Quantitation of deoxynucleoside triphosphates by click reactions.

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.

New detection method for nucleoside triphosphates based on carbon dots: The distance-dependent singlet oxygen trapping.

The distance-dependent based sensing mechanism, such as fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) absorption of gold nanoparticles, has been used widely in visual detection. In this work, we report another distance-dependent detection method for nucleoside triphosphates (NTPs) based on carbon dots (CDs) (1O2 donor) and 9, 10-diphenylanthracene-2-boronic acid (DABA, 1O2 acceptor). The CDs can generate singlet oxygen (1O2) which allows diffusion within 200 nm. Thus, the distance between CDs and DABA decreased through binding of NTPs (<200 nm), leading to absorption changes of DABA under light irradiation due to 1O2 trapping. This sensing system (CDs@DABA) has high selectivity for the detection of NTPs due to the double molecular recognition and a linear response in the 0-80 µM concentration range was accomplished with the detection limit as low as 4.35 µM.

Development and validation of an LC-MS/MS quantitative method for endogenous deoxynucleoside triphosphates in cellular lysate.

The endogenous deoxynucleoside triphosphate (dNTP) pool includes deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (TTP). The endogenous dNTP pool is regulated by complex enzymatic pathways that can be targeted by drugs, such as antimetabolites. In addition, these components compete with antiviral nucleos(t)ide analog triphosphates, contributing to the mechanism of pharmacologic action. This communication describes the development and validation of a sensitive method to quantify the intracellular dNTP pool in cellular lysates. The extraction process was optimized for dNTPs using an indirect strategy - the separation of mono-, di- and triphosphate moieties by strong anion exchange, dephosphorylation of target fractions to molar equivalent nucleosides - followed by sensitive quantitation using liquid chromatography-tandem mass spectrometry. The validated analytical range was 50-2500 fmol/sample for each dNTP. The assay was used to quantify dNTPs in peripheral blood mononuclear cells from 40 clinical research participants (n = 279 samples). Median (interquartile range) concentrations were 143 (116, 169) for dATP, 737 (605, 887) for dCTP, 237 (200, 290) for dGTP and 315 (220, 456) for TTP, in femtomoles per million cells. This method allows for future studies of endogenous dNTP disposition in subjects receiving antimetabolites or nucleos(t)ide analogs, or for other clinical scenarios.

Increased deoxythymidine triphosphate levels is a feature of relative cognitive decline.

Mitochondrial bioenergetics, mitochondrial reactive oxygen species (ROS) and cellular levels of nucleotides have been hypothesized as early indicators of Alzheimer's disease (AD). Utilizing relative decline of cognitive ability as a predictor of AD risk, we evaluated the correlation between change of cognitive ability and mitochondrial bioenergetics, ROS and cellular levels of deoxyribonucleotides. Change of cognitive abilities, scored at ages of approximately 20 and 57 was determined for a cohort of 1985 male participants. Mitochondrial bioenergetics, mitochondrial ROS and whole-cell levels of deoxyribonucleotide triphosphates were measured in peripheral blood mononuclear cells (PBMCs) from a total of 103 selected participants displaying the most pronounced relative cognitive decline and relative cognitive improvement. We show that relative cognitive decline is associated with higher PBMC content of deoxythymidine-triphosphate (dTTP) (20%), but not mitochondrial bioenergetics parameters measured in this study or mitochondrial ROS. Levels of dTTP in PBMCs are indicators of relative cognitive change suggesting a role of deoxyribonucleotides in the etiology of AD.

A new strategy for the detection of adenosine triphosphate by aptamer/quantum dot biosensor based on chemiluminescence resonance energy transfer.

We designed an aptasensor for the detection of adenosine triphosphate (ATP) based on chemiluminescence resonance energy transfer (CRET). An adenosine aptamer was cut into two pieces of ssDNA, which were attached to quantum dots (QDs) and horse radish peroxidase (HRP), respectively. They could reassemble into specific structures in the presence of ATP and then decrease the distance of HRP and QDs. ATP detection can be easily realized according to the fluorescent intensity of QDs, which is excited by CRET between luminol and QDs. Results show that the concentration of ATP is linear relation with the fluorescent intensity of the peak of QDs emission and the linear range for the linear equation is from 50 µM to 231 µM and the detection limit was 185 nM. When the concentration of ATP was 2 mM, the efficiency of CRET is 13.6%. Good specificity for ATP had been demonstrated compared to thymidine triphosphate (TTP), cytidine triphosphate (CTP) and guanosine triphosphate (GTP), when 1 mM of each was added, respectively. This method needs no external light source and can avoid autofluorescence and photobleaching, and ATP can be detected selectively, specifically, and sensitively in a low micromolar range, which means that the strategy reported here can be applicable to the detection of several other target molecules.

