Simultaneous Determination of Deoxyribonucleoside in the Presence of Ribonucleoside Triphosphates in Human Carcinoma Cells by High-Performance Liquid Chromatography

Décosterd, Laurent A.; Cottin, E; Chen, X.; Lejeune, Ferdy J.; Mirimanoff, RenéO.; Biollaz, J; Coucke, Philippe

doi:10.1006/abio.1999.4066

Cited by 45 publications

(44 citation statements)

References 27 publications

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“…This suggested that the dNTP content measurement with the dNTP samples extracted from cells should be performed within the linear dNTP ranges (4 -64 fmol of dNTP or 2-32% of primer extension). The minimum detection of the dNTP content in this HIV-1 RT-based assay (4 fmol) is 25-and 500-fold more sensitive than the KF (0.1 pmol) (21) and high pressure liquid chromatography-based assays (Ͼ2 pmol) (25)(26)(27)(28), respectively, that have been used previously for dNTP content measurement.…”

Section: Determination Of Dntp Levels In Hiv-1 Target Cells Humanmentioning

confidence: 94%

Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

Diamond

Roshal

Jamburuthugoda³

et al. 2004

Journal of Biological Chemistry

273

423

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Section: Determination Of Dntp Levels In Hiv-1 Target Cells Humanmentioning

confidence: 94%

Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

Diamond

Roshal

Jamburuthugoda³

et al. 2004

Journal of Biological Chemistry

273

423

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“…Aliquots (10 l) were injected onto a 4.6 ϫ 250 mm Zorbax 300SB-C18 5-m column (Hewlett Packard) at 30°C. Chromatography conditions were as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

Cloning, Expression, and Characterization of a Human Inosine Triphosphate Pyrophosphatase Encoded by the ITPAGene

Lin

McLennan

Kong

et al. 2001

Journal of Biological Chemistry

129

156

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ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC 3.6.1.19). Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before. At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low. K m values for ITP, dITP, and xanthosine 5-triphosphate were 0.51, 0.31, and 0.57 mM, respectively, and k cat values were 580, 360, and 640 s ؊1 , respectively. A divalent cation was absolutely required for activity. The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized. A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals. The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.

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“…Table 3 shows the concentration of Investigation of energy charge changes in cerebral ischemia 633 ORIGINAL RESEARCH acids. Tetrabutylammonium (Werner, 1993;Decosterd et al, 1999;Di Pierro et al, 1995;Claire, 2000) and triethylammonium (Uesugi et al, 1997;Reichelova et al, 1996) are the cationic ion species commonly used to form ion-pair complexes. The neutralized sample was stable at room temperature for at least 48 h. The choice of extraction procedure for the HPLC of adenine nucleotides from mouse cerebral tissue and Neuro-2a cells is important for accurate quantification.…”

Section: Analysis Of Samplesmentioning

confidence: 99%

Development of an ion‐pair HPLC method for investigation of energy charge changes in cerebral ischemia of mice and hypoxia of Neuro‐2a cell line

Chen

Xing

Wang

et al. 2007

Biomedical Chromatography

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The determination of adenine nucleotides and energy charge (EC) has great importance in the characterization of cerebral ischemic injury and post-ischemic recovery. An IP-HPLC method was developed for the quantification of AMP, ADP, ATP and EC in cerebral ischemia and hypoxia of the Neuro-2a cell line. The chromatographic conditions were: a Zorbax SB-C18 reversed-phase column; mobile phase 100 mM KH(2)PO(4), 1 mM tetrabutylammonium hydroxide, and 2.5% acetonitrile, brought to pH 7.0 with potassium hydroxide (4 M), filtered through a 0.45 microm Millipore filter and degassed prior to use. The flow-rate was 1.0 mL/min. The injection volume was 20 microL. Detection was performed at a wavelength of 254 nm under a constant temperature (27 +/- 1 degrees C). The method was validated by means of linearity, using calibration curves constructed with five concentration levels of each compound. The limit of detection was also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. Cerebral tissue was homogenized (4 degrees C) in 1 mL of an ice-cold 6% trichloroacetic acid that contained ATPase inhibitor and obtained good recovery (>90%). The results show that the described method for the determination of adenine nucleotides by HPLC has good linearity, limit of detection, precision and specificity, and is simple and rapid to perform.

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Simultaneous Determination of Deoxyribonucleoside in the Presence of Ribonucleoside Triphosphates in Human Carcinoma Cells by High-Performance Liquid Chromatography

Abstract: Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in cells by HPLC is an

Cited by 45 publications

References 27 publications

Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

Cloning, Expression, and Characterization of a Human Inosine Triphosphate Pyrophosphatase Encoded by the ITPAGene

Development of an ion‐pair HPLC method for investigation of energy charge changes in cerebral ischemia of mice and hypoxia of Neuro‐2a cell line

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