Uracil Research Articles

Disruption of URA-3 gene of a paclitaxel-producing fungus N8

Objective In order to get genetic markers,an auxotrophic paclitaxel-producing fungus named Pestalotiopsis malicola N8 strain was isolated by genetic modification.Methods Based on the homologous recombination,URA-3 which is the key gene for uracil synthetic route of Pestalotiopsis malicola N8 strain was knocked out.The transformants were screened by minimal medium with the combination of 5-fluoroorotic acid (5-FOA) and uracil.Results The results showed that the uracil auxotrophic strain was able to grow in the minimal medium containing 5-FOA and uracil while the wild type strain was not.Conclusions The uracil auxotrophic strain can be used as a new selection marker for future gene function studies of N8 strain. Key words: Paclitaxel-producing fungus; URA-3; Gene knock-out

Clinical characteristics and analysis of mass spectrometric data in patients with ornithine transcarbamylase deficiency

To explore the clinical manifestations and biochemical characteristics of patients with ornithine transcarbamylase deficiency (OTCD) so as to increase the clinician awareness for this disease. The clinical manifestations, blood ammonia levels, citrulline levels, urinary orotic acid and uracil levels were analyzed for 40 patients with OTCD from 2005 to 2013. And comparisons were made with 25 healthy children. Among them, the median age of onset was 1.4 years (3 days-29 years). The major clinical manifestations were feeding difficulties, persistent vomiting, convulsions, unconsciousness and hyperammonemia, etc. The blood levels of citrulline in these patients were significantly lower than those of the control group (6.35 (1.84-21.11) vs 13.65 (10.23-24.52) µmol/L, P < 0.05) . The urinary levels of orotic acid and uracil in these patients were significantly higher than those in the control group (167.77 (1.21-1 650.45) vs 0.25 (0-2.32) mmol/molCr, 52.67 (3.50-338.64) vs 0.69 (0-2.87) mmol/molCr, P < 0.05) . For patients with hyperammonemia, the decreased levels of citrulline in blood tested by tandem mass spectrometry and increased orotic acid and uracil in urine on gas chromatography-mass spectrometry may aid the diagnosis OTCD.

Microglia P2Y₆ receptors mediate nitric oxide release and astrocyte apoptosis.

BackgroundDuring cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis.MethodsCo-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining.ResultsUTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia.ConclusionsIn LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.

Thin Layer Chromatography of Pesticides and Their Residues

Recent progress in the analysis of organo-phosphorus, organo-chlorine, carbamate, urea, uracil pesticides and their residues by thin layer chromatorgraphic methods employing chemical and enzymatic methods is reviewed.

Ultrasound microbubble-mediated cytosine deaminsae: uracil phosphoribosyltransferase suicide gene for esophageal cancer in vitro

Objective To explore the transfection efficiency of cytosine deaminsae:uracil phosphoribosyltransferase/5-flurocytosine (CD:UPRT/5-FC) suicide gene system mediated by ultrasound microbubble (UM) for esophageal cancer in vitro,and indentify the effects on the cell viability.Methods EC9706 cells(l × 108/L) were seeded in the 12-well plates,and randomly divided into 4 groups:A.blank control group; B.CD:UPRT gene only; C.UM only; D.CD:UPRT gene and UM.After transfection,the expression of green fluorescent protein (gfp) was observed under the fluorescent microscopy and quantified by flow cytometry.Western blotting was used to detect the expression of CD protein.Different concentrations (0-160 mg/L) of 5-FC were added into the culture medium,then cells were cultured for another 36 h.The viability of EC9706 cells was measured by CCK-8 assay.Results (1) Gene fluorescence intensity and tansfection efficiency in group D were higher than other groups.The gene transfection efficiency in groups A,B,C,D and E was 0％,1.42％,0％ and 32.60％ respectively.The cell viability had no significant difference among groups before addition of 5-FC ; (2) CD protein in group D was higher than other groups (P ＜ 0.05).(3) 5-FC had strong antitumor effect on successfully transfected esophageal cancer cells,almost all EC9706 cells in group D were killed when the concentration of 5-FC was above 80 mg/L.Conclusion Ultrasound microbubble can enhance the efficiency of gene transfection without obvious damage to cell viability in EC9706 cells.The method can also enhance the killing effect of CD:UPRT/5-FC suicide gene system in vitro. Key words: Ultrasound microbubble ; Esophageal cancer; Cytosine deaminsae : uracil phosphoribosyltransferase/5-flurocytosine ; Gene therapy

