A AuNP-capped cage fluorescent biosensor based on controlled-release and cyclic enzymatic amplification for ultrasensitive detection of ATP.

Gold nanodevices have attracted extensive interest in the detection of specific targets within cells. However, constructing gold sensing devices that can be activated by the simulation of remote applications remains a huge challenge. Here, we report a Au nanoparticle (AuNP)-capped cage fluorescent biosensor based on controlled-release and Exonuclease III (Exo III) assisted cyclic enzymatic amplification that can be activated by adenosine triphosphate (ATP). In the system, AuNPs were used as the building blocks to cap the pores of Au nanocages (AuNCs) loaded with Rhodamine B (RhB) molecules through the hybridization of DNA. The RhB fluorescent molecules were finally released with the help of Exo III in the presence of ATP for detection purposes. Ultimately, the biosensor leads to a wide linear ATP detection range from 1.0 × 10-9 to 1.0 × 10-7 M with a limit of detection (LOD) down to 0.88 nM. In addition, it also has good selectivity for ATP to distinguish between ATP and ATP analogues such as cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP). Therefore, as a convenient and sensitive biosensor, it is expected to be widely used in the biomedical field.

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.

Target-triggered transcription machinery for ultra-selective and sensitive fluorescence detection of nucleoside triphosphates in one minute.

Nucleoside triphosphates (NTPs) play important roles in living organisms. However, no fluorescent assays are currently available to simply and rapidly detect multiple NTPs with satisfactory selectivity, sensitivity and low cost. Here we demonstrate for the first time a target-triggered in-vitro transcription machinery for ultra-selective, sensitive and instant fluorescence detection of multiple NTPs. The machinery assembles RNA polymerase, DNA template and non-target NTPs to convert the target NTP into equivalent RNA signal sequences which are monitored by the fluorescence enhancement of molecular beacon. The machinery offers excellent selectivity for the target NTP against NDP, NMP and dNTP. Notably, to accelerate the kinetics of the machinery while maintain its high specificity, we investigated the sequence of DNA templates systematically and established a set of guidelines for the design of the optimum DNA templates, which allowed for instant detection of the target NTP at fmol level in less than 1min. Furthermore, the machinery could be transformed into logic gates to study the coeffects of two NTPs in biosynthesis and real-time monitoring systems to reflect the distribution of NTP in nucleotide pools. These results provide very useful and low-cost tools for both biochemical tests and point-of-care analysis.

Novel one-pot ATP regeneration system based on three-enzyme cascade for industrial CTP production.

OBJECTIVES: To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production. RESULTS: We developed a new one-pot PPK system by applying a three-enzyme cascade (CMK, NDK and PPK) with an in vitro polyP-based ATP regeneration system. The PPK was selected from twenty sources, and was made solvable by fusion expressing with soluble protein and constructing polycistronic plasmids, or co-expressing with molecular chaperones GroES/EL. Activities of other enzymes were optimized by employing fusion expression, tac-pBAD system, Rosetta host and codon optimization. After 24 h, the concentration of CDP and CTP reached 3.8 ± 0.2 and 6.9 ± 0.3 mM l-1 respectively with a yield of approximately 79%. The molar conversion rate of CTP was 51%, and its yield and conversion rate increased 100% from the traditional system. CONCLUSIONS: A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.

Simultaneous quantification of energetically important metabolites in various cell types by CZE.

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.

A terbium(III)-organic framework for highly selective sensing of cytidine triphosphate.

Highly selective sensing of cytidine triphosphate (CTP) against other triphosphate nucleosides including ATP, GTP and UTP is successfully achieved with a luminescent terbium(III)-organic framework (TbOF) of [Tb(2)(2,3-pzdc)(2)(ox)(H(2)O)(2)](n) (2,3-pzdc(2-) = 2,3-pyrazinedicarboxylate, ox(2-) = oxalate).

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.

Fluorescence detection of adenosine triphosphate through an aptamer-molecular beacon multiple probe.

An aptamer-molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.

Fluorescent and colorimetric chemosensors for detection of nucleotides, FAD and NADH: highlighted research during 2004-2010.

Due to the biological importance of nucleotides and related species, such as XNP (where X = adenosine (A), uridine (U), cytidine (C), guanosine (G), and N = mono, di, tri), FAD and NADH, the development of optical probes for these molecules has recently been an active area of research. This tutorial review focuses on the contributions between 2004-2010 concerning the fluorescent or colorimetric sensors for these biomolecules, and is organized according to their target molecule's structural classification.

