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Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with viral genome replication. In this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can initially bind in a comparatively stabilized state to the viral RdRp active site, as it primarily forms base stacking with the template uracil nucleotide (nt +1), which under freely fluctuations supports a low free energy barrier of the RDV-TP insertion (∼1.5 kcal mol-1). In comparison, the corresponding natural substrate ATP binds initially to the RdRp active site in Watson-Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (∼2.6 kcal mol-1), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be specifically stabilized by motif C-S759, S682 (near motif B) with the base, and motif G-K500 with the template backbone. Although the RDV-TP insertion can be hindered by motif F-R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif A-D623, respectively.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes divided into two subgroups (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) and (P2Y12, P2Y13, and P2Y14). The P2Y receptors are expressed in various cell types and play important roles in physiology and pathophysiology including inflammatory responses and neuropathic pain. The antagonism of P2Y12 receptors is used in pharmacotherapy for the prevention and therapy of cardiovascular events. The nucleoside analogue ticagrelor and active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel inhibit platelet P2Y12 receptors and reduce thereby platelet aggregation. The P2Y2 receptor agonist diquafosol is used for the treatment of the dry eye syndrome. The P2Y receptor subtypes differ in their amino acid sequences, their pharmacological profiles and their signaling transduction pathways. Recently, selective receptor ligands have been developed for all subtypes. The published crystal structures of the human P2Y1 and P2Y12 receptors as well as receptor models will facilitate the development of novel drugs for pharmacotherapy.

GPR17 is a G (i)-coupled dual receptor, linked to P2Y and CysLT receptors stimulated by uracil nucleotides and cysteinyl leukotrienes, respectively. Recent evidence has demonstrated that GPR17 inhibition ameliorates the progression of cerebral ischemic injury by regulating neuronal death and microglial activation. The present study aimed to assess the detailed regulatory roles of this receptor in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemia-like injury in vitro and explore the underlying mechanism. The results demonstrated that OGD/R induced ischemic neuronal injury and microglial activation, including enhanced phagocytosis and increased inflammatory cytokine release in neuron‑glial mixed cultures of cortical cells. GPR17 upregulation during OGD/R was spatially and temporally correlated with neuronal injury and microglial activation. In addition, GPR17 knockdown inhibited OGD/R-induced responses in neuron-glial mixed cultures. GPR17 knockdown also attenuated cell injury induced by the agonist leukotriene D4 (LTD4) or uridine 5′-diphosphate (UDP) in neuron-glial mixed cultures. However, GPR17 knockdown did not affect OGD/R-induced ischemic neuronal injury in primary cultures of neurons. In primary astrocyte cultures, neither GPR17 nor OGD/R induced injury. By contrast, GPR17 knockdown ameliorated OGD/R-induced microglial activation, boosting phagocytosis and inflammatory cytokine release in primary microglia cultures. Finally, the results demonstrated that the conditioned medium of microglia pretreated with OGD/R induced neuronal death, and the neuronal injury was significantly inhibited by GPR17 knockdown. These findings suggested that GPR17 may mediate ischemia-like neuronal injury and microglial activation in vitro; however, the protective effects on ischemic neuronal injury might depend upon microglial activation. Whether GPR17 regulates neuronal injury mediated by oligodendrocyte linkage remains to be investigated.

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7–9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 μM) > ATP (EC50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (

Sensitive evaluation of the uracil-DNA glycosylase (UDG) activity is greatly significant in both fundamental biochemical process studies and disease prognosis. In this study, a simple but sensitive UDG activity-sensing strategy was designed on the basis of UDG-triggered rolling circle amplification (RCA) reaction. In this strategy, two oligonucleotides were used. The hairpin-like structure of the oligonucleotide containing a uracil nucleotide is destroyed in the presence of UDG, and then can be employed to form the circular template of RCA and initiate the subsequent RCA reaction. The participation of a nicking endonuclease makes the RCA reaction proceed in an exponential amplification mode. The amplification product may fold into a G-quadruplex structure, which can be specifically combined with thioflavin T to generate a fluorescence signal without any extra label. This UDG activity-sensing strategy was demonstrated to work well in both end-point and real-time detection modes with high sensitivity and excellent specificity. As low as 5.5 × 10−5 U mL−1 UDG could be detected. The advantages of simple operation, short RCA time and automatic measurement using commercial instruments make the real-time detection mode suitable for high-throughput detection with reduced risk of amplification product carryover contamination, and its application feasibility in real samples was demonstrated by UDG activity analysis of cell lysate.

Nucleosides, nucleotides and 2'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxy-methyl) uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside mono-phosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).

Professor Geoffrey Burnstock proposed the concept of purinergic signaling via P1 and P2 receptors. P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular adenine and uracil nucleotides. Eight mammalian P2Y receptor subtypes have been identified. They are divided into two subgroups (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) and (P2Y12, P2Y13, and P2Y14). P2Y receptors are found in almost all cells and mediate responses in physiology and pathophysiology including pain and inflammation. The antagonism of platelet P2Y12 receptors by cangrelor, ticagrelor or active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel reduces the ADP-induced platelet aggregation in patients with thrombotic complications of vascular diseases. The nucleotide agonist diquafosol acting at P2Y2 receptors is used for the treatment of the dry eye syndrome. Structural information obtained by crystallography of the human P2Y1 and P2Y12 receptor proteins, site-directed mutagenesis and molecular modeling will facilitate the rational design of novel selective drugs.

An efficient P(V)-N activation method for the synthesis of 5-carboxy-2'-deoxyuridine and 5-carboxy-2'-deoxycytidine triphosphates directly from the corresponding phosphoropiperidate precursors has been developed.

To evaluate the effectiveness of 3% diquafosol tetrasodium for treating dry eye after cataract surgery.Among patients who underwent bilateral cataract surgery, 34, who met the diagnostic criteria for dry eye syndrome 1 week postoperatively, were enrolled. Patients were randomly assigned to receive 3.0% diquafosol tetrasodium ophthalmic solution in one eye and 0.9% saline in the other eye four times daily for 8 weeks. Dry eye severity was measured at 1, 5, and 9 postoperative weeks using the Schirmer 1 test (SIT), tear film breakup time (TBUT), and fluorescein corneal staining. tear meniscus height (TMH), tear meniscus depth (TMD), and tear meniscus area (TMA) measured using Fourier-domain optical coherence tomography and symptom questionnaire scores.TBUT and corneal staining significantly improved 8 weeks postoperatively in eyes treated with 3.0% diquafosol tetrasodium (p0.01, p0.01) and were better than normal saline-treated eyes (p0.01, p0.01). SIT did not improve (p = 0.26). TMH, TMD, and TMA did not improve at 4 and 8 weeks. All symptom questionnaire scores improved in eyes treated with 3.0% diquafosol tetrasodium (all p0.01).The 3.0% diquafosol tetrasodium treatment improved tear film stability and subjective symptoms of dry eye after cataract surgery. Increased mucin production as a result of diquafosol treatment may have caused these results.

