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- Bazzurro, V., et al.
(author)
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Antisecretory Factor Modulates GABAA Receptor Activity in Neurons
- 2018
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In: Journal of Molecular Neuroscience. - : Springer Science and Business Media LLC. - 0895-8696 .- 1559-1166. ; 64:2, s. 312-320
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Journal article (peer-reviewed)abstract
- The antisecretory factor is an endogenous protein found in all mammalian tissues investigated so far. It acts by counteracting intestinal hypersecretion and various forms of inflammation, but the detailed mechanism of antisecretory factor (AF) action is unknown. We tested neuronal GABAA receptors by means of AF-16, a potent AF peptide derived from amino acids 36–51 from the NH2 part of AF. Cultured rat cerebellar granule cells were used, and the effects on the GABA-mediated chloride currents were determined by whole-cell patch clamp. Both the neurotransmitter GABA and AF-16 were added by perfusion of the experimental system. A 3-min AF-16 preincubation was more efficacious than 30s in significantly elevating the rapidly desensitizing GABA-activated chloride current. No effect was found on the tonic, slowly desensitizing current. The GABA-activated current increase by AF-16 demonstrated a low k of 41pM with a maximal increase of 37% persisting for some minutes after AF washout, independent from GABA concentration. This indicates an effect on the maximal stimulation (E%Max) excluding an altered affinity between GABA and its receptor. An immunocytochemical fluorescence approach with anti γ2 subunit antibodies demonstrated an increased expression of GABAA receptors. Thus, both the electrophysiological and the immunofluorescence approach indicate an increased appearance of GABAA receptors on the neuronal membrane. The rationale of the experiments was to test the effect of AF on a defined neuronal population of GABAA receptors. The implications of the results on the impact of AF on the enteric nervous system or on brain function are discussed. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.
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| 2. |
- Bazzurro, V., et al.
(author)
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Involvement of GABA(A) receptors containing alpha(6) subtypes in antisecretory factor activity on rat cerebellar granule cells studied by two-photon uncaging
- 2022
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In: European Journal of Neuroscience. - : Wiley. - 0953-816X .- 1460-9568. ; 56:5, s. 4505-4513
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Journal article (peer-reviewed)abstract
- The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABA(A) receptor populations in cerebellar granule cells, we took advantage of the two-photon uncaging method as this technique allows to stimulate the cell locally in well-identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF-16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABA(A) receptors, containing the alpha(6) subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell.
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| 3. |
- Niborski, Leticia L., et al.
(author)
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Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system
- 2021
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In: EBioMedicine. - : Elsevier. - 2352-3964. ; 63
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Journal article (peer-reviewed)abstract
- Background: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. Methods: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein targets orthologues, focusing mainly on post-translational modifications. Findings: A2R1 recognizes a parasite protein of similar to 50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. Interpretation: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.
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