Acycloguanosyl 5'-thymidyltriphosphate, a thymidine analogue prodrug activated by telomerase, reduces pancreatic tumor growth in mice.

BACKGROUND & AIMS: Gemcitabine is the standard of care for metastatic and nonresectable pancreatic tumors. Phase II and III trials have not demonstrated efficacy of recently developed reagents, compared with gemcitabine alone; new chemotherapic agents are needed. Ninety percent of pancreatic tumors have telomerase activity, and expression correlates with tumor stage. We developed a thymidine analogue prodrug, acycloguanosyl 5'-thymidyltriphosphate (ACV-TP-T), that is metabolized by telomerase and releases the active form of acyclovir. We investigated the antitumor efficacy of ACV-TP-T in vitro and in vivo. METHODS: We evaluated proliferation and apoptosis of human pancreatic cancer cells (PANC-1, MiaPaca2, BxPc3, PL45, and Su.86.86) incubated with ACV-TP-T. The presence of ACV-TP-T and its metabolite inside the cells were analyzed by mass spectrometry. In vivo efficacy was evaluated in nude mice carrying PANC-1 or MiaPaca2 pancreatic xenograft tumors. RESULTS: The prodrug of ACV-TP-T was actively metabolized inside pancreatic cancer cells into the activated form of acyclovir; proliferation was reduced, apoptosis was increased, and the cell cycle was altered in pancreatic cancer incubated with ACV-TP-T, compared with controls. Administration of ACV-TP-T to mice reduced growth, increased apoptosis, and reduced proliferation and vascularization of pancreatic xenograft tumors. CONCLUSIONS: ACV-TP-T, a thymidine analogue that is metabolized by telomerase and releases the active form of acyclovir, reduces proliferation and induces apoptosis of human pancreatic cancer cell lines in vitro and pancreatic xenograft tumors in mice.

Decomposition of 3'-azido-2',3'-dideoxythymidine 5'-monophosphate (AZTMP) prodrugs in biological media studied by on-line solid-phase extraction coupled to liquid chromatography mass spectrometry.

Using a column-switching HPLC method previously described, we studied the behavior of some mononucleotide prodrugs (pronucleotides) of 3'-azido-2',3'-dideoxythymidine in various biological media. From UV data, this method allowed quantification of transient metabolites resulting from prodrug bioconversion. The kinetic data related to the successive steps were calculated according to pseudo-first-order kinetic models and optimized using mono- or poly-exponential regressions. Various metabolites were identified by co-injection with authentic samples and/or ESI-MS coupling. The results led us to propose, for each considered pronucleotide, a global decomposition pathway ending in the selective delivery of the corresponding mononucleotide. Associated to the determination of other parameters (lipophilicity, aqueous solubility), the present study contributes to the search of suitable pharmacological properties for further in vivo evaluations.

Methylenetetrahydrofolate reductase deficiency and low dietary folate reduce tumorigenesis in Apc min/+ mice.

