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71.
FJ Northington RJ Traystman RC Koehler JD Rothstein LJ Martin 《Canadian Metallurgical Quarterly》1998,85(4):1183-1194
Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate which can have either neurotrophic or neurotoxic effects in the developing brain, depending on its concentration. Using immunoblotting and immunocytochemistry, we tested the hypotheses that expression of neuronal and glial glutamate transporter proteins was regionally and temporally regulated in the developing ovine brain and that expression of the glial isoform early in development was not cell-type specific. Immunoblots for the neuronal glutamate transporter EAAC1 revealed a major band of immunoreactivity at 69,000 nmol. wt, whereas glial glutamate transporter-1 (GLT1) immunoreactivity was observed as 73,000 and 146,000 mol. wt proteins. EAAC1 and GLT1 are regulated differently during development, with EAAC1 immunoreactivity being most abundant at 60 and 71 days completed gestation (term=145 days) and dissipating thereafter, while GLT1 immunoreactivity was most abundant at 136 days gestation. By immunocytochemistry EAAC1 expression is neuronal throughout gestation with intense labelling of dendrites within the telencephalon evident at 60 days. Neuropil, neuronal cell bodies and processes are EAAC1-immunoreactive throughout gestation with no evidence of astrocytic or oligodendroglial immunoreactivity. In contrast, GLT1 is expressed by neuronal and non-neuronal cell types during midgestation with astrocyte selectivity developing by 136 days. During midgestation, GLT1 is transiently expressed in neurons of the subplate, cranial nerve nuclei, basal ganglia, and cerebellar cortex. The major finding of this study, that GLT1 is transiently expressed in various neuronal populations at midgestation demonstrates that the cell-type specificity of the GLT1 phenotype is developmentally regulated and depends on brain maturity. 相似文献
72.
Exposure to apparently subtoxic doses of chlorpyrifos during late stages of brain development affects cell acquisition through a mixture of cholinergic and noncholinergic mechanisms. In the current study, we modeled these effects in vitro using rat pheochromocytoma (PC12), a cell line that, upon nerve-growth factor (NGF)-induced differentiation, develops the appearance and function of cholinergic target neurons, including the expression of cholinergic receptors. In the undifferentiated state (no NGF), chlorpyrifos evoked an immediate (1 h), robust, concentration-dependent inhibition of DNA synthesis as evaluated by [3H]thymidine incorporation, with a threshold of 0.5-1.5 microg/ml. Continuous exposure for up to 24 h maintained the same degree of inhibition. The effects were selective for DNA synthesis, as much smaller inhibitions were found for synthesis of RNA or protein. In contrast, direct cholinergic stimulation of the cells by 100 microM nicotine had much smaller effects on DNA synthesis. Moreover, the effects of chlorpyrifos on DNA synthesis could not be blocked by nicotinic or muscarinic antagonists, confirming that the effects were not mediated primarily through cholinergic hyperstimulation consequent to cholinesterase inhibition or to direct receptor-mediated effects. When PC12 cells underwent NGF-induced differentiation, the rate of cell replication fell dramatically and neurite extension was evident both from morphological examination and from biochemical markers (increased protein:DNA ratio). After introduction of NGF, chlorpyrifos maintained its ability to inhibit DNA synthesis acutely. However, the ability to inhibit RNA and protein synthesis initially intensified and then disappeared, indicating a shift in macromolecular targets as differentiation proceeded. We also tested the effects of long-term exposure to chlorpyrifos during the process of NGF-induced differentiation. Continuous chlorpyrifos exposure resulted in severe reductions in macromolecule synthesis and a deficit in the total number of cells, effects similar to those seen with chlorpyrifos treatment in vivo. At the highest concentrations, neurite extension was also inhibited. Our results suggest that chlorpyrifos can interact directly with developing neural cells to inhibit replication and neuritic outgrowth. 相似文献
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75.