Cytokine and sex hormone effects on zidovudine- and lamivudine-triphosphate concentrations in vitro.

INTRODUCTION: Elevated zidovudine- and lamivudine-triphosphates have been observed in peripheral blood mononuclear cells (PBMCs) from females versus males and in patients with high inflammatory states versus lower inflammatory states. Consistent with high triphosphate exposures, these same patient groups also experience elevated rates of toxicity, including lipoatrophy. The purpose of this study was to evaluate the effects of oestradiol, progesterone and testosterone as well as tumour necrosis factor (TNF)-alpha and interferon (IFN)-alpha on zidovudine- and lamivudine-triphosphates in PBMCs and, for the cytokines, in 3T3-L1 adipocytes. METHODS: Multiple replicates of adipocytes and human PBMCs were incubated with experimental versus control conditions using several cytokine and sex hormone doses. Zidovudine- and lamivudine-triphosphate concentrations were determined with validated LC-MS-MS assays. A mixed effects, cell means model that accounted for experiment number was used to evaluate the effects of experimental conditions relative to control. RESULTS: In adipocytes, TNF-alpha doses below 2 ng/mL increased zidovudine-triphosphate by 18% (5-31%). Lamivudine-triphosphate was not detectable in adipocytes. In PBMCs, pooled IFN-alpha doses (0.1-10 U/mL) decreased zidovudine-triphosphate 26% (10-42%); 100 and 1000 ng/mL of progesterone decreased lamivudine-triphosphate by 22% (1-43%) and 47% (25-68%), respectively. Pooled testosterone doses (10-1000 ng/mL) decreased lamivudine-triphosphate by 24% (7-41%). No other statistically significant effects were observed. CONCLUSIONS: We found evidence that sex hormones and cytokines influence zidovudine-triphosphate and lamivudine-triphosphate slightly in PBMCs and adipocytes in vitro. These findings provide insight and scientific direction to address inflammation-dependent and sex-dependent phosphorylation and responses in patients.

Cyclopentenyl cytosine increases gemcitabine radiosensitisation in human pancreatic cancer cells.

The deoxycytidine analogue 2',2'-difluoro-2'-deoxycytidine (dFdC, gemcitabine) is a potent radiosensitiser, but has limited efficacy in combination with radiotherapy in patients with pancreatic cancer due to acute toxicity. We investigated whether cyclopentenyl cytosine (CPEC), targetting the 'de novo' biosynthesis of cytidine triphosphate (CTP), could increase dFdC cytotoxicity alone or in combination with irradiation in a panel of human pancreatic cancer cells (Panc-1, Miapaca-2, BxPC-3). To investigate the role of deoxycytidine kinase (dCK), the rate-limiting enzyme in the activation of dFdC, human lung cancer cells without (dFdC-resistant SWg) and with an intact dCK gene (dFdC-sensitive SWp) were included. We found that CPEC (100-1000 nmol l(-1)) specifically reduced CTP levels in a dose-dependent manner that lasted up to 72 h in all cell lines. Preincubation with CPEC resulted in a dose-dependent increase in dFdC incorporated into the DNA only in dFdC-sensitive cells. Consequently, CPEC increased the effectiveness of dFdC (300 nmol l(-1) for 4 h) only in dFdC-sensitive cells, which was accompanied by an increase in apoptosis. We also found that CPEC enhanced the radiosensitivity of cells treated with dFdC (30-300 nmol l(-1) for 4 h). These results indicate that CPEC enhances the cytotoxicity of dFdC alone and in combination with irradiation in several human tumour cell lines with an intact dCK gene.

Extracellular inosine modulates ERK 1/2 and p38 phosphorylation in cultured Sertoli cells: possible participation in TNF-alpha modulation of ERK 1/2.

Extracellular ATP and adenosine modulation of MAPKs is well described in different cells types, but few studies have addressed the effects of extracellular inosine on these kinases. Previous results showed that hydrogen peroxide and TNF-alpha increase extracellular inosine concentration in cultured Sertoli cells and this nucleoside protects Sertoli cells against hydrogen peroxide induced damage and participates in TNF-alpha induced nitric oxide production. In view of the fact that MAPKs are key mediators of the cellular response to a large variety of stimuli, we investigated the effect of extracellular inosine on the phosphorylation of ERK 1/2 and p38 MAPKs in cultured Sertoli cells. The involvement of this nucleoside in the activation of ERK 1/2 by TNF-alpha was also investigated. Inosine and the selective A1 adenosine receptor agonist R-PIA increases the phosphorylation of ERK 1/2 and p38, and this was blocked by the selective A1 adenosine receptors antagonists, CPT and DPCPX. These antagonists also inhibited TNF-alpha increase in the phosphorylation of ERK 1/2. TNF-alpha also rapidly augmented extracellular inosine concentration in cultured Sertoli cells. These results show that extracellular inosine modulates ERK 1/2 and p38 in cultured Sertoli cells, possible trough A1 adenosine receptor activation. This nucleoside also participates in TNF-alpha modulation of ERK 1/2.