BACKGROUND: Clinical studies suggest that mild methylenetetrahydrofolate reductase (MTHFR) deficiency and high dietary folate may reduce the risk for colorectal cancer. There is concern, however, that high folate intake (a consequence of food fortification) may enhance tumour growth in individuals with pre-existing tumours or genetic predisposition to tumorigenesis. AIM: To determine if Mthfr deficiency and low dietary folate influence tumorigenesis in mice genetically predisposed to form numerous intestinal adenomas (Apc(min/+)). METHODS: Male Apc(min/+) mice were mated with Mthfr(+/-) and/or Mthfr(+/+) females. Diets with variable folate content were administered either pre-natally or at weaning; tumours were counted in offspring at 10 weeks of age. Plasma homocysteine and levels of apoptosis, DNA methylation and nucleotide ratios (dUTP:dTTP) in normal (pre-neoplastic) intestine were measured. RESULTS: Apc(min/+) mice fed high folate diets from weaning developed more adenomas than those fed the folic acid-deficient diet (FADD) or the control diet (CD); Mthfr deficiency did not affect adenoma number. However, when the FADD and CD were administered to dams prior to conception, throughout pregnancy and continued in offspring post-weaning, Apc(min/+) offspring fed FADD developed fewer adenomas than those fed CD. Mthfr(+/-) genotype of the mother or of the offspring also reduced adenoma numbers in the Apc(min/+) offspring. Adenoma number was inversely correlated with plasma homocysteine (r = -0.49, p<0.005, intestinal dUTP/dTTP ratios (r = -0.42, p = 0.05), and levels of intestinal apoptosis (r = -0.36, p = 0.08). CONCLUSIONS: Low dietary folate and Mthfr deficiency reduce adenoma formation in mice predisposed to tumorigenesis, possibly through increased apoptosis consequent to hyperhomocysteinaemia and nucleotide imbalances.

Depletion of dimeric all-alpha dUTPase induces DNA strand breaks and impairs cell cycle progression in Trypanosoma brucei.

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is responsible for the control of intracellular levels of dUTP thus controlling the incorporation of uracil into DNA during replication. Trypanosomes and certain eubacteria contain a dimeric dUTP-dUDPase belonging to the recently described superfamily of all-alpha NTP pyrophosphatases which bears no resemblance with typical eukaryotic trimeric dUTPases and presents unique properties regarding substrate specificity and product inhibition. While the biological trimeric enzymes have been studied in detail and the human enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies, little is known regarding the biological function of dimeric proteins. Here, we show that in Trypanosoma brucei, the dimeric dUTPase is a nuclear enzyme and that down-regulation of activity by RNAi greatly reduces cell proliferation and increases the intracellular levels of dUTP. Defects in growth could be partially reverted by the addition of exogenous thymidine. dUTPase-depleted cells presented hypersensitivity to methotrexate, a drug that increases the intracellular pools of dUTP, and enhanced uracil-DNA glycosylase activity, the first step in base excision repair. The knockdown of activity produces numerous DNA strand breaks and defects in both S and G2/M progression. Multiple parasites with a single enlarged nucleus were visualized together with an enhanced population of anucleated cells. We conclude that dimeric dUTPases are strongly involved in the control of dUTP incorporation and that adequate levels of enzyme are indispensable for efficient cell cycle progression and DNA replication.

DNA polymerase kappa produces interrupted mutations and displays polar pausing within mononucleotide microsatellite sequences.

Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T](11) microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol kappa) inserts dGMP and dCMP within the [T](11) mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase beta produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol kappa and polymerase alpha-primase within a [T](11) allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.

Quantification of zidovudine and its monophosphate in cell extracts by on-line solid-phase extraction coupled to liquid chromatography.

A simple and rapid analytical method for the simultaneous quantification of zidovudine (AZT) and its monophosphate (AZTMP) in cell extracts has been developed using high-performance liquid chromatography (HPLC) with on-line solid-phase extraction and 2-aminoethyl-3'-azido-2',3'-dideoxythymidin-5'-yl phosphodiester sodium salt as internal standard (IS). The cell extract samples were directly injected on a short reversed-phase precolumn using an aqueous buffer containing an ion-pairing reagent as a mobile phase. Under these conditions, the analytes were retained on the precolumn whereas the proteins were discarded. The analytes were then transferred onto the analytical column by increasing the strength of the eluent. The calibration curve was linear over a concentration range of 0.5-100 microg/ml. Inter- and intra-day accuracy and precision results satisfied the accepted criteria for bioanalytical validation. This method was used to study the decomposition pathway of a model pronucleotide in an in vitro approach.

Concentrations of zidovudine- and lamivudine-triphosphate according to cell type in HIV-seronegative adults.

