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- Enmark, Martin, 1984-, et al.
(author)
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Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides
- 2019
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In: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 411:15, s. 3383-3394
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Journal article (peer-reviewed)abstract
- This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.
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| 2. |
- Haseeb, Abdul, et al.
(author)
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Method development for the acquisition of adsorption isotherm of ion pair reagents Tributylamine and Triethylamine in ion pair chromatography
- 2023
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In: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1687
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Journal article (peer-reviewed)abstract
- Tributylamine (TBuA) and triethylamine (TEtA) are the most commonly used ion pair reagents in ion pair chromatography especially for the analysis of oligonucleotides. In order to improve the understanding of the retention and separation mechanism of oligonucleotides in ion pair chromatography, it is important to understand the retention mechanism and the nature of interaction of these ion pair reagents with the stationary phase in the chromatographic column. Adsorption isotherm is helpful in evaluating such interactions, and subsequently predicting the retention mechanism. Alkylamines are very polar molecules which lack suitable chromophore and are commonly present in charged forms. Therefore, their determination and the subsequent acquisition of their adsorption isotherms using traditional liquid chromatography is very difficult. In this study, we first developed an analytical method for the determination of TBuA and TEtA in a typical chromatographic mobile phase (acetonitrile-water) and then used the same method to acquire the adsorption isotherms for tributylammonium acetate (TBuAA) and triethylammonium acetate (TEtAA). This method started with the conversion of the alkylammonium ions to free neutral forms by treating the sample with a strong base, followed by pentane-mediated extraction and finally the analysis of the extracts using gas chromatography-flame ionization detector (GC-FID). This three-step method was validated for parameters like range, linearity, intra-day and inter-day precision and accuracy, limit of detection and limit of quantitation. For the adsorption isotherms, the C18 column was first equilibrated with the solutions having different concentrations of alkylammonium ions and then stripped with eluent devoid of alkylammonium ions. Several stripping eluents were investigated and it was discovered that the eluent requirement could be decreased by the addition of sodium chloride. The effluents from the stripping phase were collected and analyzed using the developed analytical method to acquire the adsorption data. Under the investigated conditions, adsorption of TBuAA and TEtAA showed type III and type I isotherm behavior respectively.
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| 6. |
- Hellberg Lindqvist, Miriam, 1982-, et al.
(author)
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Expression of Chlorite Dismutase and Chlorate Reductase in the Prescence of Oxygen and/or Chlorate as the Terminal Electron Acceptor in Ideonella dechloratans
- 2012
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In: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 78:12, s. 4380-4385
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Journal article (peer-reviewed)abstract
- The ability of microorganisms to perform dissimilatory (per)chlorate reduction is, for most species, known to be oxygen sensitive. Consequently, bioremediation processes for the removal of oxochlorates will be disturbed if oxygen is present. We measured the expression of chlorite dismutase and chlorate reductase in the presence of different terminal electron acceptors in the chlorate reducer Ideonella dechloratans. Enzyme activity assays and mRNA analyses by real-time quantitative reverse transcription (qRT)-PCR were performed on cell extracts from cells grown aerobically with and without chlorate and on cells grown anaerobically in the presence of chlorate. Our results showed that both chlorite dismutase and chlorate reductase are expressed during aerobic growth. However, transfer to anaerobic conditions with chlorate resulted in significantly enhanced enzyme activities and mRNA levels for both enzymes. Absence of oxygen was necessary for the induction to occur, since chlorate addition under aerobic conditions produced neither increased enzyme activities nor higher relative levels of mRNA. For chlorite dismutase, the observed increase in activity was on the same order of magnitude as the increase in the relative mRNA level, indicating gene regulation at the transcriptional level. However, chlorate reductase showed about 200 times higher enzyme activity in anaerobically induced cells, whereas the increase in mRNA was only about 10-fold, suggesting additional mechanisms influence the enzyme activity.
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| 7. |
- Hellberg Lindqvist, Miriam, 1982-, et al.
(author)
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Expression of the gene cluster for chlorate metabolism in the chlorate-respiring bacterium Ideonella dechloratans
- 2012
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In: Biochimica et Biophysica Acta - Bioenergetics. - Frankfurt : Elsevier. - 0005-2728 .- 1879-2650. ; 1817, s. 157-158, s. S157-S158
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Journal article (peer-reviewed)abstract
- Ideonella dechloratans is a facultative anaerobe able to use chlorate as a terminal electron acceptor under anaerobic conditions. Two enzymes are necessary for the decomposition of chlorate to chloride and molecular oxygen; chlorate reductase (Clr) and chlorite dismutase (Cld). The genes for these two enzymes are close to each other in the genome and form, together with a cytochrome c and a mob B gene, a gene cluster for chlorate metabolism. The localization of the cyt c gene suggests a function in electron transport during chlorate reduction but the corresponding protein has not been found. We have addressed the questions of how the expression of Cld and Clr is regulated during the aerob/anaerob switch and if the cyt c gene is expressed in I. dechloratans. The enzyme activities of Cld and Clr were measured in extracts from cells grown at different conditions; aerobically or anaerobically [1]. Both enzymes were found to be active in all samples and the activity increased upon transfer of the cells from aerobic to anaerobic conditions, by five times for Cld and more than 200 times for Clr. Relative mRNA levels of Cld and Clr were determined by qRT-PCR in RNA preparations from cells grown under the same conditions as for the enzyme activity measurements. mRNA from both genes was detected in all preparations but with ten times higher levels in samples from anaerobic conditions. This increase in mRNA level is on the same scale as the increase in enzyme activity for Cld but accounts for less than a tenth of the activity enhancement seen for Clr. A possible effect of chlorate was tested by the addition of chlorate under aerobic conditions but this resulted in neither increased enzyme activities nor increased mRNA levels. qRT-PCR was performed with primers specific for the cyt c gene and this gene was also found to be expressed at both aerobic and anaerobic conditions. In summary, the results show that chlorate respiration is activated by anaerobiosis but not by chlorate in I. dechloratans and that this activation occurs at the transcriptional level. Due to the much larger increase in enzyme activity compared to the increase in mRNA level, the activity of Clr also seems to be effected by other mechanisms. Detection of cyt c mRNA suggests that its gene product can be found and the function investigated.
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| 8. |
- Nilsson, Thomas, 1955-, et al.
(author)
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Microbial metabolism of oxochlorates : A bioenergetic perspective
- 2013
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In: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier. - 0005-2728 .- 1879-2650. ; 1827:2, s. 189-197
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Research review (peer-reviewed)abstract
- The microbial metabolism of oxochlorates is part of the biogeochemical cycle of chlorine. Organisms capable of growth using perchlorate or chlorate as respiratory electron acceptors are also interesting for applications in biotreatment of oxochlorate-containing effluents or bioremediation of contaminated areas. In this review, we discuss the reactions of oxochlorate respiration, the corresponding enzymes, and the relation to respiratory electron transport that can contribute to a proton gradient across the cell membrane. Enzymes specific for oxochlorate respiration are oxochlorate reductases and chlorite dismutase. The former belong to DMSO reductase family of molybdenum-containing enzymes. The heme protein chlorite dismutase, which decomposes chlorite into chloride and molecular oxygen, is only distantly related to other proteins with known functions. Pathways for electron transport may be different in perchlorate and chlorate reducers, but appear in both cases to be similar to pathways found in other respiratory systems.
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