P2Y receptors (P2YRs) are a family of G protein-coupled receptors activated by extracellular nucleotides. Physiological P2YR agonists include purine and pyrimidine nucleoside di- and triphosphates, such as ATP, ADP, UTP, UDP, nucleotide sugars, and dinucleotides. Eight subtypes exist, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14, which represent current or potential future drug targets. Here we provide a comprehensive overview of ligands for the subgroup of the P2YR family that is activated by uracil nucleotides: P2Y2 (UTP, also ATP and dinucleotides), P2Y4 (UTP), P2Y6 (UDP), and P2Y14 (UDP, UDP-glucose, UDP-galactose). The physiological agonists are metabolically unstable due to their fast hydrolysis by ectonucleotidases. A number of agonists with increased potency, subtype-selectivity and/or enzymatic stability have been developed in recent years. Useful P2Y2R agonists include MRS2698 (6-01, highly selective) and PSB-1114 (6-05, increased metabolic stability). A potent and selective P2Y2R antagonist is AR-C118925 (10-01). For studies of the P2Y4R, MRS4062 (3-15) may be used as a selective agonist, while PSB-16133 (10-06) is a selective antagonist. Several potent P2Y6R agonists have been developed including 5-methoxyuridine 5'-O-((Rp)α-boranodiphosphate) (6-12), PSB-0474 (3-11), and MRS2693 (3-26). The isocyanate MRS2578 (10-08) is used as a selective P2Y6R antagonist, although its reactivity and low water-solubility are limiting. With MRS2905 (6-08), a potent and metabolically stable P2Y14R agonist is available, while PPTN (10-14) represents a potent and selective P2Y14R antagonist. The radioligand [3H]UDP can be used to label P2Y14Rs. In addition, several fluorescent probes have been developed. Uracil nucleotide-activated P2YRs show great potential as drug targets, especially in inflammation, cancer, cardiovascular and neurodegenerative diseases.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1 , A2A , A2B , and A3 ) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'-di- and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union. J. Cell. Physiol. 231: 1852-1861, 2016. © 2016 Wiley Periodicals, Inc.

Adenine and uracil nucleotides play key functions in the autonomic nervous system (ANS). For instance, ATP acts as a neurotransmitter, co-transmitter and neuromodulator in the ANS. The purinergic system encompasses (1) receptors that respond to extracellular purines, which are designated as P1 and P2 purinoceptors, (2) purine release and uptake, and (3) a cascade of enzymes that regulate the concentration of purines near the cell surface. Ectonucleotidases and adenosine deaminase (ADA) are enzymes responsible for the hydrolysis of ATP (and other nucleotides such as ADP, UTP, UDP, AMP) and adenosine, respectively. Accordingly, these enzymes are expected to play an important role in the control of neuro-effector transmission in tissues innervated by both the sympathetic and parasympathetic divisions of the ANS. Indeed, ectonucleotidases have the ability to either terminate P2 receptor responses initiated by nucleoside triphosphates (ATP and UTP), and/or to favor the activation of ADP (e.g. P2Y1,12,13) and UDP (e.g. P2Y6) and/or adenosine (P1) specific receptors. In addition, ectonucleotidases can also importantly protect some P2 receptors from desensitization (e.g. P2X1, P2Y1). In this review, we present the (putative) roles of ectonucleotidases and ADA in the ANS with a focus on their regulatory activity at neuro-effector junctions in the following tissues: heart, vas deferens, urinary bladder, salivary glands, blood vessels and the intestine. We also present their implication in nociceptive transmission.

UDP-sugar 4-epimerase (GalE) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins and is one of enzymes in the Leloir pathway. They have been shown to be important virulence factors in a number of Gram-negative pathogens and to be involved in the biosynthesis of different polysaccharide structures. The metabolic disease type III galactosemia is caused by detrimental mutations in the human GalE. GalE and related enzymes display unusual enzymologic, chemical, and stereochemical properties; including irreversible binding of the cofactor NAD and uridine nucleotide-induced activation of this cofactor. These epimerases have been found active on UDP-hexoses, the N-acetylated and uronic acid forms thereof as well as UDP-pentoses. As they are involved in different pathways and functions, a deeper understanding of the enzymes, and their substrate promiscuity and/or selectivity, could lead to drug and vaccine design as well as antibiotic and probiotic development. This review summarizes the research performed on UDP-sugar 4-epimerases' structure, mechanism and substrate promiscuity.

The P2Y14 receptor (P2Y14R) is a Gi protein-coupled receptor that is activated by uracil nucleotides UDP and UDP-glucose. The P2Y14R structure has yet to be solved through X-ray crystallography, but the recent agonist-bound crystal structure of the P2Y12R provides a potentially suitable template for its homology modeling for rational structure-based design of selective and high-affinity ligands. In this study, we applied ligand docking and molecular dynamics refinement to a P2Y14R homology model to qualitatively explain structure-activity relationships of previously published synthetic nucleotide analogues and to probe the quality of P2Y14R homology modeling as a template for structure-based design. The P2Y14R model supports the hypothesis of a conserved binding mode of nucleotides in the three P2Y12-like receptors involving functionally conserved residues. We predict phosphate group interactions with R253(6.55), K277(7.35), Y256(6.58) and Q260(6.62), nucleobase (anti-conformation) π-π stacking with Y102(3.33) and the role of F191(5.42) as a means for selectivity among P2Y12-like receptors. The glucose moiety of UDP-glucose docked in a secondary subpocket at the P2Y14R homology model. Thus, P2Y14R homology modeling may allow detailed prediction of interactions to facilitate the design of high affinity, selective agonists as pharmacological tools to study the P2Y14R.

The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for oligo(U) RNA-binding factors. Here, we demonstrate that NEXT displays preference for U-rich pyrimidine sequences and this RNA binding is mediated by the RNA recognition motif (RRM) of the RBM7 subunit. We solved the structure of RBM7 RRM and identified two phenylalanine residues that are critical for interaction with RNA. Furthermore, we showed that these residues are required for the NEXT interaction with snRNAs in vivo. Finally, we show that depletion of components of the NEXT complex alone or together with exosome nucleases resulted in the accumulation of mature as well as extended forms of snRNAs. Thus, our data suggest a new scenario in which the NEXT complex is involved in the surveillance of snRNAs and/or biogenesis of snRNPs.