INTRODUCTION: Concentrations of zidovudine (ZDV)- and lamivudine (3TC)-triphosphates (TP) have been quantified in unfractionated peripheral blood mononuclear cells (PBMC) from HIV+ patients. The objective of this study was to determine whether concentrations of ZDV-TP and 3TC-TP in PBMC reflect the concentrations within CD4 T cells in HIV-seronegative adults. METHODS: Volunteers had taken 300 mg of ZDV plus 150 mg of 3TC twice daily for > or = 7 days. Blood (60 mL) was collected 2 or 5 h post observed dose. PBMC were processed into three cell fractions using CD4 magnetic immunobeads: CD4-purified cells; unfractionated PBMC; and CD4-depleted PBMC. TP were determined in each cell fraction with liquid chromatography-mass spectrometry and compared across cell types by non-parametric analyses. RESULTS: Six males and two females participated. The median (range) percentage of CD4 T cells (CD4%) in each fraction were: CD4-purified, 99%; unfractionated, 63% (range, 53-70); and CD4-depleted, 14% (range, 4-29). Corresponding median (range) ZDV-TP concentrations were 8.0 (5.3-10.3), 26.5 (12.9-42.2), and 34.2 (16.4-52.2) fmol/1 x 10 cells (Friedman P = 0.0008). The 3TC-TP values were 4.6 (2.3-6.7), 4.8 (3.5-8.8), and 6.8 (4.0-13.1) pmol//1 x 10 cells (Friedman P = 0.01). In mixed model analyses: ZDV-TP (fmol/1 x 10 cells) = 42-0.32 (CD4%); P < 0.001 and 3TC-TP (pmol/1 x 10 cells) = 7.3-0.03(CD4%); P = 0.003. CONCLUSIONS: In HIV-seronegative volunteers, 3TC-TP concentrations in PBMC reflected the concentrations within CD4 T cells, but ZDV-TP concentrations were more than 70% lower in CD4 T cells than in PBMC. Thus, TP concentrations differ according to cell type in vivo with corresponding efficacy and toxicity implications for cells with low or high triphosphates.

Separation of nucleotides with an aqueous mobile phase using pH- and temperature-responsive polymer modified packing materials.

A new method for the qualitative analysis of adenosine nucleotides (AMP, ADP, and ATP) and synthetic oligonucleotides has been proposed, utilizing a pH- and temperature-responsive polymer of N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and N,N-dimethylaminopropylacrylamide (DMAPAAm) as the stationary phase of HPLC. In the chromatographic system using the copolymer with ionizable groups of modified packing materials, we investigated how to separate adenosine nucleotides and oligonucleotides by temperature. The properties of the surface of the copolymer-grafted stationary phase altered from hydrophilic to hydrophobic and from charged to non-charged due to changes in the temperature and in the pH, respectively. In addition, it is possible to exhibit and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena result from changes in the charge and hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface with the controlling temperature. A pH- and temperature-responsive chromatography would be greatly useful for biopolymer and nucleotide separation and purification.

Analysis and validation of the phosphorylated metabolites of two anti-human immunodeficiency virus nucleotides (stavudine and didanosine) by pressure-assisted CE-ESI-MS/MS in cell extracts: sensitivity enhancement by the use of perfluorinated acids and alcohols as coaxial sheath-liquid make-up constituents.

A CE method utilizing triple quadrupole electrospray (ES) MS (MS/MS) detection was developed and validated for the simultaneous measurement of nucleoside 5'-triphosphate and 5'-monophosphate anabolites of the anti-HIV (human immunodeficiency virus) didanosine (ddAMP, ddATP) and stavudine (d4TMP, d4TTP), among a pool of 14 endogenous 5'-mono-, di-, and triphosphate nucleosides. These compounds were spiked and extracted from peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. An acetic acid/ammonia buffer (pH 10, ionic strength of 40 mM) was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of +30 kV and an overimposed pressure of 28 mbar (0.4 psi). The application of pressure assistance was needed to provide stable ES conditions for successful coupling. The coupling was carried out with a modified sheath-flow interface, with one uninterrupted capillary (80 cmx 50 microm id; 192 microm od) in a dimension that fits into the ESI needle to get a stable ion spray. Some CE-MS parameters such as overimposed pressure, sheath-liquid composition, sheath-liquid and sheath-gas flow rates, ES voltage, and the CE capillary position were optimized in order to obtain an optimal sensitivity. The use of perfluorinated alcohols and acids in the coaxial sheath-liquid make-up (2,2,2-trifluoroethanol + 0.2 mM tridecafluoroheptanoic acid) appeared to provide the best MS sensitivity and improve the stability of spray. The linearity of the CE-MS and CE-MS/MS methods was checked under these conditions. Validation parameters such as accuracy, intraday and interday precision, and LOQs were determined in CE-MS/MS mode. Finally, the quantitation of d4T-TP and ddA-TP was validated in this CE-MS/MS system.