Effect of oral CDP-choline on plasma choline and uridine levels in humans.

Twelve mildly hypertensive but otherwise normal fasting subjects received each of four treatments in random order: CDP-choline (citicoline; 500, 2000, and 4000 mg) or a placebo orally at 8:00 a.m. on four different treatment days. Eleven plasma samples from each subject, obtained just prior to treatment (8:00 a.m.) and 1-12 hr thereafter, were assayed for choline, cytidine, and uridine. Fasting terminated at noon with consumption of a light lunch that contained about 100 mg choline. Plasma choline exhibited dose-related increases in peak values and areas under the curves (AUCs), remaining significantly elevated, after each of the three doses, for 5, 8, and 10 hr, respectively. Plasma uridine was elevated significantly for 5-6 hr after all three doses, increasing by as much as 70-90% after the 500 mg dose, and by 100-120% after the 2000 mg dose. No further increase was noted when the dose was raised from 2000 to 4000 mg. Plasma cytidine was not reliably detectable, since it was less than twice blank, or less than 100 nM, at all of the doses. Uridine is known to enter the brain and to be converted to UTP; moreover, we found that uridine was converted directly to CTP in neuron-derived PC-12 cells. Hence, it seems likely that the circulating substrates through which oral citicoline increases membrane phosphatide synthesis in the brains of humans involve uridine and choline, and not cytidine and choline as in rats.

The intracellular activation of lamivudine (3TC) and determination of 2'-deoxycytidine-5'-triphosphate (dCTP) pools in the presence and absence of various drugs in HepG2 cells.

AIMS: Lamivudine (3TC, 2'-deoxy-3'-thiacytidine) requires intracellular metabolism to its active 5'-triphosphate, 3TC-5'-triphosphate (3TCTP), to inhibit the replication of hepatitis B virus (HBV). We have investigated the activation of 3TC, in the presence and absence of a range of compounds, in HepG2 cells. The intracellular levels of the endogenous competitor of 3TCTP, 2'-deoxycytidine-5'-triphosphate (dCTP), were also determined and 3TCTP/dCTP ratios calculated. METHODS: The effects of a number of compounds on 3TC (3H; 1 microM) phosphorylation were investigated by radiometric h.p.l.c. dCTP levels were determined using a template primer extension assay. 3TCTP/dCTP ratios were calculated from these results. RESULTS: The phosphorylation of 3TC was significantly increased in the presence of either hydroxyurea (HU), methotrexate (MTX), or fludarabine (FLU). For example, at 100 microM HU, control 3TCTP levels were increased to 361% of control, whereas at 100 microM FLU, control 3TCTP levels were increased to 155%. dCTP pools were significantly reduced in the presence of HU and FLU, at 100 microM concentrations only. However, for all the above three compounds investigated, the ratio of 3TCTP/dCTP was favourably enhanced (e.g. at 1 microM MTX, 255% of control). Neither ganciclovir (GCV), lobucavir (LCV), penciclovir (PCV), adefovir dipivoxil (ADV), nor foscarnet (FOS) had any significant effects on 3TC phosphorylation or dCTP pools. CONCLUSIONS: These results suggest that the activity of 3TC may be potentiated when combined with one of the modulators studied. The lack of an interaction between 3TC and the other anti-HBV agents is reassuring. These in vitro studies can be used as an initial screen to examine potential interactions at the phosphorylation level.

Induction of human high K(M) 5'-nucleotidase in cultured 293 cells.

Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.

Control of lymphoproliferative and autoimmune disease in MRL-lpr/lpr mice by brequinar sodium: mechanisms of action.

