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  • Identification and biochemical characterization of unique secretory nucleases of the human enteric pathogen, Entamoeba histolytica. 17766245

    The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these secretory enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.
    Document Type:
    Reference
    Product Catalog Number:
    ECM600
    Product Catalog Name:
    uPA Activity Assay Kit

  • P2X2 purinoreceptor protein in hypothalamic neurons associated with the regulation of food intake. 20736052

    Purines such as ATP act as extracellular messengers through specific purinergic receptors. Three different classes of purinergic receptors have been identified and termed P1, P2X and P2Y. The purinergic receptor subunit P2X2 is a ligand-gated ion channel that is widely expressed by neurons in the central nervous system. The aim of this study was to study the cellular localization and to identify the chemical phenotypes of ionotropic P2X2 receptor (P2X2R)-containing neurons in the rat mediobasal hypothalamus by immunohistochemistry using three different P2X2R antisera, with special reference to neurons that influence food intake and body weight. P2X2R immunoreactivity was mainly observed in cell bodies and neural extensions located in the ventromedial part of the hypothalamic arcuate nucleus, a subregion of the nucleus with a weak blood-brain barrier (BBB). At the subcellular level, P2X2R immunoreactivity was located to the periphery of individual cells, likely representing the plasma membrane. Many P2X2R-immunoreactive cell bodies in the arcuate nucleus contained the orexigenic peptides neuropeptide Y (NPY) and agouti-related protein (AgRP), and the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). In contrast, P2X2R immunoreactive cell bodies of the arcuate nucles only occasionally contained the anorexigenic peptides α-melanocyte-stimulating hormone (α-MSH) or cocaine- and amphetamine-regulated transcript (CART), or the opioid peptide dynorphin (DYN). There was no evidence for colocalization of P2X2R with somatostatin or neuronal nitric oxide synthase (nNOS) in neurons of the arcuate nucleus. In the parvocellular part of the paraventricular nucleus, P2X2R was demonstrated in some corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH) and CART-containing neurons. In some cell bodies of the lateral hypothalamic area P2X2R was colocalized with DYN. The presence of P2X2R immunoreactivity in primarily orexigenic NPY/AgRP/GABA-containing neurons of the arcuate nucleus suggests that extracellular ATP has a regulatory action on this neuronal population located in a strategic position of the brain.
    Document Type:
    Reference
    Product Catalog Number:
    AB5087
    Product Catalog Name:
    Anti-Melanocyte Stimulating Hormone α Antibody

  • Chemical phenotypes of P2X2 purinoreceptor immunoreactive cell bodies in the area postrema. 22038573

    Purines such as adenosine 5'-triphosphate (ATP) act as extracellular messengers through specific purinergic receptors. Three different classes of purinergic receptors have been identified and termed P1, P2X, and P2Y. The purinergic receptor subunit P2X2 is a ligand-gated ion channel that is widely expressed by neurons in the CNS. In the brainstem medulla oblongata, the ionotropic P2X2 receptor (P2X2R) is enriched in the area postrema (AP). Two different antisera to P2X2R were used to determine the chemical nature of P2X2R immunoreactive cell bodies in the rat AP, an area lacking a blood-brain barrier. Subcellularly, P2X2R immunoreactivity was located to the periphery of individual cell bodies. The majority of P2X2R-immunoreactive cells were shown to contain tyrosine hydroxylase (TH) (63.5 ± 7.7%) and dopamine β-hydroxylase (61.5 ± 5.1%). Phenylethanolamine N-methyltransferase (PNMT)-containing cells were not detected in the AP, supporting a noradrenergic nature of P2X2R cells in the AP. There were no P2X2R-immunoreactive cells in the AP that contained the GABA-synthesizing enzyme glutamic acid decarboxylase 65. Only single vesicular glutamate transporter 2-immunoreactive cell bodies that were not P2X2R-positive were demonstrated in the AP. Some P2X2R-positive cells in the AP were immunoreactive for the neuropeptides substance P and pituitary adenylate cyclase-activating polypeptide, whereas dynorphin-, enkephalin-, or cholecystokinin-positive cells were not P2X2R-immunoreactive. Presence of P2X2R in a majority of noradrenergic cells of the AP implies that ATP may have a regulatory action on neuronal noradrenaline release from the AP, a circumventricular organ with a strategic position enabling interactions between circulating substances and the central nervous system.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids. 3876384

    Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Biosensor measurement of purine release from cerebellar cultures and slices. 21103217