Simultaneous determination of thymidylate and thymidine diphosphate by capillary electrophoresis as a rapid monitoring tool for thymidine kinase and thymidylate kinase activities.

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of thymidylate (TMP) and thymidine 5'-diphosphate (TDP) in enzyme assays without using radioactive-labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. The separation of micromolar TMP and TDP from millimolar adenosine 5'-triphosphate (ATP) was performed at 25 degrees C using sodium tetraborate as the background electrolyte. Under the optimal condition, a good separation with high efficiency was achieved in 6 min. Several parameters affecting the separation were studied, including the pH of electrolyte, the applied voltage, and acetonitrile-salt sample stacking. The fronting of the ATP peak resulting from the interference of magnesium ion in the enzyme assay buffer was suppressed by the addition of sodium ethylenediaminetetraacetate to the sample solution. Using deoxyadenylate as an internal standard, the linear range of the method was 5-200 microM, and the concentration limits of detection of TMP and TDP were 2.6 and 3.8 microM, respectively. Application of the proposed method for simultaneous determination of TMP and TDP in enzyme assays was demonstrated by the activity assays of thymidine kinase and thymidylate kinase from white spot syndrome virus. This is a sensitive, nonradioactive method for thymidine kinase and thymidylate kinase assays.

Simultaneous quantitation of zidovudine and zidovudine monophosphate from plasma, amniotic fluid and tissues by micellar capillary electrophoresis.

Zidovudine (AZT) therapy given during pregnancy has been shown to reduce the vertical transmission of the human immunodeficiency virus (HIV) from mother to fetus. In order to investigate the efficacy of AZT, it is important to know the concentration of its active phosphorylated metabolites. We have developed the first CE method for the simultaneous quantitation of AZT and zidovudine monophosphate (AZT-MP) from rat plasma, amniotic fluid and fetal tissues. Sample extractions were performed by protein precipitation using acetonitrile for the plasma and amniotic fluids, while in fetal tissues solid phase extraction using Waters Oasis HLB extraction cartridges was used. Recoveries ranged from 78 to 92% for AZT, AZT-MP and 3'-azidouridine (internal standard, AZDU), in the three matrices. The optimum separation conditions were achieved using a 40 mm sodium dodecylsulfate (SDS) in 50 mm phosphate buffer (pH 7) with a run voltage of 15 kV. The CE system consists of a 75 microm i.d., 50 cm effective length uncoated fused silica capillary. The method was validated over the range 0.5-100 microg/ml ( micro g/g for tissues). Intra-day precision (RSD) and accuracy (%error) for AZT ranged from 0.13 to 11 and 0.68 to 11.1%, respectively, while for AZT-MP it ranged from 2.05 to 11.1 and 4.22 to 11.7%. Inter-day precision and accuracy for AZT ranged from 3.82 to 11.2 and 3.14 to 9.01%, while for AZT-MP it ranged from 3.9 to 9.32 and 3.44 to 9.37%, respectively. We also report the enzymatic dephosphorylation of AZT-MP in the placental tissue of rats. This new enzymatic pathway provides increased understanding of the mechanism of anti-viral transport in the rat during pregnancy.

Intracellular metabolism and pharmacokinetics of 5'-hydrogenphosphonate of 3'-azido-2',3'-dideoxythymidine, a prodrug of 3'-azido-2',3'-dideoxythymidine.

5'-Hydrogenphosphonate of 3'-azido-2',3'-dideoxythymidine (HpAZT), a novel anti-HIV drug approved for the treatment of HIV-infected patients in Russia, displays some clinical advantages over azidothymidine (AZT). Metabolism in the HL-60 cell culture and pharmacokinetics in mice of [6-3H]-HpAZT (in comparison with [6-3H-AZT) were studied to elucidate the metabolic basis of its lower clinical toxicity. Accumulation of [6-3H]-HpAZT-derived products in cells with time, distribution of its radioactive metabolites among blood and different mouse organs and dependence of drug accumulation on the route of administration were investigated. The rate of accumulation of [3H]-HpAZT metabolites in cells was slower than the rate of accumulation of [3H]-AZT metabolites. [3H]-AZTMP was the dominating metabolite at all time points, achieving the level of 15 +/- 3 pmol/10(6) cells after 25 h incubation. After oral or intravenous administrations of [3H]-HpAZT, the (radioactive) metabolites were rapidly distributed among blood, stomach, intestine and liver and were not found in brain, muscles and spleen. [3H]-HpAZT underwent rapid and extensive metabolism, [3H]-AZT being the dominating product at all time points. Administration of 180 nmol of [3H]-HpAZT resulted in an AZT concentration in blood of 1-3 microM after 5 min, which remained practically constant during the next 25 min and did not depend on the route of administration.