Supplemental digital content is available in the text., Purpose Two new drugs with mucin-inducing and secretion-promotive effects, rebamipide and diquafosol, were recently approved as topical dry-eye treatments. We report two cases in which the long-term use of mucin-inducing eye drops improved chronic ocular graft-versus-host disease (cGVHD)–related dry eye and ocular cicatricial pemphigoid (OCP)-like disease. Case Reports Case 1. A 61-year-old woman had cGVHD-related dry eye that resisted traditional medications. Next, we use topical diquafosol in addition to conventional treatments. The patient used diquafosol for 6 months without experiencing any side effects. The symptoms, including dry-eye sensation, ocular pain, foreign body sensation, and photophobia, as well as ocular surface findings including fluorescein and rose bengal scores and tear break-up time (TBUT), partly improved. To further improve the clinical signs and symptoms and decrease chronic inflammation, rebamipide was added to diquafosol. The symptoms, TBUT, and fluorescein and rose bengal scores markedly improved after long-term dual treatment without any side effects for 6 months. Case 2. A 77-year-old woman had OCP-like disease with dry eye. The patient did not improve using the currently available conventional treatments. Next, we use topical rebamipide in addition to conventional treatments. Symptoms including asthenopia, dry-eye sensation, ocular pain, and dull sensation, as well as fluorescein and rose bengal scores and TBUT, partly improved. Specifically, functional visual acuity was markedly improved after commencement of rebamipide. To further improve the clinical signs and symptoms and increase tear film stability and tear film volume, diquafosol was added to rebamipide. The combination of diquafosol and rebamipide worked for the patient. Improvements were seen in several symptoms, fluorescein and rose bengal scores, Schirmer test value, and TBUT without any side effects for 12 months. Conclusions Long-term treatment with topical rebamipide and diquafosol can improve dry eye in patients with cGVHD or OCP-like disease.

The interactions of the nucleobases thymine (C5H6N2O2) and uracil (C4H4N2O2) with Cr-doped C20 fullerene (C19Cr) are investigated by performing density functional theory calculations. The adsorption of these nucleobases on C19Cr leads to two distinct geometries (P1 and P2) differing in the orientation of the nucleobases. The interaction of the nucleobases with the C19Cr nanocluster is highly exothermic, revealing that they are chemically adsorbed on C19Cr. The results show that the binding energy of the thymine–C19Cr complex is slightly higher than that of the uracil–C19Cr complex. In addition, the P2 geometry is more stable compared to P1 due to the higher binding energy in the former configuration. However, based on the results of natural bond orbital and frontier molecular orbitals analyses, the C19Cr nanocage has higher reactivity with the nucleobases in P1 geometry in comparison with P2 due to the larger charge transfer and orbital hybridization in the former geometry. Moreover, the band gap of the C19Cr nanocage decreases after interaction with the nucleobases, and interestingly the impact is more pronounced for P1 geometry, confirming the higher sensitivity of C19Cr to the nucleobases in P1 geometry. Our findings reveal the promising potential of C19Cr as an organometallic carrier for nucleobases thymine and uracil.

The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3' oligo-uridylation activity of TUT4 and TUT7 (refs 10-12). The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3l2 (refs 13, 14), a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition we determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28-let-7 pathway.

P2Y(6) nucleotide receptor (P2Y(6)-R) plays important physiological roles, such as insulin secretion and reduction of intraocular pressure. However, this receptor is still lacking potent and selective agonists to be used as potential drugs. Here, we synthesized uracil nucleotides and dinucleotides, substituted at the C5 and/or P(α) position with methoxy and/or borano groups, 18-22. Compound 18A, R(p) isomer of 5-OMe-UDP(α-B), is the most potent and P2Y(6)-R selective agonist currently known (EC(50) 0.008 μM) being 19-fold more potent than UDP and showing no activity at uridine nucleotide receptors, P2Y(2)- and P2Y(4)-R. Analogue 18A was highly chemically stable under conditions mimicking gastric juice acidity (t(1/2) = 16.9 h). It was more stable to hydrolysis by nucleotide pyrophosphatases (NPP1,3) than UDP (15% and 28% hydrolysis by NPP1 and NPP3, respectively, vs 50% and 51% hydrolysis of UDP) and metabolically stable in blood serum (t(1/2) = 17 vs 2.4, 11.9, and 21 h for UDP, 5-OMe-UDP, and UDP(α-B), respectively). This newly discovered highly potent and physiologically stable P2Y(6)-R agonist may be of future therapeutic potential.

To assess the additive effect of diquafosol tetrasodium on sodium hyaluronate monotherapy in patients with dry eye syndrome.This study evaluated 64 eyes of 32 patients (age: 62.6±12.8 years (mean±SD)) in whom treatment with 0.1% sodium hyaluronate was insufficiently responsive. The eyes were randomly assigned to one of the two regimens in each patient: topical administration of sodium hyaluronate and diquafosol tetrasodium in one eye, and that of sodium hyaluronate in the other. Before treatment, and 2 and 4 weeks after treatment, we determined tear volume, tear film break-up time (BUT), fluorescein and rose bengal vital staining scores, subjective symptoms, and adverse events.We found a significant improvement in BUT (P=0.049, Dunnett test), fluorescein and rose bengal staining scores (P=0.02), and in subjective symptoms (P=0.004 for dry eye sensation, P=0.02 for pain, and P=0.02 for foreign body sensation) 4 weeks after treatment in the diquafosol eyes. On the other hand, we found no significant change in these parameters after treatment in the control eyes.In dry eyes, where sodium hyaluronate monotherapy was insufficient, diquafosol tetrasodium was effective in improving objective and subjective symptoms, suggesting its viability as an option for the additive treatment of such eyes.

G protein-coupled receptor 17 (GPR17), the new P2Y-like receptor, is phylogenetically related to the P2Y and cysteinyl leukotriene receptors, and responds to both uracil nucleotides and cysteinyl leukotrienes. GPR17 has been proposed to be a damage sensor in ischemic stroke; however, its role in brain inflammation needs further detailed investigation. Here, we extended previous studies on the spatiotemporal profiles of GPR17 expression and localization, and their implications for brain injury after focal cerebral ischemia. We found that in the ischemic core, GPR17 mRNA and protein levels were upregulated at both 12-24 h and 7-14 days, but in the boundary zone the levels increased 7-14 days after reperfusion. The spatiotemporal pattern of GPR17 expression well matched the acute and late (subacute/chronic) responses in the ischemic brain. According to previous findings, in the acute phase, after ischemia (24 h), upregulated GPR17 was localized in injured neurons in the ischemic core and in a few microglia in the ischemic core and boundary zone. In the late phase (14 days), it was localized in microglia, especially in activated (ED1-positive) microglia in the ischemic core, but weakly in most microglia in the boundary zone. No GPR17 was detectable in astrocytes. GPR17 knockdown by a small interfering RNA attenuated the neurological dysfunction, infarction, and neuron loss at 24 h, and brain atrophy, neuron loss, and microglial activation at 14 days after reperfusion. Thus, GPR17 might mediate acute neuronal injury and late microgliosis after focal cerebral ischemia.