ATP released via pannexin-1 hemichannels mediates bladder overactivity triggered by urothelial P2Y6 receptors

In contrast to the well-known signaling role of urothelial ATP to control bladder function, the hypothesis that uracil nucleotides (UTP and/or UDP) also exert autocrine/paracrine actions only recently gained experimental support. Urothelial cells express UDP-sensitive P2Y6 receptors, yet their role in the control of bladder activity has been mostly neglected. This study was designed to investigate the ability of PSB0474, a stable UDP analogue which exhibits selectivity for P2Y6 receptors, to modulate urodynamic responses in the anaesthetized rat in vivo. Instillation of PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions. PSB0474-induced bladder overactivity was prevented by the selective P2Y6 antagonist, MRS2578. The increase in the VF produced by PSB0474 was also blocked by inhibitors of pannexin-1 hemichannels, 10Panx or carbenoxolone, when these drugs were applied inside the bladder lumen but not when they were administered intravenously. Reduction of hemichannels pore permeability with H1152 also prevented PSB0474-induced bladder overactivity, but the exocytosis inhibitor, Exo-1, was inactive. PSB0474 increased by 3-fold the urinary ATP content. Implication of hemichannels permeability on PSB0474-induced ATP release was demonstrated by real-time fluorescence video-microscopy measuring the uptake of propidium iodide by intact urothelial cells in the absence and in the presence of MRS2578 or carbenoxolone. Confocal microscopy studies confirmed the co-localization of pannexin-1 and P2Y6 receptors in the rat urothelium. Data indicate that activation of P2Y6 receptors causes bladder overactivity in the anaesthetized rat indirectly by releasing ATP from the urothelium via pannexin-1 hemichannels.

Enigmatic GPCR Finds a Stimulating Drug

GPR17 is an orphan G protein-coupled receptor involved in orchestration of oligodendrocyte differentiation and myelination in the central nervous system. In this issue of Science Signaling, Hennen et al. used a signaling pathway-unbiased screen to identify two small molecule activators of this receptor. One of these, MDL29951, was carried forward to illustrate GPR17-dependent activation of Gαi- and Gαq-promoted signaling pathways in cell lines expressing recombinant GPR17, whereas no effect was observed with previously proposed but dubitable agonists (uracil nucleotides and cysteinyl leukotrienes) of this receptor. Conversely, MDL29951 did not activate any of the known uracil or adenine nucleotide-activated P2Y receptors or cysteinyl leukotriene receptors. Gαi- and Gαq-dependent signaling responses also were observed in primary rat oligodendrocytes in the presence of MDL29951. Moreover, MDL29951 diminished myelination in primary oligodendrocytes isolated from heterozygous mice but had no effect on myelination in oligodendrocytes from GPR17 knockout mice. Effects of a small-molecule GPR17 agonist observed during oligodendrocyte differentiation support the idea that development of antagonists of GPR17 is a rational goal for elaboration of pharmacotherapies in demyelinating diseases.

Dual role of P2Y6 receptors in the human urinary bladder: On the role of ATP released from the urothelium

ATP released from urothelium during bladder distention initiates the micturition reflex through the activation of P2X3 receptors on suburothelial sensory nerve fibers. Pathological activation of this pathway underlies bladder pain sensation and detrusor hyperactivity. ATP can be also released as a co-transmitter with acetylcholine from parasympathetic motor nerves; ATP-sensitive P2X1 receptors prevail on the detrusor smooth muscle. Contrasting to the compelling evidence for the extracellular signaling role of ATP, the hypothesis that uracil nucleotides exert autocrine/paracrine actions has recently gained experimental support.

