Morphometric characterization of the neuromuscular junction of rodents intoxicated with 2,4-dithiobiuret: evidence that nerve terminal recycling processes contribute to muscle weakness.
ABSTRACT 2,4-Dithiobiuret (DTB) causes ascending motor weakness when given chronically to rodents. In muscles of animals with DTB-induced weakness, quantal release of acetylcholine (ACh) is impaired. We examined in detail the structural changes that occurred at neuromuscular junctions and their associated Schwann cells of extensor digitorum longus (EDL) muscles of male rats treated with DTB to the onset of muscle weakness, 5-8 days. Our objective was to assess the involvement of the Schwann cells and to determine the most likely primary targets of DTB. At the onset of muscle weakness, nerve terminals exhibited some enlarged regions, but did not sprout. Terminal Schwann cells became flatter and expanded to cover most of the endplate. The extent of invasion of the synaptic cleft by Schwann cell processes was not significantly different from controls; extension of Schwann cell sprouts away from the junction was not seen. Thus, the morphology of the Schwann cells, although clearly affected by DTB, does not suggest that they contribute directly to the physiological defects of DTB-treated terminals. Abnormal tubulovesicular structures or tangles of neurofilaments were clustered in the centers of about 25% of treated terminals. Fewer synaptic vesicles occupied the region opposite the postsynaptic folds. Vesicle volumes were variable and included some very large vesicles, corresponding with the variable MEPP amplitudes reported previously for terminals of DTB-treated rodents. The postsynaptic area stained by rhodamine-labeled alpha-bungarotoxin expanded with terminal swelling, apparently by unpleating of the postsynaptic folds. No loss of ACh receptors or spread of ACh receptors beyond terminal boundaries was detected. Morphometric data are consistent with the conclusion that DTB affects, either directly or indirectly, vesicular release of ACh and the subsequent vesicular recycling process.
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ABSTRACT: In several literature reports biuret and its sulfur analogs are reported to exist in their diketo form with general formula H2NCXNHCYNH2 (X = O, Y = O, biuret; X = Y = S, dithiobiuret; and X = O, Y = S, thiobiuret). On the other hand, recently reported results on the electronic structure of biguanide analogs (X = Y = NH)demonstrated that a form equivalent to diketo is not the preferred structure. Thus, a systematic ab initio study on the tautomeric preferences of biuret and its sulfur analogs (dithiobiuret and thiobiuret) has been carried out. The results indicate that an interplay of conjugative stabilization and intramolecular hydrogen bonding to play a role in tautomeric preferences. Energy and geometric parameters, natural bond orbital analyses have been employed to understand the chemistry of the title compounds. The results indicate that unlike biguanides, these compounds prefer diketo forms containing hydrogen on the bridging nitrogen (N4) and in a trans-arrangement (1a–4a). However, tautomerization of these keto forms to the corresponding enol isomers was also found to be highly probable. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008International Journal of Quantum Chemistry 02/2008; 108(7):1277 - 1286. · 1.17 Impact Factor
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ABSTRACT: The identification of signaling pathways involved in megakaryocytopoiesis is essential for development of novel therapeutics to treat hematological disorders. Following our previous findings that megakaryocytes express functional channel-forming N-methyl-D-aspartate-type glutamate receptors, here we aimed to determine the glutamate release capacity in undifferentiated and differentiated megakaryocytes and the role of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins that are known to be associated with vesicular exocytosis. Using the megakaryocytic cell line MEG-01, primary megakaryocytes, and tissue sections of bone marrow, reverse transcription polymerase chain reaction, Western blot analysis, and immunolocalization were employed to detect factors required for vesicular glutamate release. Vesicle recycling was monitored by acridine orange and FM1-43 staining and glutamate release activity was assessed by an enzyme-linked fluorimetric assay. Genetically modified MEG-01 cells, with deletion or overexpression of SNARE and vesicular proteins, were also examined for glutamate release activity. We demonstrated that megakaryocytes express numerous proteins required for vesicular glutamate release, including core SNARE proteins, vesicle-associated membrane protein, soluble N-ethyl maleimide-sensitive factor attachment protein-23, and syntaxin, as well as specific glutamate-loading vesicle proteins, VGLUT1 and VGLUT2. Moreover, active vesicle recycling and differentiation-dependent glutamate release were observed in megakaryocytes. Vesicle-associated membrane protein-deficient MEG-01 cells, which are impaired in vesicle recycling, showed a 30% decrease in released glutamate, whereas overexpression of VGLUT1 exhibited up to a 2.2-fold increase in glutamate release. These data show that glutamate release from megakaryocytes occurs in a SNARE-dependent, exocytotic manner and is increased during differentiation, suggesting that manipulation of glutamate signaling could influence megakaryocytopoiesis and, therefore, offer a suitable target for the treatment of thrombosis and other hematological disorders.Experimental hematology 03/2010; 38(6):504-15. · 3.11 Impact Factor
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ABSTRACT: STOP (Stable Tubulin-Only Polypeptide) null mice show behavioral deficits, impaired synaptic plasticity, decrease in synaptic vesicular pools and disturbances in dopaminergic transmission, and are considered a neurodevelopmental model of schizophrenia. Olfactory neurons highly express STOP protein and are continually generated throughout life. Experimentally-induced loss of olfactory neurons leads to epithelial regeneration within two months, providing a useful model to evaluate the role played by STOP protein in adult olfactory neurogenesis. Immunocytochemistry and electron microscopy were used to study the structure of the glomerulus in the main olfactory bulb and neurogenesis in the neurosensorial epithelia. In STOP null mice, olfactory neurons showed presynaptic swellings with tubulovesicular profiles and autophagic-like structures. In olfactory and vomeronasal epithelia, there was an increase in neurons turnover, as shown by the increase in number of proliferating, apoptotic and immature cells with no changes in the number of mature neurons. Similar alterations in peripheral olfactory neurogenesis have been previously described in schizophrenia patients. In STOP null mice, regeneration of the olfactory epithelium did not modify these anomalies; moreover, regeneration resulted in abnormal organisation of olfactory terminals within the olfactory glomeruli in STOP null mice. In conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia.PLoS ONE 01/2010; 5(9):e12753. · 3.53 Impact Factor