The P2Y-like receptor GPR17 has been reported to respond to both uracil nucleotides and cysteinyl-leukotrienes (cysLTs), such as UDP-glucose and LTD(4). Our previous data suggest a potential role for GPR17 in regulation of both cell viability and differentiation state of central nervous system cells. On this basis, in the present paper we investigated the effect of GPR17 receptor ligands on PC12 cell viability, following induction of morphological differentiation by nerve growth factor (NGF). In addition, the role of GPR17 ligands, either alone or in combination with growth factors, on the degree of PC12 cell differentiation was investigated. GPR17, which was not basally expressed in undifferentiated PC12 cells, was specifically induced by a 10day-treatment with NGF, suggesting a role in the control of neuronal specification. Both UDP-glucose and LTD(4), agonists at the nucleotide and cysLT GPR17 binding sites, respectively, induced a significant pro-survival effect on PC12 cells after priming with NGF. By in vitro silencing experiments with specific small interfering RNAs and by using receptor antagonists, we confirmed that the agonist effects are indeed mediated by the selective activation of GPR17. We also demonstrated that GPR17 agonists act, both alone and synergistically with NGF, to promote neurite outgrowth in PC12 cells. In addition, GPR17 ligands were able to confer an NGF-like activity to the epidermal growth factor (EGF), that, under these experimental conditions, also promoted cell differentiation and neurite elongation. Finally, we show that GPR17 ligands activate the intracellular phosphorylation of both ERK 1/2 and p38 kinases, that have been identified as important signalling pathways for neurotrophins in PC12 cells. Our results establish GPR17 as a neurotrophic regulator for neuronal-like cells and suggest a possible interplay between endogenous uracil derivatives, cysLTs and NGF in the signalling pathways involved in neuronal survival and differentiation. They also represent the first direct demonstration, in a native system, that GPR17 can indeed be activated by uracil nucleotides and cysLTs, in line with what previously demonstrated in recombinant expression systems.

The side-by-side interactions of nucleobases contribute to the organization of RNA, forming the planar building blocks of helices and mediating chain folding. Dinucleotide platforms, formed by side-by-side pairing of adjacent bases, frequently anchor helices against loops. Surprisingly, GpU steps account for over half of the dinucleotide platforms observed in RNA-containing structures. Why GpU should stand out from other dinucleotides in this respect is not clear from the single well-characterized H-bond found between the guanine N2 and the uracil O4 groups. Here, we describe how an RNA-specific H-bond between O2'(G) and O2P(U) adds to the stability of the GpU platform. Moreover, we show how this pair of oxygen atoms forms an out-of-plane backbone 'edge' that is specifically recognized by a non-adjacent guanine in over 90% of the cases, leading to the formation of an asymmetric miniduplex consisting of 'complementary' GpUpA and GpA subunits. Together, these five nucleotides constitute the conserved core of the well-known loop-E motif. The backbone-mediated intrinsic stabilities of the GpU dinucleotide platform and the GpUpA/GpA miniduplex plausibly underlie observed evolutionary constraints on base identity. We propose that they may also provide a reason for the extreme conservation of GpU observed at most 5'-splice sites.

Although great progress has been made in identifying microRNAs (miRNAs) and their functions, their essential functional features remain largely unknown. In this study, we systemically investigated the nucleotide and thermodynamic folding distribution characteristics of 3853 miRNAs currently reported for metazoans. We determined that uracil is the dominant nucleotide in both mature and precursor sequences, and that it is particularly enriched at three sites in mature miRNAs: the first, ninth, and the five terminal 3' nucleotides. The location of these enriched uracil nucleotides is particularly interesting because positions one and nine are the edges of the "seed region", which is responsible for targeting mRNAs for gene regulation. The prevalence of U residues at these sites may contribute to the mechanism whereby miRNAs target and bind to their corresponding mRNAs. A comparison of the overall lengths of metazoan pre-miRNAs revealed that they ranged from 53 to 215 nt in length with an average of 88.10+/-14.14 nt, significantly higher than previously reported. Comparisons of miRNA diversity at different taxonomic levels revealed that the 12 features investigated in this study varied significantly among miRNAs represented by different phyla, with particularly high levels of divergence in platyhelminths relative to nematodes, arthropods or vertebrates. By comparison, lower levels of diversity were observed at lower taxonomic levels such that there was a direct relationship between divergence in miRNA features and taxonomic level. We conclude that large-scale genome analysis shows that miRNAs have many more unique features than previously reported. In particular, the distribution of nucleotides suggests an important role for uracil at the boundaries of the 'seed' region and at their termini. These results will facilitate the design of new computational programs for identifying novel miRNAs and investigating the mechanism of miRNA-mediated gene regulation.

Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca 2+ ] i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca 2+ -free external medium moderately decreased, whereas a depletion of the intracellular Ca 2+ storage sites by cyclopiazonic acid markedly depressed the [Ca 2+ ] i transients induced by either ATP or UTP. Further, the P2Y 1 receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y 1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y 1,2,4 -immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y 2 receptor-activation and thereby causes [Ca 2+ ] i transients by stimulating a P2Y 1 -like receptor.

Adsorption of poly(rA) on a single-walled carbon nanotube surface in aqueous suspension and the subsequent hybridization of this polymer with free poly(rU) is studied. A comparison of the temperature dependence of the absorbance of free poly(rA) and poly(rA) adsorbed on the nanotube surface [poly(rA)(NT)] at nu(max)= 38,500 cm(-1) shows that the thermostability of the adsorbed polymer is higher. Molecular dynamics simulations demonstrate that more than half of the adenines are not stacked on the tube surface and some of them undergo self-stacking. After addition of a complementary poly(rU) to the poly(rA)(NT) suspension, a double-stranded polymer is formed as confirmed by the characteristic S-like form of its melting curve. However, the melting temperature of this polymer is lower than that of the free poly(rA)poly(rU) duplex. This result indicates that poly(rU) hybridization with poly(rA)(NT) occurs with defects along the whole length of the polymer because of pi-pi stacking between nitrogen bases and the nanotube surface, which hinders the usual hybridization process. Computer modeling demonstrates different possible structures of hybridized polymers on the nanotube surface.