Is GPR17 a P2Y/Leukotriene Receptor? Examination of Uracil Nucleotides, Nucleotide Sugars, and Cysteinyl Leukotrienes as Agonists of GPR17

The orphan receptor GPR17 has been reported to be activated by UDP, UDP-sugars, and cysteinyl leukotrienes, and coupled to intracellular Ca(2+) mobilization and inhibition of cAMP accumulation, but other studies have reported either a different agonist profile or lack of agonist activity altogether. To determine if GPR17 is activated by uracil nucleotides and leukotrienes, the hemagglutinin-tagged receptor was expressed in five different cell lines and the signaling properties of the receptor were investigated. In C6, 1321N1, or Chinese hamster ovary (CHO) cells stably expressing GPR17, UDP, UDP-glucose, UDP-galactose, and cysteinyl leukotriene C4 (LTC4) all failed to promote inhibition of forskolin-stimulated cAMP accumulation, whereas both UDP and UDP-glucose promoted marked inhibition (>80%) of forskolin-stimulated cAMP accumulation in C6 and CHO cells expressing the P2Y14 receptor. Likewise, none of these compounds promoted accumulation of inositol phosphates in COS-7 or human embryonic kidney 293 cells transiently transfected with GPR17 alone or cotransfected with Gαq/i5, which links Gi-coupled receptors to the Gq-regulated phospholipase C (PLC) signaling pathway, or PLCε, which is activated by the Gα12/13 signaling pathway. Moreover, none of these compounds promoted internalization of GPR17 in 1321N1-GPR17 cells. Consistent with previous reports, coexpression experiments of GPR17 with cysteinyl leukotriene receptor 1 (CysLTR1) suggested that GPR17 acts as a negative regulator of CysLTR1. Taken together, these data suggest that UDP, UDP-glucose, UDP-galactose, and LTC4 are not the cognate ligands of GPR17.

REACTION OF 5,6-DIAMINO-1,3-DIMETHYLURACIL WITH ARYLIDENEACETONES AND ARYLIDENECYCLANONES

The reaction of 5,6-diamino-1,3-dimethyluracil dihydrochloride with mono- and diarylideneacetones and with cyclohexanones give 2,4-disubstituted 1,7,9-triazaspiro[4,5]dec-1-ene-6,8,10-triones. It was found that arylidenecyclopentanones form only 5-azomethines with retention of the amino group at position 6 of the uracil ring. Authors: N. N. Kolos, V. A. Chebanov, and V. D. Orlov. English Translation in Chemistry of Heterocyclic Compounds , 1999, 35 (9), pp 1085-1088 http://link.springer.com/article/10.1007/BF02251801

Uracil nucleotides enhance the degranulation of human basophils induced by anti-IgE antibody via a purinergic receptor (P6016)

Abstract The P2Y receptors (P2YRs) are a family of G protein-coupled purinergic receptors that are stimulated by ATP, ADP, UTP, UDP, and UDP-G, and their interaction induces a wide range of biological effects in various cells through phospholipase C activation, inositol triphosphate generation, and Ca2+ release from intracellular stores. In our previous study, we demonstrated that in neutrophils, ATP regulates chemotaxis and phagocytosis via P2YR. In the present study, we examined the distribution and function of P2YR in another innate immune cell type, basophils, the degranulation of which is associated with type I allergy. To date, P2YR have been subdivided into 8 different subclasses: P2Y1, 2, 4, 6, 11, 12, 13, and 14. Of these 8 subclasses, our RT-PCR results showed the predominant expression of P2Y2, P2Y4, and particularly P2Y6, which are known as uracil nucleotide-sensitive P2YRs. Interestingly, HPLC analysis indicated that basophils spontaneously secrete UTP, the ligand for P2Y2 and 4, whereas UDP, the ligand for P2Y6, is probably generated from UTP in a metabolic pathway. Although the addition of UTP and UDP did not directly induce basophil degranulation, these nucleotides clearly enhanced the degranulation induced by anti-IgE cross-linking. These results suggest that uracil nucleotides act as potent inducers of histamine release in basophils.

