 wt% n−9 LnA and 6.3 wt% MA, and acylglycerols containing 10.7 wt% n−9 LnA and 23.7 wt% MA. The FFA fraction was used for preparation of n−9 LnA‐rich FFA. After removal of saturated FA, the FFA were esterified with lauryl alcohol (LauOH) using C. rugosa lipase. Two selective esterifications increased the n−9 LnA content to 54.0 wt% with 38.2% recovery of the initial content of TGM17 oil. The acylglycerol fraction obtained in the hydrolysis with P. aeruginosa lipase was used for preparation of MA‐rich FFA. The acylglycerol fraction was hydrolyzed under alkaline conditions, and saturated FA were eliminated by urea adduct fractionation. Two selective esterifications of the FFA with LauOH increased the MA content to 60.2 wt% with 53.5% recovery. Thus, the two‐step enzymatic process was effective for fractionation and enrichment of n−9 LnA and MA.

DNA binding by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition and nucleotide excision repair of monofunctional adducts.

Antitumor effects of cis-diamminedichloroplatinum(II) (cisplatin) and the clinical inactivity of its trans isomer (transplatin) have been considered a paradigm for the classical structure-activity relationships of platinum drugs. However, several new analogues of transplatin which exhibit a different spectrum of cytostatic activity including activity in tumor cells resistant to cisplatin have been recently identified. Analogues containing the planar amine ligand of the general structure trans-[PtCl(2)(NH(3))(L)], where L = planar amine, represent an example of such compounds. DNA is believed to be the major pharmacological target of platinum compounds. To contribute to the understanding of mechanisms underlying the activation of trans geometry in transplatin analogues containing planar amine ligands, various biochemical and biophysical methods were employed in previous studies to analyze the global modifications of natural DNA by trans-[PtCl(2)(NH(3))(L)]. These initial studies have revealed some unique features of the DNA binding mode of this class of platinum drugs. As the monofunctional lesions represent a significant fraction of stable adducts formed in DNA by bifunctional antitumor trans-platinum compounds with planar ligands, we analyzed in the present work short DNA duplexes containing the single, site-specific monofunctional adduct of a representative of this class of platinum drugs, antitumor trans-[PtCl(2)(NH(3))(thiazole)]. It has been shown that, in contrast to the adducts of monodentate chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH(3))(3)]Cl, the monofunctional adduct of trans-[PtCl(2)(NH(3))(thiazole)] inhibits DNA synthesis and creates a local conformational distortion similar to that produced in DNA by the major 1,2-GG intrastrand CL of cisplatin, which is considered the lesion most responsible for its anticancer activity. In addition, the monofunctional adducts of trans-[PtCl(2)(NH(3))(thiazole)] are recognized by HMGB1 domain proteins and removed by the nucleotide excision repair system similarly as the 1,2-GG intrastrand CL of cisplatin. The results of the present work further support the view that the simple chemical modification of the structure of an inactive platinum compound alters its DNA binding mode into that of an active drug and that processing of the monofunctional DNA adducts of the trans-platinum analogues in tumor cells may be similar to that of the major bifunctional adducts of "classical" cisplatin.

Purification of Ethyl Docosahexaenoate through Selective Alcoholysis with Immobilized Rhizomucor miehei Lipase

Ethyl docosahexaenoate (E-DHA) was efficiently enriched by selective alcoholysis of ethyl esters from tuna oil with lauryl alcohol (LauOH) using immobilized Rhizomucor miehei lipase. We thus attempted the development of an enzymatic process applicable to the industrial purification of E-DHA. The amount of LauOH was found the most important factor in the alcoholysis, and a larger amount of LauOH was effective for the enrichment of E-DHA. In this study, the amount was fixed at 7 molar equivalents for ethyl esters originating from tuna oil (E-DHA55; E-DHA content, 54.6mol%). A substrate mixture of E-DHA55/LauOH was introduced into a column packed with 8.0g of immobilized Rhizomucor lipase (22×63mm) at 30°C and a flow rate of 10mL/h (8.3g/h). E-DHA content increased to 87mol% with 58% alcoholysis. Even after 150 d, E-DHA content increased to 85mol%, although alcoholysis decreased to 48%. The half life of the lipase was determined as 150 d based on decrease in alcoholysis in the batch reaction. The reaction mixture flowing from the column was applied to film distillation, and unreacted ethyl esters were recovered in 82% yield. The ethyl ester fraction was contaminated with 2.4wt% LauOH and 6.3wt% lauryl esters, and lauryl esters could be completely removed by urea adduct fractionation. Through a series of purifications, E-DHA content was raised to 88wt% in 52% yield of the initial content in E-DHA55.