Thymine-DNA glycosylases (TDGs) initiate base excision repair by debasification of the erroneous thymine or uracil nucleotide in G.T and G.U mispairs which arise at high frequency through spontaneous or enzymatic deamination of methylcytosine and cytosine, respectively. Human TDG has furthermore been shown to have a functional role in transcription and epigenetic regulation through the interaction with transcription factors from the nuclear receptor superfamily, transcriptional coregulators, and a DNA methyltransferase. The TDG N-terminus encodes regulatory functions, as it assures both G.T versus G.U specificity and contains the sites for interaction and posttranslational modification by transcription-related activities. While the molecular function of the evolutionarily conserved central catalytic domain of TDG in base excision repair has been elucidated by determination of its three-dimensional structure, the mechanisms by which the N-terminus exerts its regulatory roles, as well as the function of TDG in transcription regulation, remain to be understood. We describe here the residual structure of the TDG N-terminus in both contexts of the isolated domain and the entire protein. These studies lead to the characterization of a small structural domain in the TDG N-terminal region preceding the catalytic core and coinciding with the region of functional regulation of TDG's activities. This regulatory domain exhibits a small degree of organization and is implicated in dynamic molecular interactions with the catalytic domain and nonselective interactions with double-stranded DNA, providing a molecular explanation for the evolutionarily acquired G.T mismatch processing activity of TDG.

We previously showed that the human heart expresses all known P2X and P2Y receptors activated by extra-cellular adenine or uracil nucleotides. Despite evidence that, both in humans and rodents, plasma levels of ATP and UTP markedly increase during myocardial infarction, the differential effects mediated by the various adenine- and uracil-preferring myocardial P2 receptors are still largely unknown. Here, we studied the effects of adenine and uracil nucleotides on murine HL-1 cardiomyocytes. RT-PCR analysis showed that HL-1 cardiomyocytes express all known P2X receptors (except for P2X(2)), as well as the P2Y(2,4,6,14) subtypes. Exposure of cardiomyocytes to adenine nucleotides (ATP, ADP or BzATP) induced apoptosis and necrosis, as determined by flow-cytometry. Cell death was exacerbated by tumour necrosis factor (TNF)-alpha, a cytokine implicated in chronic heart failure progression. Conversely, uracil nucleotides (UTP, UDP and UDPglucose) had no effect 'per se', but fully counteracted the deleterious effects induced by adenine nucleotides and TNF-alpha, even if added to cardiomyocytes after beginning exposure to these cell death-inducing agents. Thus, exposure of cardiomyocytes to elevated concentrations of ATP or ADP in the presence of TNF-alpha contributes to cell death, an effect which is counteracted by uracil-preferring P2 receptors. Cardiomyocytes do not need to be 'primed' by uracil nucleotides to become insensitive to adenine nucleotides-induced death, suggesting the existence of a possible 'therapeutic' window for uracil nucleotides-mediated protection. Thus, release of UTP during cardiac ischaemia and in chronic heart failure may protect against myocardial damage, setting the basis for developing novel cardioprotective agents that specifically target uracil-preferring P2Y receptors.

We examined the metabolic fate of [2- 14 C]uridine, [2- 14 C]cytidine, [2- 14 C]uracil and [2- 14 C]dihydrouracil in leaf disks of Bruguiera sexangula in 0 mM or 250 mM NaCl. Uridine was readily converted to uracil nucleotides, and cytidine was converted to cytosine nucleotides and also uracil nucleotides, possibly after conversion to uridine. These nucleotides were utilised for synthesis of RNA and UDP-glucose 3 h after administration of labelled precursors. Degradation of uridine and cytidine was extremely limited. In contrast, salvage of [2- 14 C]uracil was low, and up to 45% of uracil was degraded to CO 2 . [2- 14 C]Dihydrouracil, which is a possible intermediate in the reductive pathway of pyrimidine catabolism, was converted to β-ureidopropionic acid and CO 2 . No inhibitory effect of 250 mM NaCl was apparent on pyrimidine metabolism of B. sexangula , although RNA synthesis was reduced slightly. The metabolic fate of [2- 14 C]uridine was surveyed in seven other mangrove species, Bruguiera gymnorrhiza , Kandelia candel , Rhizophora stylosa , Sonneratia alba , Avicennia marina , Lumnitzera racemosa and Pemphis acidula . The general profile of [2- 14 C]uridine in these mangrove leaves was similar to that in B. sexangula ; almost all uridine was salvaged to nucleotides, UDP-glucose and RNA, and catabolism was very low. No or very little inhibitory effect of 250 mM NaCl on uridine salvage was found in leaf disks of mangrove species, except L. racemosa . A stimulatory effect of 250 mM NaCl on uridine salvage was found in P. acidula . In contrast, uptake and metabolism of [2- 14 C]uridine, especially a salvage pathway, in the leaves of the non-halophyte plant Populus alba , were drastically impaired by 250 mM NaCl.

Purine and pyrimidine nucleotides have been identified as potent extracellular signalling molecules, acting at two classes of cell surface receptors, ionotropic P2X and metabotropic P2Y receptor (-R) types. Hitherto eight subtypes of the P2Y-R family have been cloned from mammalian species that exhibit sensitivity to the adenine nucleotides ATP/ADP (P2Y(1,11,12,13)), the uracil nucleotides UTP/UDP (P2Y(2,4,6) or UDP-glucose in the case of P2Y(14)) or both adenine and uracil nucleotides (P2Y(2)). The P2Y-Rs are G protein-coupled receptors activating phospholipase C via Galpha(q/11) protein and stimulating or inhibiting adenylyl cyclase via Galpha(s) and Galpha (i/o) proteins, respectively. These receptors may activate distinct signalling cascades. Although classical models predict that P2Y-Rs exist in the cell membrane as monomers, homo- or heterodimeric assemblies may be generated. Interactions with certain ion channels or ligand-gated receptors as well as the co-localization of several receptor subtypes in the same cell provide the basis for a high functional diversity. The proteins for various P2Y-Rs are expressed early in the embryonic brain and are broadly distributed on both, neurons and astroglial cells. P2Y-R involvement in the regulation of normal physiological processes on the cellular level or in vivo, such as modulation of transmitter release, generation of astroglial Ca(2+) waves, in diverse effects on behavioural functions and in the etiopathology of neurodegenerative diseases, are discussed and own data are presented. However, the exact understanding of the role of individual P2Y-R subtypes is still limited. Concerning the potentially important functions of P2Y-Rs, there is a strong need to develop stable, lipophilic and subtype-selective P2Y-R ligands, which may open new therapeutic strategies.