    We have previously described an action-potential and Ca(2+)-dependent form of adenosine release in the molecular layer of cerebellar slices. The most likely source of the adenosine is the parallel fibres, the axons of granule cells. Using microelectrode biosensors, we have therefore investigated whether cultured granule cells (from postnatal day 7-8 rats) can release adenosine. Although no purine release could be detected in response to focal electrical stimulation, purine (adenosine, inosine or hypoxanthine) release occurred in response to an increase in extracellular K(+) concentration from 3 to 25 mM coupled with addition of 1 mM glutamate. The mechanism of purine release was transport from the cytoplasm via an ENT transporter. This process did not require action-potential firing but was Ca(2+)dependent. The major purine released was not adenosine, but was either inosine or hypoxanthine. In order for inosine/hypoxanthine release to occur, cultures had to contain both granule cells and glial cells; neither cellular component was sufficient alone. Using the same stimulus in cerebellar slices (postnatal day 7-25), it was possible to release purines. The release however was not blocked by ENT blockers and there was a shift in the Ca(2+) dependence during development. This data from cultures and slices further illustrates the complexities of purine release, which is dependent on cellular composition and developmental stage.
    Document Type:
    Reference
    Product Catalog Number:
    AB5541
    Product Catalog Name:
    Anti-Glial Fibrillary Acidic Protein Antibody

  • Expression of P2Y receptors in the rat anterior pituitary. 21567129

    In this study, the distribution patterns of P2Y(1), P2Y(2) P2Y(4), P2Y(6), P2Y(12), and P2Y(13) receptors in the anterior pituitary cells of rat were studied with double-labeling immunofluorescence and Western blot. The results showed that P2Y receptors were widely expressed in the anterior pituitary. P2Y(1) and P2Y(4) receptors were found to be expressed in the majority of gonadotrophs and thyrotrophs, P2Y(2) receptors were expressed in a small subpopulation of lactotrophs and almost all the folliculo-stellate cells, that were also stained with S100 protein immunoreactivity. P2Y(6) receptors were expressed in macrophages. P2Y(13) receptors were expressed in a small subpopulation of cells in the rat anterior pituitary, the identity of which needs to be clarified. P2Y(1) and P2Y(4) receptors are co-expressed in some gonadotrophs and thyrotrophs. Corticotrophs and somatotrophs were found not to express P2Y receptors in this study. FSH and TSH were shown to coexist in the same endocrine cells in rat anterior pituitary. The present data suggests that purines and/or pyrimidines could be involved in regulating the functions of gonadotrophs and thyrotrophs via P2Y(1) and P2Y(4) receptors, some lactotrophs via P2Y(2) receptors, and folliculo-stellate cells via P2Y(2) receptors in the rat anterior pituitary.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Lack of endogenous adenosine tonus on sympathetic neurotransmission in spontaneously hypertensive rat mesenteric artery. 25158061

    Increased sympathetic activity has been implicated in hypertension. Adenosine has been shown to play a role in blood flow regulation. In the present study, the endogenous adenosine neuromodulatory role, in mesenteric arteries from normotensive and spontaneously hypertensive rats, was investigated.The role of endogenous adenosine in sympathetic neurotransmission was studied using electrically-evoked [3H]-noradrenaline release experiments. Purine content was determined by HPLC with fluorescence detection. Localization of adenosine A1 or A2A receptors in adventitia of mesenteric arteries was investigated by Laser Scanning Confocal Microscopy. Results indicate a higher electrically-evoked noradrenaline release from hypertensive mesenteric arteries. The tonic inhibitory modulation of noradrenaline release is mediated by adenosine A1 receptors and is lacking in arteries from hypertensive animals, despite their purine levels being higher comparatively to those determined in normotensive ones. Tonic facilitatory adenosine A2A receptor-mediated effects were absent in arteries from both strains. Immunohistochemistry revealed an adenosine A1 receptors redistribution from sympathetic fibers to Schwann cells, in adventitia of hypertensive mesenteric arteries which can explain, at least in part, the absence of effects observed for these receptors.Data highlight the role of purines in hypertension revealing that an increase in sympathetic activity in hypertensive arteries is occurring due to a higher noradrenaline/ATP release from sympathetic nerves and the loss of endogenous adenosine inhibitory tonus. The observed nerve-to-glial redistribution of inhibitory adenosine A1 receptors in hypertensive arteries may explain the latter effect.
    Document Type:
    Reference
    Product Catalog Number:
    MAB318
    Product Catalog Name:
    Anti-Tyrosine Hydroxylase Antibody, clone LNC1