FT-IR product study of the photo-oxidation of dimethyl sulfide: Temperature and O2 partial pressure dependence

The products of the OH-radical initiated oxidation of dimethyl sulfur (DMS) have been investigated under NOx free conditions using the photolysis of H2O2 as the OH radical source and FT-IR spectroscopy to monitor reactants and products at 1000 mbar total pressure (N2+O2). The products were investigated as a function of the temperature (284, 295 and 306±2 K) and the O2 partial pressure (20, 200 and 500 mbar). Dimethyl sulfoxide (DMSO), SO2 and methane sulfonic acid (MSA: CH3SO3H) were the major sulfur-containing products and evidence has been found for the formation of methane sulfinic acid (MSIA: CH3S(O)OH) in considerable yield. The variation of the formation of DMSO and SO2 with temperature and O2 partial pressure is consistent with a mechanism involving both addition and abstraction channels. The molar formation yields of DMSO support that a major fraction of the OH-DMS adduct formed in the addition channel reacts with O2 to form DMSO. Due to the uncertainty in the literature rate coefficients used for the corrections in the formation yield of DMSO the possibility of other channels cannot be completely excluded. High overall molar formation yields of SO2 have been measured, suggesting that further oxidation of the products in both the addition and abstraction channels result mainly in SO2 production under the NOx free conditions employed in the experiments. Consideration is made of the implications of the results for the atmospheric oxidation of DMS under conditions of low NOx such as occur in the remote marine boundary layer.

Large‐scale purification of γ‐linolenic acid by selective esterification using Rhizopus delemar lipase

Abstractγ‐Linolenic acid (GLA) is a physiologically valuable fatty acid, and is desired as a medicine, but a useful method available for industrial purification has not been established. Thus, large‐scale purification was attempted by a combination of enzymatic reactions and distillation. An oil containing 45% GLA (GLA45 oil) produced by selective hydrolysis of borage oil was used as a starting material. GLA45 oil was hydrolyzed at 35°C in a mixture containing 33% water and 250 U/g‐reaction mixture of Pseudomonas sp. lipase; 91.5% hydrolysis was attained after 24 h. Film distillation of the dehydrated reaction mixture separated free fatty acids (FFA; acid value 199) with a recovery of 94.5%. The FFA were selectively esterified at 30°C for 16 h with two molar equivalents of lauryl alcohol and 50 U/g of Rhizopus delemar lipase in a mixture containing 20% water. The esterification extent was 52%, and the GLA content in the FFA fraction was raised to 89.5%. FFA and lauryl esters were not separated by film distillation, but the FFA‐rich fraction contaminated with 18% lauryl esters was recovered by simple distillation. To further increase the GLA content, the FFA‐rich fraction was selectively esterified again under similar conditions. As a result, the GLA content in the FFA fraction was raised to 97.3% at 15.2% esterification. After simple distillation of the reaction mixture, lauryl esters contaminating the FFA‐rich fraction were completely eliminated by urea adduct fractionation. When 10 kg of GLA45 oil was used as a starting material, 2.07 kg of FFA with 98.6% GLA was obtained with a recovery of 49.4% of the initial content.

Adduct distributions in piscine DNA: South-Eastern black sea

The south-eastern Black Sea is relatively unpolluted and contains polynuclear aromatic hydrocarbons (PAHs) only of the order of 1 μg 1 −1. Pelagic and benthic fish were monitored during three successive winters from 1993 to 1996 for PAHs and for DNA adducts. The fish caught ( Mugil sp., Mullus barbatus, Alosa fallax, Merlangius merlangus, Sprattus sprattus and Platichthys flesus) varied annually in length, weight and species composition. Although their livers were oily and contained significant concentrations of PAHs (11–56 μg g −1), 32P-postlabelling relatively low levels of hydrophobic DNA .adducts (3–20 per 10 8 nucleotides) in either fish livers or blood. A significant fraction of the DNA adducts in the blood samples, unlike those in the livers, were dephosphorylated by nuclease-P1.