Background and purpose: The uracil nucleotides UDP and UTP have been reported to activate P2Y2, P2Y4 and P2Y6 receptors to cause vasoconstriction. We have performed a comparative analysis of these receptors in endothelium-denuded smooth muscle from porcine isolated coronary and ear arteries, using pharmacological and molecular tools. Experimental approach: Tissue segments were used to construct non-cumulative concentration response curves for UTP and UDP, in the absence and presence of the P2 receptor antagonists PPADS or suramin. RT-PCR and immunoblot analyses were employed to define gene expression and immunoreactivity for P2Y2, P2Y4 and P2Y6 receptors. Key results: In the coronary artery, UTP-evoked contractile responses were reduced in the presence of suramin, but not PPADS, while the smaller responses to UDP were unaffected by either antagonist. In the ear artery, contractile responses to UDP were much smaller than those to UTP; responses to UTP were inhibited by both PPADS and suramin. RT-PCR suggested predominant expression of P2Y2 receptors in the coronary artery, while P2Y4 and P2Y6 receptor gene expression appeared equivalent in both tissues. Immunoblot analyses provided evidence for P2Y6 receptors in both tissues, with equivocal evidence of P2Y2 and P2Y4 receptor immunoreactivities. Conclusions and implications: We conclude that UTP-evoked contraction of porcine coronary artery smooth muscle appears to be predominantly P2Y2-mediated, while the ear artery appears to express a uracil nucleotide-sensitive P2 receptor(s) which fails to fit readily into the current classification. British Journal of Pharmacology (2007) 150, 604–612. doi:10.1038/sj.bjp.0707120

The biosynthesis of brain membrane phosphatides, e.g., phosphatidylcholine (PtdCho), may utilize three circulating compounds: choline, uridine (a precursor for UTP, CTP, and CDP-choline), and a PUFA (e.g., docosahexaenoic acid); moreover, oral administration of the uridine source uridine-5'-monophosphate (UMP) can significantly increase levels of the phosphatides throughout the rodent brain. Since PtdCho can provide choline for acetylcholine (ACh) synthesis, we determined whether UMP administration also affects ACh levels in striatum and striatal extracellular fluid, in aged and young rats. Among aged animals consuming a UMP-containing diet (2.5%, w/w) for 1 or 6 weeks, baseline ACh levels in striatal dialysates rose from 73 fmol/min to 148 or 197 fmol/min (P

To evaluate the clinical efficacy of 3 % diquafosol sodium ophthalmic solution for dry eye, and to analyze the concentration of tear proteins and mucin-like substances after the treatment. Fifty eyes of 25 patients with dry eye syndrome were prospectively enrolled. The patients were treated with diquafosol solution at a dose of 1 drop in each eye 6 times daily for 4 weeks. The parameters of clinical efficacy were tear osmolarity, tear breakup time (BUT), fluorescein staining scores for the cornea and conjunctiva, Schirmer test values, and subjective symptoms evaluated using the ocular surface disease index (OSDI). Tears collected with Schirmer test strips were analyzed by high-performance liquid chromatography, and the concentrations of the total protein and the 4 major tear proteins, namely, secretory IgA, lactoferrin, lipocalin-1, lysozyme, and N-acetyl-neuraminic acid (Neu5Ac), were measured. Neu5Ac is a major sialic acid, a marker of secretory mucins. The BUT, keratoconjunctival staining scores, and Schirmer test values were improved with statistical significance after the treatment with diquafosol solution, while changes in the other parameters, including tear osmolarity, corneal staining scores, and OSDI scores were not significant. The Neu5Ac concentration was significantly increased, which was not accompanied by changes in tear proteins. Topical application of diquafosol significantly improved the clinical parameters of the BUT, keratoconjunctival staining scores, and Schirmer test values and was accompanied by increased sialic acid content in the tears of patients with dry eye.

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.

MicroRNA (miRNA) is one class of newly identified, small, non-coding RNAs that play versatile and important roles in post-transcriptional gene regulation. All miRNAs have similar secondary hairpin structures; many of these are evolutionarily conserved. This suggests a powerful approach to predict the existence of new miRNA orthologs or homologs in other species. We developed a comprehensive strategy to identify new miRNA homologs by mining the repository of available ESTs. A total of 481 miRNAs, belonging to 37 miRNA families in 71 different plant species, were identified from more than 6 million EST sequences in plants. The potential targets of the EST-predicted miRNAs were also elucidated from the EST and protein databases, providing additional evidence for the real existence of these miRNAs in the given plant species. Some plant miRNAs were physically clustered together, suggesting that these miRNAs have similar gene expression patterns and are transcribed together as a polycistron, as observed among animal miRNAs. The uracil nucleotide is dominant in the first position of 5' mature miRNAs. Our results indicate that many miRNA families are evolutionarily conserved across all major lineages of plants, including mosses, gymnosperms, monocots and eudicots. Additionally, the number of miRNAs discovered was directly related to the number of available ESTs and not to evolutionary relatedness to Arabidopsis thaliana, indicating that miRNAs are conserved and little phylogenetic signal exists in the presence or absence of these miRNAs. Regulation of gene expression by miRNAs appears to have existed at the earliest stages of plant evolution and has been tightly constrained (functionally) for more than 425 million years.

Pyrimidine nucleotides, including UTP, UDP and UDP-glucose, are important signaling molecules which activate G protein-coupled membrane receptors (GPCRs) of the P2Y family. Four distinct pyrimidine nucleotide-sensitive P2Y receptor subtypes have been cloned, P2Y2, P2Y4, P2Y6 and P2Y14. P2Y2 and P2Y4 receptors are activated by UTP (the P2Y2, and the rat but not the human P2Y4 receptor are also activated by ATP), the P2Y6 receptor is activated by UDP, and the P2Y14 receptor by UDP-glucose. Furthermore, non-P2Y GPCRs, the cysteinylleukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinylleukotrienes. While P2Y2, P2Y4, and P2Y6 receptor activation results in stimulation of phospholipase C, the P2Y14 receptor is coupled to inhibition of adenylate cyclase. Derivatives and analogs of the physiological nucleotides UTP, UDP and ATP have been synthesized and evaluated in order to obtain enzymatically stable, subtype-selective agonists. The P2Y2 receptor agonists diuridine tetraphosphate (diquafosol) and the uracil-cytosine dinucleotide denufosol are currently undergoing clinical trials for dry eye disease, retinal detachment disease, upper respiratory tract symptoms, and cystic fibrosis, respectively. The first antagonists for P2Y2 and P2Y6 receptors that appear to be selective versus other P2Y receptor subtypes have recently been described. Selective antagonists for P2Y4 and P2Y14 receptors are still lacking. Uracil nucleotide-sensitive P2Y receptor subtypes may constitute future targets for the treatment of certain cancer types, vascular diseases, inflammatory diseases, and immunomodulatory intervention. They have also been proposed to play a role in neurodegenerative diseases. This article is an updated version of "P2-Pyrimidinergic Receptors and Their Ligands" by C. E. Müller published in Curr. Pharm. Des. 2002, 8, 2353-2369.

