Histochemical Approach for Simultaneous Detection of Ectonucleotidase and Alkaline Phosphatase Activities in Tissues.
Losenkova K1, Paul M1, Irjala H2, Jalkanen S1, Yegutkin GG3.
Author information
1
MediCity Research Laboratory, University of Turku, Turku, Finland.
2
Department of Otorhinolaryngology, Head and Neck Surgery, Turku University Hospital and Turku University, Turku, Finland.
3
MediCity Research Laboratory, University of Turku, Turku, Finland. gennady.yegutkin@utu.fi.
Abstract
Studies on pathophysiology and the therapeutic potential of extracellular ATP and other purines represent an important and rapidly evolving field. The integral response of the cell is determined by multiple factors, including the release of endogenous ATP, co-expression of different types of nucleotide- and adenosine-selective receptors, as well as the specific makeup of ectoenzymes governing the duration and magnitude of purinergic signaling. Current findings support the presence of an extensive network of purine-converting ectoenzymes that are co-expressed to a variable extent among the mammalian tissues and share similarities in substrate specificity. Here, we describe a histochemical approach for simultaneous detection of ecto-nucleotidase and tissue-nonspecific alkaline phosphatase (TNAP) activities in the same tissue slice. Further employment of this technique for staining human palatine tonsil cryosections revealed selective distribution of the key ectoenzymes within certain tonsillar structures, including germinal centers and connective tissues (ecto-5'-nucleotidase/CD73), as well as interfollicular area (TNAP and NTPDase1/CD39).

KEYWORDS:
Ecto-5′-nucleotidase/CD73; Enzyme histochemistry; Human tonsils; NTPDase1/CD39; Tissue-nonspecific alkaline phosphatase

PMID: 31646483 DOI: 10.1007/978-1-4939-9717-6_7
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22.
Methods Mol Biol. 2020;2041:87-106. doi: 10.1007/978-1-4939-9717-6_6.
Developmental Expression of Ectonucleotidase and Purinergic Receptors Detection by Whole-Mount In Situ Hybridization in Xenopus Embryos.
Blanchard C1,2,3, Massé K4,5.
Author information
1
Univ. de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France.
2
CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France.
3
INSERM, U1215, Neurocentre Magendie, Univ. de Bordeaux, Bordeaux, France.
4
Univ. de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France. karine.masse@u-bordeaux.fr.
5
CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France. karine.masse@u-bordeaux.fr.
Abstract
Xenopus embryos are one of the most used animal models in developmental biology and are well suited for apprehending functions of signaling pathways during embryogenesis. To do so, it is necessary to be able to detect expression pattern of the key genes of these signaling pathways. Here we describe the whole-mount in situ hybridization technique to investigate the expression pattern of ectonucleotidases and purinergic receptors during embryonic development.

KEYWORDS:
Ectonucleotidases; Expression pattern; P2X receptors; Purinergic signaling pathway; Whole-mount in situ hybridization; Xenopus embryo

PMID: 31646482 DOI: 10.1007/978-1-4939-9717-6_6
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23.
Methods Mol Biol. 2020;2041:77-86. doi: 10.1007/978-1-4939-9717-6_5.
Using RNA Interference for Purinoceptor Knockdown In Vivo.
Amorim RP1, Lopes Cendes IT2, da Silva Fernandes MJ3,4.
Author information
1
Departamento de Neurologia e Neurocirurgia, Disciplina de Neurociência, Universidade Federal de São Paulo-UNIFESP, São Paulo, Brazil.
2
Departamento de Genetica Médica, Escola de Ciências Médicas da Universidade de Campinas-UNICAMP, Campinas, São Paulo, Brazil.
3
Departamento de Neurologia e Neurocirurgia, Disciplina de Neurociência, Universidade Federal de São Paulo-UNIFESP, São Paulo, Brazil. mjsfernandes19@unifesp.br.
4
Escola Paulista de Medicina, Universidade Federal de SÐo Paulo (EPM/UNIFESP), São Paulo, Brazil. mjsfernandes19@unifesp.br.
Abstract
RNA interference (RNAi) is a powerful post-transcriptional gene silencing (PTGS) induced by small double-stranded RNA (dsRNA). The method allows silencing of genes of interest by translation inhibition or by mRNA degradation. In this chapter, we provide a brief overview of the mechanisms involved in each step of gene silencing. A nonviral infusion of short siRNA into ventricular system of rats was used to study purinoceptor in the rat brain.

KEYWORDS:
Brain; Hippocampus; Purinergic receptor; RNAi; Rat

PMID: 31646481 DOI: 10.1007/978-1-4939-9717-6_5
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24.
Methods Mol Biol. 2020;2041:65-75. doi: 10.1007/978-1-4939-9717-6_4.
Homology Modeling of P2X Receptors.
Stavrou A1, Dayl S2, Schmid R3,4.
Author information
1
Department of Molecular and Cell Biology, University of Leicester, Leicester, UK.
2
Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq.
3
Department of Molecular and Cell Biology, University of Leicester, Leicester, UK. R.Schmid@le.ac.uk.
4
Leicester Institute of Structural and Chemical Biology (LISCB), University of Leicester, Leicester, UK. R.Schmid@le.ac.uk.
Abstract
Since the X-ray structure of the zebra fish P2X4 receptor in the closed state was published in 2009 homology modeling has been used to generate structural models for P2X receptors. In this chapter, we outline how to use the MODELLER software to generate such structural models for P2X receptors whose structures have not been solved yet.