Brequinar sodium (BQR) was originally developed as an antitumor drug and subsequently as an immunosuppressant for controlling transplant rejection. It has been widely accepted that the antitumor and immunosuppressive activities of BQR are dependent on its ability to inhibit the enzymatic activity of dihydroorotate dehydrogenase, the fourth enzyme in the de novo pyrimidine synthesis pathway. Recently, we discovered that BQR has the ability to inhibit protein tyrosine phosphorylation in anti-CD3-stimulated murine T lymphocytes and to inhibit the activity of src-related protein tyrosine kinases, p56lck and p59fyn. We examined the in vivo activities of BQR in MRL-lpr/lpr mice. We report that the dose of BQR (10 mg/kg/day) that induced anemia, controlled lymphadenopathy and inhibited autoantibody production, also selectively reduced the pyrimidine nucleotide levels in the bone marrow and in the lymph nodes. Coadministration of uridine (1000 mg/kg/day) with BQR completely normalized pyrimidine nucleotide levels in the bone marrow and lymph nodes, and prevented BQR-induced anemia. However, coadministration of uridine with BQR only partially reversed the anti-proliferative effects of BQR, and did not antagonize the inhibitory effect of BQR on autoantibody production. Finally, we report that BQR markedly reduced protein tyrosine phosphorylation in lymph nodes of MRL-lpr/lpr mice. These results collectively suggest that the control of lymphadenopathy and autoantibody production in MRL-lpr/lpr mice by BQR is only partially dependent on inhibition of pyrimidine nucleotide synthesis, and suggest a critical role for in vivo inhibition of protein tyrosine phosphorylation.

Effects of dual combinations of antifolates with atovaquone or dapsone on nucleotide levels in Plasmodium falciparum.

The triazine antifolates, cycloguanil and 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-[(2,4,5-trichlorophenoxy)propy loxy]-1,3,5-triazine hydrobromide (WR99210), and their parent biguanide compounds, proguanil and N-[3-(2,4,5-trichlorophenoxy)propyloxy]-n-(1-methylethyl)-imido dicarbonimidic-diamine hydrochloride (PS-15), were tested in combination with a series of antimalarial drugs for synergism against Plasmodium falciparum growing in erythrocytic culture. Four synergistic combinations were found: cycloguanil dapsone, WR99210-dapsone, proguanil-atovaquone, and PS-15-atovaquone. Cycloguanil-dapsone or WR99210-dapsone had a profound suppressive effect on the concentration of dTTP in parasites while that of dATP increased. Depletion of dTTP is consistent with cycloguanil or WR99210 inhibiting dihydrofolate reductase and dapsone inhibiting dihydropteroate synthase. For the combinations proguanil-atovaquone and PS-15-atovaquone, the levels of nucleoside triphosphates (NTPs) and dNTPs were generally suppressed, suggesting that inhibition is not through nucleotide pathways but probably through another metabolic mechanism(s). Combinations of two synergistic pairs of antimalarial drugs, (proguanil-atovaquone)-(cycloguanil-dapsone) and (PS-15-atovaquone)-(WR99210-dapsone), were tested, and it was found that NTPs and dNTPs decreased much more than for a single synergistic combination. Dual synergistic combinations could play an important role in the therapy of multidrug-resistant malaria, just as combination chemotherapy is used to treat cancer.

Analysis of individual purine and pyrimidine nucleoside di- and triphosphates and other cellular metabolites in PCA extracts by using multinuclear high resolution NMR spectroscopy.

This work demonstrates that individual purine and pyrimidine NDP and NTP can be assigned in high resolution 31P NMR spectra from tissue extracts. To the best of our knowledge, it is shown for the first time that ATP, GTP, UTP, CTP, and the corresponding diphosphates can be quantitated in cell extracts without using HPLC or other biochemical methods. This work provides the basis for further optimization of nucleotide quantitation by 31P NMR spectroscopy, and for a full assessment of this method. Furthermore, a new technique was developed for 1H, 31P, and 13C NMR signal assignment and quantitation in cell extracts by using the same external reference capillary for all three nuclei. This allows for efficient, quantitative, multinuclear NMR spectroscopy without extract contamination by standard material.

Characterization of fluorescent nucleoside triphosphates by capillary electrophoresis with laser-induced fluorescence detection: action of alkaline phosphatase and DNA polymerase.

A method of analysis of fluor-labeled nucleoside triphosphates based on alkaline phosphatase-catalyzed sequential cleavage of phosphate groups with monitoring of all fluorescent species by capillary electrophoresis with laser-induced fluorescence detection is presented. The method allows determination of the purity of the triphosphate samples as well as the relative amounts of the lower phosphate contaminants. The ability of one of the fluor-labeled nucleoside triphosphates to serve as polymerase substrate was verified by labeling DNA restriction fragments by the method of filling recessed 3'-ends using DNA polymerase Klenow fragment.