Cardiomyocytes express one or more subtypes of P2 receptors for extracellular nucleotides. P2 purinoceptors, which are activated by nucleotides, are classified as P2X or P2Y: P2X receptors are ligand-gated intrinsic ion channels, and P2Y receptors are G protein-coupled receptors. Extracellular pyrimidine and purine nucleotides are released from the heart during hypoxia. Although the cardioprotective effects of purines acting via purinoceptors were studied intensively, the physiological role of uracil nucleotide-responsive P2Y2, P2Y4, P2Y6, and P2Y14 receptors is still unclear, especially in the cardiovascular system. This study revealed that uridine-5′-triphosphate (UTP) protected cultured rat cardiomyocytes during hypoxia and explored the UTP signaling pathway leading to this cardioprotection. We found that UTP, but not UDP or uridine, significantly reduced cardiomyocyte death induced by hypoxia. Incubation with UTP for 1 h, before exposure to hypoxic conditions, protected the cells 24 h later. The cardioprotective effect of UTP was reduced in the presence of the P2 antagonist suramin. In addition, UTP caused a transient increase of [Ca2+]i in cardiomyocytes. Pyridoxal-5′-phosphate-6-azophenyl-2,4-disulfonate (PPADS) or Reactive blue 2 (RB-2), other antagonists of P2 receptors, abolished the [Ca2+]i elevation caused by UTP. We used various inhibitors of the Ca2+ signaling pathway to show that UTP elevated levels of [Ca2+]i, originating from intracellular sources, via activation of phospholipase C and the IP3 receptor. Interestingly, these inhibitors of the Ca2+ signaling pathway did not prevent the immediate protective effect caused by UTP. Although mitochondrial KATP channels are involved in other preconditioning mediator pathways, the involvement of these channels in the cardioprotective effect induced by UTP was ruled out, because 5-hydroxydecanoic acid (5-HD), a specific inhibitor of these channels, did not prevent the protection.

Summary During hypoxia, ATP was released from type I (glomus) cells in the carotid bodies. We studied the action of ATP on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of type I cells dissociated from rat carotid bodies using a Ca(2+) imaging technique. ATP did not affect the resting [Ca(2+)](i) but strongly suppressed the hypoxia-induced [Ca(2+)](i) elevations in type I cells. The order of purinoreceptor agonist potency in inhibiting the hypoxia response was 2-methylthioATP > ATP > ADP >> alpha, beta-methylene ATP > UTP, implicating the involvement of P2Y(1) receptors. Simultaneous measurements of membrane potential and [Ca(2+)](i) show that ATP inhibited the hypoxia-induced Ca(2+) signal by reversing the hypoxia-triggered depolarization. However, ATP did not oppose the hypoxia-mediated inhibition of the oxygen-sensitive TASK-like K(+) background current. Neither the inhibition of the large-conductance Ca(2+)-activated K(+) (maxi-K) channels nor the removal of extracellular Na(+) could affect the inhibitory action of ATP. Under normoxic condition, ATP caused hyperpolarization and increase in cell input resistance. These results suggest that the inhibitory action of ATP is mediated via the closure of background conductance(s) other than the TASK-like K(+), maxi-K or Na(+) channels. In summary, ATP exerts strong negative feedback regulation on hypoxia signaling in rat carotid type I cells.

A method for the enzymatic synthesis of [1-14C-N-acetyl, P18O2] cytidine monophosphate neuraminic acid (CMP-NeuAc) is described. Central to the synthesis of [1-14C-N-acetyl, P18O2]CMP-NeuAc was the enzymatic preparation of [γ-P18O3]ATP for use in a reaction with uridine kinase and cytidine to provide 5′-[P18O3]CMP. The [1-14C-N-acetyl, P18O2]CMP-NeuAc isotopomer was then synthesized from a reaction involving nucleoside monophosphate kinase, pyruvate kinase and CMP-NeuAc synthetase. The isolated reaction yield was 35%. Copyright © 2004 John Wiley & Sons, Ltd.

Although adenosine or ATP is known to control various physiological functions in the brain, including synaptic transmission, neuronal cell death, and neurite outgrowth via P1 or P2 purinergic receptors in the nervous system, little is known about the functions of many other purine derivatives. We examined the effects of various purines on survival in the cerebellar cortex of Purkinje cells with large cell bodies and highly branched dendrites, and it was found that some purine and pyrimidine derivatives influence Purkinje cell survival. Treatment with adenine, guanine, guanosine, guanine nucleotides, and uracil nucleotides protected Purkinje cells from cell death in the cerebellar primary cultures. Among the effective compounds, adenine had the most potent survival activities on Purkinje cells. Other adenine-based purines such as adenosine, AMP, ADP, and ATP did not promote Purkinje cell survival. Furthermore, metabolic inhibitors of adenine had no effect on the protective ability of adenine for Purkinje cells, suggesting that adenine itself, not adenine metabolites, maintains Purkinje cell survival. These results suggest that adenine is involved in the control of Purkinje cell survival in cerebellar primary cultures via a novel adenine-dependent mechanism.

Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The 96 to 1 region mediated UTP-induced OPN overexpression (27660%). Sequence analysis of this region revealed a potential site for AP-1 located at 76. When this AP-1 site was deleted, UTP-induced activation of the 96 to 1 region was totally inhibited. Thus, this AP-1 (76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation. (Circ Res. 2003;93:674-681.)