KEYWORDS:
Homology modeling; Ion channel; MODELLER software; P2X receptor; Protein structure prediction

PMID: 31646480 DOI: 10.1007/978-1-4939-9717-6_4
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25.
Methods Mol Biol. 2020;2041:45-64. doi: 10.1007/978-1-4939-9717-6_3.
Agonists and Antagonists for Purinergic Receptors.
Müller CE1, Baqi Y2, Namasivayam V3.
Author information
1
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany. christa.mueller@uni-bonn.de.
2
Department of Chemistry, Sultan Qaboos University, Muscat, Oman.
3
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany.
Abstract
Membrane receptors that are activated by the purine nucleoside adenosine (adenosine receptors) or by purine or pyrimidine nucleotides (P2Y and P2X receptors) transduce extracellular signals to the cytosol. They play important roles in physiology and disease. The G protein-coupled adenosine receptors comprise four subtypes: A1, A2A, A2B, and A3. The G-protein-coupled P2Y receptors are subdivided into eight subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14, while the P2X receptors represent ATP-gated homomeric or heteromeric ion channels consisting of three subunits; the most important subunits are P2X1, P2X2, P2X3, P2X4, and P2X7. This chapter provides guidance for selecting suitable tool compounds for studying these large and important purine receptor families.

KEYWORDS:
Adenosine receptors; Agonists; Allosteric modulators; Antagonists; Binding site; Ligands; P2X receptors; P2Y receptors; Purine receptors; Structure; Tool compounds

PMID: 31646479 DOI: 10.1007/978-1-4939-9717-6_3
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26.
Methods Mol Biol. 2020;2041:17-43. doi: 10.1007/978-1-4939-9717-6_2.
Knockout and Knock-in Mouse Models to Study Purinergic Signaling.
Rumney RMH1, Górecki DC2,3.
Author information
1
School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK.
2
School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK. darek.gorecki@port.ac.uk.
3
Military Institute of Hygiene and Epidemiology, Warsaw, Poland. darek.gorecki@port.ac.uk.
Abstract
Purinergic signaling involves extracellular purines and pyrimidines acting upon specific cell surface purinoceptors classified into the P1, P2X, and P2Y families for nucleosides and nucleotides. This widespread signaling mechanism is active in all major tissues and influences a range of functions in health and disease. Orthologs to all but one of the human purinoceptors have been found in mouse, making this laboratory animal a useful model to study their function. Indeed, analyses of purinoceptors via knock-in or knockout approaches to produce gain or loss of function phenotypes have revealed several important therapeutic targets. None of the homozygous purinoceptor knockouts proved to be developmentally lethal, which suggest that either these receptors are not involved in key developmental processes or that the large number of receptors in each family allowed for functional compensation. Different models for the same purinoceptor often show compatible phenotypes but there have been examples of significant discrepancies. These revealed unexpected differences in the structure of human and mouse genes and emphasized the importance of the genetic background of different mouse strains. In this chapter, we provide an overview of the current knowledge and new trends in the modifications of purinoceptor genes in vivo. We discuss the resulting phenotypes, their applications and relative merits and limitations of mouse models available to study purinoceptor subtypes.

KEYWORDS:
Genetically modified animals; Knock-in; Knockout; Purinergic signaling; Purinoceptor

PMID: 31646478 DOI: 10.1007/978-1-4939-9717-6_2
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27.
Methods Mol Biol. 2020;2041:1-15. doi: 10.1007/978-1-4939-9717-6_1.
Introduction to Purinergic Signaling.
Burnstock G1.
Author information
1
Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, VIC, Australia. gburnstock@unimelb.edu.au.
Abstract
Purinergic signaling was proposed in 1972, after it was demonstrated that adenosine 5'-triphosphate (ATP) was a transmitter in nonadrenergic, noncholinergic inhibitory nerves supplying the guinea-pig taenia coli. Later, ATP was identified as an excitatory cotransmitter in sympathetic and parasympathetic nerves, and it is now apparent that ATP acts as a cotransmitter in most, if not all, nerves in both the peripheral nervous system and central nervous system (CNS). ATP acts as a short-term signaling molecule in neurotransmission, neuromodulation, and neurosecretion. It also has potent, long-term (trophic) roles in cell proliferation, differentiation, and death in development and regeneration. Receptors to purines and pyrimidines have been cloned and characterized: P1 adenosine receptors (with four subtypes), P2X ionotropic nucleotide receptors (seven subtypes) and P2Y metabotropic nucleotide receptors (eight subtypes). ATP is released from different cell types by mechanical deformation, and after release, it is rapidly broken down by ectonucleotidases. Purinergic receptors were expressed early in evolution and are widely distributed on many different nonneuronal cell types as well as neurons. Purinergic signaling is involved in embryonic development and in the activities of stem cells. There is a growing understanding about the pathophysiology of purinergic signaling and there are therapeutic developments for a variety of diseases, including stroke and thrombosis, osteoporosis, pain, chronic cough, kidney failure, bladder incontinence, cystic fibrosis, dry eye, cancer, and disorders of the CNS, including Alzheimer's, Parkinson's. and Huntington's disease, multiple sclerosis, epilepsy, migraine, and neuropsychiatric and mood disorders.

KEYWORDS:
ATP; Adenosine; Cotransmission; Cough; Development; Neurodegenerative diseases; Pain; Purinoceptor; Stem cells; Thrombosis

PMID: 31646477 DOI: 10.1007/978-1-4939-9717-6_1
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