FG Boess FJ Monsma V Meyer C Zwingelstein AJ Sleight 《Canadian Metallurgical Quarterly》1997,52(3):515-523
We examined the ligand-binding site of the 5-hydroxytryptamine6 (5-HT6) receptor using site-directed mutagenesis. Interactions with residues in two characteristic positions of trans-membrane region V are important for ligand binding in several bioamine receptors. In the 5-HT6 receptor, one of these residues is a threonine (Thr196), whereas in most other mammalian 5-HT receptors, the corresponding residue is alanine. After transient expression in human embryonic kidney 293 cells, we determined the effects of the mutation T196A on [3H]d-lysergic acid diethylamide (LSD) binding and adenylyl cyclase stimulation. This mutation produced a receptor with a 10-fold reduced affinity for [3H]LSD and a 6-fold reduced affinity for 5-HT. The potency of both LSD and 5-HT for stimulation of adenylyl cyclase was also reduced by 18- and 7-fold, respectively. The affinity of other N1-unsubstituted ergolines (e.g., ergotamine, lisuride) was reduced 10-30 fold, whereas the affinity of N1-methylated ergolines (e.g., metergoline, methysergide, mesulergine) and other ligands, such as methiothepine, clozapine, ritanserin, amitriptyline, and mainserin, changed very little or increased. This indicates that in wild-type 5-HT6 receptor, Thr196 interacts with the N1 of N1-unsubstituted ergolines and tryptamines, probably forming a hydrogen bond. Based on molecular modeling, a serine residue in transmembrane region IV of the 5-HT2A receptor has previously been proposed to interact with the N1-position of 5-HT. When the corresponding residue of the 5-HT6 receptor (Ala154) was converted to serine, no change in the affinity of twelve 5-HT6 receptor ligands or in the potency of 5-HT and LSD could be detected, suggesting that this position does not contribute to the ligand binding site of the 5-HT6 receptor. 相似文献
76.
FJ Overdyk PM Gramling-Babb JR Handy NI Faller MJ Miller 《Canadian Metallurgical Quarterly》1997,84(1):213-215
Robertsonian translocations, although relatively common as a constitutional genetic aberration, are rarely encountered in leukaemia. We report a case of acute myeloid leukaemia which showed an acquired Robertsonian translocation in the form of der(14;21) by cytogenetic analysis of leukaemic cells. This was confirmed by the PHA-stimulated culture of peripheral blood lymphocytes. A review of the literature identifies only eight reported cases of acquired Robertsonian translocations in leukaemia. In the majority of cases the Robertsonian translocation occurs as a secondary change in a complex abnormal clone, whereas in two out of nine patients reported, including ours, it is found as a sole karyotypic abnormality. 相似文献
77.
A series of variants of the neuroactive 17-residue gamma-carboxyglutamate-(Gla)-containing polypeptide, conantokin-G (con-G), were synthesized with the intention of determining those features that were important for its N-methyl-D-aspartate (NMDA) receptor-targeted antagonist activity and for adoption of its divalent cation-dependent alpha-helical conformation. Employing the binding of [3H]dizolcipine (MK-801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con-G (IC50 = 0.48 microM), it was found that replacement by an Ala residue of Gla4 led to complete inactivation of the peptide, whereas a similar replacement of Gla3 resulted in a 20-fold decreased potency. Ala substitutions for Gla10 and Gla14 did not substantially affect [3H]MK-801 binding. This same substitution at Gla7 appeared to slightly enhance binding. Ala replacements of non-Gla residues demonstrated that four of them, viz. Glu2, Leu5, Gln9, and Ile12, possessed at least 200-fold decreases in inhibitory potency, whereas similar replacements at Gly1, Leu11, and Arg13 resulted in peptides with 8- to 12-fold increases in the IC50 values. The remaining amino acid residues tested in the single Ala replacement series showed no significant changes in the inhibitory characteristics of wild-type con-G. Additional studies with carboxyl-terminal truncated peptides revealed that the carboxyl-terminal 4 amino acids were unimportant for this activity. There was no strict correlation of inhibition of [3H]MK-801 binding with the ability of these peptides to form cation-dependent alpha-helices. Peptides with notably low alpha-helical content in the presence of these cations were lacking at least one, or both, of Gla10 and Gla14. Con-G[Gla3,4,7,10,14E] and con-G[Gla7,10,14E] were the only peptides that remained in a completely random conformation upon metal ion addition. 相似文献
78.