The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes

Base excision repair is hindered by nucleosomes. Outwardly oriented uracils near the nucleosome center are efficiently cleaved; however, polymerase β is strongly inhibited at these sites. The histone octamer presents different levels of constraints on BER, dependent on the structural requirements for enzyme activity. Chromatin remodeling is necessary to prevent accumulation of aborted intermediates in nucleosomes. Packaging of DNA into chromatin affects accessibility of DNA regulatory factors involved in transcription, replication, and repair. Evidence suggests that even in the nucleosome core particle (NCP), accessibility to damaged DNA is hindered by the presence of the histone octamer. Base excision repair is the major pathway in mammalian cells responsible for correcting a large number of chemically modified bases. We have measured the repair of site-specific uracil and single nucleotide gaps along the surface of the NCP. Our results indicate that removal of DNA lesions is greatly dependent on their rotational and translational positioning in NCPs. Significantly, the rate of uracil removal with outwardly oriented DNA backbones is 2-10-fold higher than those with inwardly oriented backbones. In general, uracils with inwardly oriented backbones farther away from the dyad center of the NCP are more accessible than those near the dyad. The translational positioning of outwardly oriented gaps is the key factor driving gap filling activity. An outwardly oriented gap near the DNA ends exhibits a 3-fold increase in gap filling activity as compared with one near the dyad with the same rotational orientation. Near the dyad, uracil DNA glycosylase/APE1 removes an outwardly oriented uracil efficiently; however, polymerase β activity is significantly inhibited at this site. These data suggest that the hindrance presented by the location of a DNA lesion is dependent on the structural requirements for enzyme catalysis. Therefore, remodeling at DNA damage sites in NCPs is critical for preventing accumulation of aborted intermediates and ensuring completion of base excision repair.

Exploring the role of the 5-substituent for the intrinsic fluorescence of 5-aryl and 5-heteroaryl uracil nucleotides: a systematic study

Derivatives of UMP (uridine monophosphate) with a fluorogenic substituent in position 5 represent a small but unique class of fluorophores, which has found important applications in chemical biology and biomolecular chemistry. In this study, we have synthesised a series of derivatives of the uracil nucleotides UMP, UDP and UTP with different aromatic and heteroaromatic substituents in position 5, in order to systematically investigate the influence of the 5-substituent on fluorescence emission. We have determined relevant photophysical parameters for all derivatives in this series, including quantum yields for the best fluorophores. The strongest fluorescence emission was observed with a 5-formylthien-2-yl substituent in position 5 of the uracil base, while the corresponding 3-formylthien-2-yl-substituted regioisomer was significantly less fluorescent. The 5-(5-formylthien-2-yl) uracil fluorophore was studied further in solvents of different polarity and proticity. In conjunction with results from a conformational analysis based on NMR data and computational experiments, these findings provide insights into the steric and electronic factors that govern fluorescence emission in this class of fluorophores. In particular, they highlight the interplay between fluorescence emission and conformation in this series. Finally, we carried out ligand-binding experiments with the 5-(5-formylthien-2-yl) uracil fluorophore and a UDP-sugar-dependent glycosyltransferase, demonstrating its utility for biological applications. The results from our photophysical and biological studies suggest, for the first time, a structural explanation for the fluorescence quenching effect that is observed upon binding of these fluorophores to a target protein.

Is GPR17 a P2Y/Leukotriene Receptor? Examination of Uracil Nucleotides, Nucleotide Sugars, and Cysteinyl Leukotrienes as Agonists of GPR17

Is GPR17 a P2Y/Leukotriene Receptor? Examination of Uracil Nucleotides, Nucleotide Sugars, and Cysteinyl Leukotrienes as Agonists of GPR17

The potentials of human adipose tissue derived mesenchymal stem cells in targeted therapy of experimental glioma