The involvement of P2Y receptors, which are activated by extracellular nucleotides, in proliferative regulation of human lung epithelial cells is unclear. Here we show that extracellular ATP and UTP stimulate bromodeoxyuridine (BrdU) incorporation into epithelial cell lines. The nucleotide efficacy profile [ATP = ADP > UDP ≥ UTP > adenosine ≥ 2-methylthioadenosine-5′-diphosphate, with α,β-methylene adenosine 5′-triphosphate, 2′,3′- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, AMP, UMP, and ATPαS inactive] and PCR analysis indicate involvement of P2Y2 and P2Y6 receptors. The signal transduction pathway, which, via the P2Y2 receptor, transmits the proliferative activity of ATP or UTP in A549 cells downstream of phospholipase C, depends on Ca2+/calmodulin-dependent protein kinase II and nuclear factor-κB, but not on protein kinase C. Signaling does not involve the mitogen-activated protein kinases extracellular signal-regulated kinases-1 and -2, the phosphatidylinositol 3-kinase pathway, or Src kinases. Thus nucleotides regulate proliferation of human lung epithelial cells by a novel pathway. The stimulatory effect of UTP, but not ATP, in A549 cells is attenuated by preincubation with interleukin-1β and interleukin-6, but not tumor ncerosis factor-α. This indicates an important role for the pyrimidine-activated P2Y receptor in the inflammatory response of lung epithelia. ATP antagonizes the antiproliferative effect of the anticancer drugs paclitaxel and etoposide, whereas it enhances the activity of cisplatin about fourfold. Thus pathways activated by extracellular nucleotides differentially control proliferation of lung epithelial tumor cells.

Leloir transferases, like UDP-d-xylosyl transferase and arabinosyl transferase, utilize nucleoside diphosphate sugars to build up plant oligo- and polysaccharides. By the described, scalable three-step synthesis a simple route is described to arrive at the respective enzyme substrates, which are otherwise difficult to obtain.

NB1011 [E-5-(2-bromovinyl)-2'-deoxyuridine-5'-(L-methylalaninyl)-phenylphosphoramidate], a phosphoramidate prodrug of E-5-(2-bromovinyl)-2'-deoxyuridine-5'-monophosphate (BVdUMP), is an investigational new anticancer drug. NB1011 targets thymidylate synthase (TS), which catalyzes the transformation of BVdUMP into cytotoxic reaction products. Due to the elevated levels of TS expression in tumor cells compared to normal cells, these cytotoxic products are preferentially generated inside tumor cells, and, as expected, NB1011 is more toxic to cells with higher levels of TS expression. Therefore, NB1011 therapy should kill tumor cells without severely damaging normal cells. Radiolabeled NB1011 was used to determine the intracellular fate of NB1011 reaction products and, possibly, the mechanism of action of this investigational new drug. We found significant incorporation of the radiolabel into cellular macromolecules. In contrast to our expectations that NB1011 product(s) would be incorporated into DNA, we discovered that cellular proteins were the labeled macromolecular fraction. Herein, we report that the intracellular transformation of NB1011 involves formation of the corresponding monophosphate, TS-dependent transformation into highly reactive intermediates, and subsequent incorporation into cellular proteins. TS itself appears to escape irreversible inactivation. Our data suggest that protein modification not DNA incorporation accounts for the therapeutic effect of NB1011. The proposed mechanism is rather unexpected for a nucleotide analogue and could lead to the discovery of new cellular protein targets for future drug design.

RNA molecules are thought to have been prominent in the early history of life on Earth because of their ability both to encode genetic information and to exhibit catalytic function. The modern genetic alphabet relies on two sets of complementary base pairs to store genetic information. However, owing to the chemical instability of cytosine, which readily deaminates to uracil, a primitive genetic system composed of the bases A, U, G and C may have been difficult to establish. It has been suggested that the first genetic material instead contained only a single base-pairing unit. Here we show that binary informational macromolecules, containing only two different nucleotide subunits, can act as catalysts. In vitro evolution was used to obtain ligase ribozymes composed of only 2,6-diaminopurine and uracil nucleotides, which catalyse the template-directed joining of two RNA molecules, one bearing a 5'-triphosphate and the other a 3'-hydroxyl. The active conformation of the fastest isolated ribozyme had a catalytic rate that was about 36,000-fold faster than the uncatalysed rate of reaction. This ribozyme is specific for the formation of biologically relevant 3',5'-phosphodiester linkages.

Folate based inhibitors of thymidylate synthase (TS) might facilitate binding of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) to TS similar to the natural reduced folate 5,10-methylenetetrahydrofolate (CH(2)-H(4)-folate). We studied the lipophilic, non-polyglutamatable antifolates Nolatrexed (NTX) and AG331 and antifolates, that can have a polyglutamate side chain like the natural folate CH(2)-H(4)-folate; GW1843U89, Raltitrexed (RTX) and Multi-targetted antifolate (MTA) and pentaglutamates (RTX-Glu(5) and MTA-Glu(5)). The capacity of these compounds to facilitate the binding of [(3)H]FdUMP to Lactobacillus casei TS and an ammoniumsulphate precipitate of human TS was investigated. Only NTX, RTX-Glu(5) and MTA-Glu(5) facilitated FdUMP binding to L. casei TS and their dissociation constant K(d) (0.2-0.7 microM) was low compared to CH(2)-H(4)-folate (2.0 microM). The small lipophilic molecule NTX was favorable to the larger AG331. Polyglutamylation, as indicated by the difference in effect of RTX vs. RTX-Glu(5) and MTA vs. MTA-Glu(5), seems to be important for a classical antifolate to facilitate binding of FdUMP to bacterial TS. Effects of antifolates on FdUMP binding to human TS were different. At a low concentration (0.05 microM) NTX, RTX-Glu(5) and MTA-Glu(5) facilitated 3-5 times higher binding of [(3)H]FdUMP to TS than CH(2)-H(4)-folate. At higher concentrations (0.3-5 microM) of NTX, RTX-Glu(5) and MTA-Glu(5) the FdUMP binding decreased. The complex remained stable in the absence of (anti)folate for at least 24hr. The K(d) values of the antifolates for human TS varied from 19 to 387 nM, while the K(d) of CH(2)-H(4)-folate for human TS was 351 nM. The Hill coefficients, which indicated the type of cooperativity of the antifolates in the binding of FdUMP to TS were positive (0.58-0.99) at low concentrations (0.3 microM) and negative (-0.35 to -0.81) at concentrations0.3 microM except for GW1843U89, which only showed negative cooperativity (-1.70). It was shown with [(14)C]NTX that when the binding of FdUMP decreased at high NTX concentrations, the binding of NTX to TS still increased. This also held for the natural substrate dUMP. The negative cooperativity of the antifolates was clearly concentration dependent. The difference between human and L. casei TS in the FdUMP binding assays with antifolates can possibly be explained by interaction of the two subunits of human TS, which was absent in L. casei TS. The binding of antifolates to one of the two subunits induced a conformational change of the other subunit. This change no longer allowed the binding of FdUMP or dUMP at the active site. In conclusion this study showed that antifolates enhanced the binding of FdUMP to TS, especially at low antifolate concentrations, that are also clinically achievable, e.g. in human plasma.