Characterization of restrictions to human immunodeficiency virus type 1 infection of monocytes.

Tissue macrophages are an important cellular reservoir for replication of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus. In vitro, the ability of macrophages to support viral replication is differentiation dependent in that precursor monocytes are refractory to infection. There is, however, no consensus as to the exact point at which infection is restricted in monocytes. We have revisited this issue and have compared the efficiencies of early HIV-1 replication events in monocytes and in differentiated macrophages. Although virus entry in monocytes was comparable to that in differentiated macrophages, synthesis of full-length viral cDNAs was very inefficient. Relative to differentiated macrophages, monocytes contained low levels of dTTP due to low thymidine phosphorylase activity. Exogenous addition of D-thymidine increased dTTP levels to that in differentiated macrophages but did not correct the reverse transcription defect. These results point to a restriction in monocytes that is independent of reverse transcription precursors and suggest that differentiation-dependent cellular cofactors of reverse transcription are rate limiting in monocytes.

A new method for analysis of AZT-triphosphate and nucleotide-triphosphates.

We have developed a new method based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for analysis of zidovudine-triphosphate and (deoxy)nucleotide-triphosphates, which ultimately can be used for nucleoside reverse transcriptase inhibitor (NRTI) treatment monitoring in HIV-1 infected children and adults. Four different matrices were compared for sensitivity and reproducibility of zidovudine-triphosphate detection and anthranilic acid mixed with nicotinic acid (AA/NA) was selected as most suitable matrix. Solutions of zidovudine-triphosphate, ATP, and dGTP were detected up to 0.5fmol per sample. Furthermore, intracellular zidovudine-triphosphate, ATP, and dGTP were detected in peripheral blood mononuclear cells (PBMCs). Zidovudine-triphosphate, ATP, and dGTP yield identical mass spectra, however MALDI-TOF post-source decay analysis can be used for discrimination between these compounds. We conclude that this method based on MALDI-TOF MS can be used for analysis of intracellular zidovudine-triphosphate and (deoxy)nucleotide-triphosphates in PBMCs.

Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli. Purification and partial characterization of its dimeric and tetrameric forms.

Human thymidine kinase 2 (hTK2) phosphorylates pyrimidine deoxyribonucleosides to the corresponding nucleoside monophosphates, using a nucleotide triphosphate as a phosphate donor. In this study, hTK2 was cloned and expressed at high levels in Escherichia coli as a fusion protein with maltose-binding protein. Induction of a heat-shock response by ethanol and coexpression of plasmid-encoded GroEL/ES chaperonins at 28 degrees C minimized the nonspecific aggregation of the hybrid protein and improved the recovery of three homooligomeric forms of the properly folded enzyme, i.e., dimer > tetramer > hexamer. The dimer and the tetramer were isolated in stable and highly purified forms after proteolytic removal of the fusion partner. Both oligomers contained a substoichiometric amount of deoxyribonucleotide triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Steady-state kinetic studies were consistent with the presence of endogenous inhibitors, and both oligomeric forms revealed a lag phase of at least approximately 5 min, which was abolished on preincubation with substrate (dThd or dCyd). The rather similar kinetic properties of the two oligomeric forms indicate that the basic functional unit is a dimer. Molecular docking experiments with a modeled hTK2 three-dimensional structure accurately predicted the binding positions at the active site of the natural substrates (dThd, dCyd, and ATP) and inhibitors (dTTP and dCTP), with highly conserved orientations obtained for all ligands. The calculated relative nonbonded interaction energies are in agreement with the biochemical data and show that the inhibitor complexes have lower stabilization energies (higher affinity) than the substrates.