In order to isolate new subtypes of P 2 purinoceptors, sets of degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P 2Y /P2Y 1 and murine neuroblastoma P 2U /P2Y 2 receptors. Their use in polymerase chain reaction (PCR) experiments on human genomic DNA amplified, among other things, a 712-base pair sequence, that was used as a probe to screen a human genomic DNA library. Several clones corresponding to a single locus were isolated, and the sequence analysis revealed an intronless 1095-base pair open reading frame. The deduced amino acid sequence is consistent with a G protein-coupled receptor and exhibits 51% identity with the human P2Y 2 receptor and 35% with the chick P2Y 1 receptor. A close comparison with the human P2Y 2 sequence reveals the conservation of histidine 262, arginine 265, lysine 289, and arginine 292, which were reported to be involved in nucleotide binding (Erb, L., Garrad, R., Wang, Y., Quinn, T., Turner, J. T., and Weisman, G. A. (1995) J. Biol. Chem. 270, 4185-4188). Northern blot analysis detected a 1.8-kilobase messenger RNA in human placenta. The coding sequence was inserted in the pcDNA3 vector in order to transfect 1321N1 human astrocytoma cells. In cells stably expressing the receptor, UTP and UDP stimulated the formation of inositol phosphates with equivalent potency and maximal effect, ATP behaved as a partial agonist, and ADP was almost inactive. We have thus cloned a new member of the G protein-coupled P 2 purinergic receptor family, which functionally behaves as a pyrimidinergic receptor.The cloning of several receptors for ATP has been reported since 1993. In keeping with the latest nomenclature proposal, these P 2 purinergic receptors can be subdivided into two classes: G protein-coupled receptors, or P2Y receptors, and receptors with intrinsic ion channel activity or P2X receptors (2). Two distinct rat P2X receptors have been cloned, respectively, from the vas deferens (3) and pheochromocytoma PC12 cells (4): they have a characteristic topology, with two hydrophobic putatively membrane-spanning segments and an ion pore motif reminiscent of potassium channels. In the P2Y family, the sequences of two subtypes, both coupled to phospholipase C, have been published: chick (5), turkey (6), bovine (7), and mouse and rat (8) P2Y 1 receptors (formerly called P 2Y ); murine (9, 10), rat (11), and human (12) P2Y 2 receptors (previously named P 2U ) on the other hand. In addition, a P2Y 3 receptor, with a preference for ADP over ATP, has been cloned from chick brain, but its sequence is not yet published (13). Furthermore, the 6H1 orphan receptor, cloned from activated chicken T lymphocytes, exhibits a significant degree of homology to the P2Y 1 and P2Y 2 receptors, suggesting that it also belongs to the P2Y family, although its responsiveness to nucleotides has not yet been demonstrated (14). In this paper, we describe the cloning of a new member of the P2Y family, tentatively called P2Y 4 , which e...

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Sets of degenerate oligonucleotide primers synthesized on the basis of the best conserved regions of the chick brain P2Y/P2Y1 and the murine neuroblastoma P2U/P2Y2 receptors were used in polymerase chain reaction experiments on human genomic DNA. An amplified fragment of 712 base pairs was then used as a probe to screen a human genomic DNA library. Several clones were isolated and sequencing revealed an intronless 1122 base pair open reading frame. The corresponding amino acid sequence revealed 83% identity with the chick brain P2Y1 receptor and 34% with the murine neuroblastoma P2Y2 receptor. In COS-7 cells transfected with the coding sequence inserted into the pcDNA3 expression vector, 2-methylthioATP and ATP produced a strong stimulation of inositol phosphates, a typical response of a P2Y1 receptor. Northern blot analysis detected a 6.7 kilobase messenger RNA in most human tissues, the strongest signals being observed in prostate and ovary.

Extracellular ATP plays an important role in the regulation of prostacyclin and nitric oxide release from vascular endothelial cells. These cellular responses to ATP are generally attributed to the stimulation of the P2y subtype of P2 purinergic receptors. However, it has recently been suggested that two types of ATP receptors might coexist on endothelial cells. To evaluate this hypothesis, we examined the effects of P2y receptor agonists 2-methylthioadenosine 5'-triphosphate (2MeSATP) and 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) and of UTP on the accumulation of inositol phosphates in bovine aortic endothelial cells. BzATP, 2MeSATP, and UTP produced a smaller maximal effect than ATP. The effects of 2MeSATP and UTP were additive, whereas the effects of ATP and either UTP or 2MeSATP were not. Prior exposure to UTP reduced the subsequent response to UTP to 12% of the control response, whereas the response to 2MeSATP was decreased to 61%. Reciprocally, preincubation with 2MeSATP reduced the subsequent response to 2MeSATP to 23% of the control response, whereas the response to UTP was reduced to 73%. Pertussis toxin pretreatment decreased the response to both ATP and UTP (65% and 70% inhibition, respectively), whereas the response to 2MeSATP was not modified. Our data support the hypothesis that two classes of receptors recognizing ATP are expressed on bovine aortic endothelial cells.

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Such a synergistic effect of noncovalent PEGylation of poly(amino methacrylate)/DNA complexes allows a new and versatile approach to tune up transfection efficiency.

BackgroundImmunotherapy with peptide hydrolysates from Lolium perenne (LPP) is an alternative treatment for seasonal allergic rhinitis with or without asthma. The aim of this study was to assess the clinical efficacy and safety of a cumulative dose of 170 μg LPP administered subcutaneously over 3 weeks.MethodsIn a randomized, double‐blind, placebo‐controlled trial, 554 adults with grass pollen rhinoconjunctivitis were randomized (1:2 ratio) to receive 8 subcutaneous injections of placebo or 170 μg LPP administered in increasing doses in 4 visits over 3 weeks. The primary outcome was the combined symptom and medication score (CSMS) measured over the peak pollen season. Reactivity to conjunctival provocation test (CPT) and quality of life (QOL) was assessed as secondary endpoints.ResultsThe mean reduction in CSMS in the LPP vs placebo group was −15.5% (P = .041) during the peak period and −17.9% (P = .029) over the entire pollen season. LPP‐treated group had a reduced reactivity to CPT (P < .001) and, during the pollen season, a lower rhinoconjunctivitis QOL global score (P = .005) compared with placebo group. Mostly mild and WAO grade 1 early systemic reaction (ESR) were observed ≤30 minutes in 10.5% of LPP‐treated patients, whereas 3 patients with a medical history of asthma (<1%) experienced a serious ESR that resolved with rescue medication.Conclusion Lolium perenne pollen peptides administered over 3 weeks before the grass pollen season significantly reduced seasonal symptoms and was generally safe and well‐tolerated.

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