Glioblastoma is the most common primary malignant brain tumor in adults. With current standard therapy which includes extensive microsurgical resection along with concurrent chemoradiotherapy and adjuvant temozolomide (TMZ), the median survival of glioblastoma patients is only 14.60 months nowadays. Recent studies demonstrated that human adipose tissue derived mesenchymal stem cells (hAT⁃ MSCs) possessed the glioma ⁃ trophic migratory capacity. The engineered hAT ⁃ MSCs expressing herpes simplex virus⁃thymidine kinase (HSV⁃tk), yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy:: UPRT), and rabbit carboxylesterase (rCE) could exert inhibitory effects on glioma when combined with prodrugs, such as ganciclovir (GCV), 5⁃fluorocytosine (5⁃FC) and irinotecan (CPT⁃11), respectively. hAT⁃ MSCs carrying the oncolytic virus or expressing tumor necrosis factor ⁃ related apoptosis ⁃ inducing ligand (TRAIL) also could inhibit the growth of glioma. This paper summarizes the recent progress in this field to pave the way for hAT⁃MSCs based targeted therapy of glioma in future. DOI：10.3969/j.issn.1672⁃6731.2012.06.005

Using partial least squares and principal component regression in simultaneous spectrophotometric analysis of pyrimidine bases

This study, for the first time, applies multivariate spectrophotometric calibration for simultaneous determination of three pyrimidine bases including uracil (URA), cytosine (CYT) and thymine (THY). Although determination of these bases is of great importance from a physiological and pharmaceutical perspective, it could be a difficult task since there is some sort of spectral overlapping. The principal component regression (PCR) and partial least-squares (PLS) model were used to overcome this problem and to construct the calibration sets containing URA, CYT and THY in the concentration range of 1.12–22.42, 1.11–27.78 and 1.26–25.22μgmL−1, respectively. The absorption spectra were recorded from 220–320nm. The results showed that the NPCs for URA, CYT and THY were 6, 5 and 4 by PCR and 4, 5 and 3 by PLS, respectively. In addition, the RMSEPs for URA, CYT and THY were 0.7067, 0.5093 and 0.6371 by PCR and 0.5469, 0.2700 and 0.5087 by PLS, respectively. The proposed method yielded recoveries ranging from 93.85 to 107.45 by PLS and 90.48 to 111.42 by PCR. The method was successful in simultaneous determination of the three bases in urine, serum and plasma.

UDP made a highly promising stable, potent, and selective P2Y6-receptor agonist upon introduction of a boranophosphate moiety

P2Y6 nucleotide receptor (P2Y6-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 Pα position with methoxy and/or borano groups, 18–22. Compound 18A, Rp isomer of 5-OMe-UDP(α-B), is the most potent and P2Y6-R selective agonist currently known (EC50 0.008μM) being 19-fold more potent than UDP and showing no activity at uridine nucleotide receptors, P2Y2- and P2Y4-R. Analogue 18A was highly chemically stable under conditions mimicking gastric juice acidity (t1/2=16.9h). 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 (t1/2=17 vs 2.4, 11.9, and 21h for UDP, 5-OMe-UDP, and UDP(α-B), respectively). This newly discovered highly potent and physiologically stable P2Y6-R agonist may be of future therapeutic potential.

Role of ecto‐NTPDases on UDP‐sensitive P2Y6 receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women

This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y(2), P2Y(4), and P2Y(6)) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca(2+)](i) in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y(6) receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y(6) receptors with MRS 2578 prevented [Ca(2+)](i) rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y(2), P2Y(4), and P2Y(6) receptors. While the expression of P2Y(6) receptors remained fairly constant (7∼21 days), P2Y(2) and P2Y(4) became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y(6) receptors coupled to increases in [Ca(2+)](i) . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.

High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS

Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC-MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C(18) stationary phase, which enabled global analysis of nucleotides and their (13)C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 minutes of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD(+), NADH, NADP(+), NADPH, cyclic nucleotides, several UDP-hexoses, and all their (13)C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U-(13)C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, (13)C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their (13)C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations.

The new P2Y-like receptor G protein-coupled receptor 17 mediates acute neuronal injury and late microgliosis after focal cerebral ischemia in rats

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.