RA Wain GL Berdejo WN Delvalle RT Lyon LA Sanchez WD Suggs T Ohki E Lipsitz FJ Veith 《Canadian Metallurgical Quarterly》1999,29(1):100-7; discussion 107-9
PURPOSE: Arteriography is the diagnostic test of choice before lower extremity revascularization, because it is a means of pinpointing stenotic or occluded arteries and defining optimal sites for the origin and termination of bypass grafts. We evaluated whether a duplex ultrasound scan, used as an alternative to arteriography, could be used as a means of accurately predicting the proximal and distal anastomotic sites in patients requiring peripheral bypass grafts and, therefore, replace standard preoperative arteriography. METHODS: Forty-one patients who required infrainguinal bypass grafts underwent preoperative duplex arterial mapping (DAM). Based on these studies, an observer blinded to the operation performed predicted what operation the patient required and the best site for the proximal and distal anastomoses. These predictions were compared with the actual anastomotic sites chosen by the surgeon. RESULTS: Whether a femoropopliteal or an infrapopliteal bypass graft was required was predicted correctly by means of DAM in 37 patients (90%). In addition, both anastomotic sites in 18 of 20 patients (90%) who had femoropopliteal bypass grafts and 5 of 21 patients (24%) who had infrapopliteal procedures were correctly predicted by means of DAM. CONCLUSION: DAM is a reliable means of predicting whether patients will require femoropopliteal or infrapopliteal bypass grafts, and, when a patient requires a femoropopliteal bypass graft, the actual location of both anastomoses can also be accurately predicted. Therefore, DAM appears able to replace conventional preoperative arteriography in most patients found to require femoropopliteal reconstruction. Patients who are predicted by means of DAM to require crural or pedal bypass grafts should still undergo preoperative contrast studies to confirm these results and to more precisely locate the anastomotic sites. 相似文献
79.
S Chávez JM Lucena JC Reyes FJ Florencio P Candau 《Canadian Metallurgical Quarterly》1999,181(3):808-813
The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 has two putative pathways for ammonium assimilation: the glutamine synthetase-glutamate synthase cycle, which is the main one and is finely regulated by the nitrogen source; and a high NADP-dependent glutamate dehydrogenase activity (NADP-GDH) whose contribution to glutamate synthesis is uncertain. To investigate the role of the latter, we used two engineered mutants, one lacking and another overproducing NADP-GDH. No major disturbances in the regulation of nitrogen-assimilating enzymes or in amino acids pools were detected in the null mutant, but phycobiline content, a sensitive indicator of the nutritional state of cyanobacterial cells, was significantly reduced, indicating that NADP-GDH plays an auxiliary role in ammonium assimilation. This effect was already prominent in the initial phase of growth, although differences in growth rate between the wild type and the mutants were observed at this stage only at low light intensities. However, the null mutant was unable to sustain growth at the late stage of the culture at the point when the wild type showed the maximum NADP-GDH activity, and died faster in ammonium-containing medium. Overexpression of NADP-GDH improved culture proliferation under moderate ammonium concentrations. Competition experiments between the wild type and the null mutant confirmed that the presence of NADP-GDH confers a selective advantage to Synechocystis sp. strain PCC 6803 in late stages of growth. 相似文献
80.
FJ Poliafico 《Canadian Metallurgical Quarterly》1999,62(1):58-60
Familial inheritance of congenital diaphragmatic hernia is uncommon. We report two siblings with identical bilateral diaphragmatic defects. 相似文献