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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 55 (1990), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 ± 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44–10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44–10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44–10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with Mab 44–10 were located immunocytochemically in sections of rat brain. 5-HT immunoreactivity was localized simultaneously in the same sections by using affinity-purified rabbit anti-5-HT antibodies and species-specific secondary antibodies coupled to a contrasting fluorophore. MAb 44–10 immunostaining involved neuronal cell bodies, neurites, and terminals. This immunostaining was intense within the nuclei of the median raphe and the B9 cell group. Coincident expression with 5-HT was observed; however, MAb 44–10 also immunostained many neurons in which 5-HT immunoreactivity was not seen. These observations may indicate that SBP is distributed more widely in the brain than 5-HT; however, because SBP immunoreactivity is not found in nonserotonergic neurons when monospecific polyclonal antibodies are used for immunocytochemistry, it seems more likely that some nonserotonergic neurons contain another protein (such as the 68-kDa SBP) that also contains an epitope recognized by MAb 44–10. Nevertheless, these data demonstrate that MAb 44–10 reacts with the 5-HT binding domain of 45-kDa and 56-kDa SBP and will be a valuable tool for analyzing these proteins.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    American Journal of Anatomy 194 (1992), S. 142-154 
    ISSN: 0002-9106
    Keywords: Neural crest ; Development ; Pancreatic innervation ; Gut ; Enteric nervous system ; Phenotypic expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Neurons in ganglia of the myenteric plexus of the duodenum and stomach have recently been demonstrated to innervate pancreatic ganglia and transsynaptically to excite acinar and islet cells. The hypothesis that crestderived cells first colonize the foregut and secondarily enter the pancreas by way of the pancreatic buds was tested. Studies were done with fetal rats (days E11-E15). Pancreatic rudiments and foregut were explanted separately and in co-culture. The development of neurons in the explants, identified by demonstrating the immunoreactivities of neurofilaments and growthassociated protein-43 (GAP-43), provided an indirect assay for the presence of neural precursors in the tissue at the time of explantation. Cells of putative neural crest origin were visualized immunocytochemically using the monoclonal antibody, NC-1. Additional markers included the immunoreactivities of dopamine-β-hydroxylase (DBH), which is expressed by vagal crest-derived cells that colonize the bowel, neuropeptides (substance P and neuropeptide Y [NPY]) found in mature pancreatic neurons, and serotonin (5-HT), which is located in the cell bodies of enteric but not pancreatic neurons. Neurons were detected in cultures of foregut, but not pancreas, when these tissues were explanted by themselves at days E11 and E12. At E11 neural precursors did not leave explants of bowel or migrate into co-cultured pancreatic rudiments. When the foregut was explanted at E12, however, neural precursors migrated away from the bowel, giving rise both to distant ganglia and to neurons within co-cultured pancreatic rudiments. Intrapancreatic ganglia developed in the co-cultures even when the pancreatic attachment to the bowel was severed. Neurons appeared in pancreatic rudiments explanted by themselves on day E13. Neurons developing in pancreatic explants expressed the immunoreactivities of DBH, substance P, and NPY, but not 5-HT. These observations support the idea that pancreatic ganglia develop from crest-derived cells that first colonize the fetal rat foregut and there acquire the ability to colonize the pancreas. A later migration into the pancreatic rudiments of a subset of the original émigrés or their progeny between days E12 and E13 gives rise to a network of pancreatic ganglia that can be regarded as an extension of the enteric nervous system. © 1992 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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