Rapid upregulation of alpha7 nicotinic acetylcholine receptors by tyrosine dephosphorylation.
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Alpha7 nicotinic acetylcholine receptors (nAChRs) modulate network activity in the CNS. Thus, functional regulation of alpha7 nAChRs could influence the flow of information through various brain nuclei. It is hypothesized here that these receptors are amenable to modulation by tyrosine phosphorylation. In both Xenopus oocytes and rat hippocampal interneurons, brief exposure to a broad-spectrum protein tyrosine kinase inhibitor, genistein, specifically and reversibly potentiated alpha7 nAChR-mediated responses, whereas a protein tyrosine phosphatase inhibitor, pervanadate, caused depression. Potentiation was associated with an increased expression of surface alpha7 subunits and was not accompanied by detectable changes in receptor open probability, implying that the increased function results from an increased number of alpha7 nAChRs. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis was shown to be a plausible mechanism for the rapid delivery of additional alpha7 nAChRs to the plasma membrane. Direct phosphorylation/dephosphorylation of alpha7 subunits was unlikely because mutation of all three cytoplasmic tyrosine residues did not prevent the genistein-mediated facilitation. Overall, these data are consistent with the hypothesis that the number of functional cell surface alpha7 nAChRs is controlled indirectly via processes involving tyrosine phosphorylation. (+info)
Platelet serotonergic markers as endophenotypes for obsessive-compulsive disorder.
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Although compelling evidence has shown that obsessive-compulsive disorder (OCD) has a strong genetic component, its genetic basis remains to be elucidated. Identifying biological abnormalities in nonaffected relatives is one of the strategies advocated to isolate genetic vulnerability factors in complex disorders. Since peripheral serotonergic disturbances are frequently observed in OCD patients, the aim of this study was to investigate if they could represent endophenotypes, by searching for similar abnormalities in the unaffected parents of OCD patients. We assessed whole blood serotonin (5-HT) concentration, platelet 5-HT transporter (5-HTT) and 5-HT2A receptor-binding characteristics, and platelet inositol trisphosphate (IP3) content in a sample of OCD probands (n = 48) and their unaffected parents (n = 65), and compared them with sex- and age-matched controls (n = 113). Lower whole blood 5-HT concentration, fewer platelet 5-HTT-binding sites, and higher platelet IP3 content were found in OCD probands and their unaffected parents compared to controls. Whole blood 5-HT concentration showed a strong correlation within families (p < 0.001). The only parameter that appeared to discriminate affected and unaffected subjects was 5-HT2A receptor-binding characteristics, with increased receptor number and affinity in parents and no change in OCD probands. The presence of peripheral serotonergic abnormalities in OCD patients and their unaffected parents supports a familial origin of these disturbances. These alterations may serve as endophenotypic markers in OCD, and could contribute to the study of the biological mechanisms and genetic underpinnings of the disorder. (+info)
Efflux of lipid from macrophages after induction of lipid accumulation by chylomicron remnants.
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The fate of cholesterol and triacylglycerol taken up and accumulated by macrophages after exposure to chylomicron remnants was investigated using macrophages derived from the human monocyte cell line THP-1 and chylomicron remnant-like particles containing human apolipoprotein (apo) E (CRLPs) as the experimental model. In THP-1 macrophages lipid loaded with CRLPs and incubated with various cholesterol acceptors for 24 h, the mass of cholesterol and cholesteryl ester found in the cells was not changed by HDL, HDL3 or lipid-free ApoA-I, although it was decreased by 38% by ApoA-I-phosphatidylcholine vesicles (ApoA-I-PC). After loading of the macrophages with [3H]cholesterol-labelled CRLPs, only about 5% of the label was effluxed in 24 h in the absence of cholesterol acceptors, and this increased to about 10% with ApoA-I or PC only, and to about 30% with apoA-I-PC. In similar experiments with [3H]triolein, only about 4% of the labelled triacylglycerol taken up by the cells was released into the medium in 24 h, and a large (>60%) and consistent proportion of the intracellular radioactivity remained associated with the triacylglycerol throughout this period. These results suggest that cholesterol and triacylglycerol derived from chylomicron remnants are not readily cleared from macrophages, and this is likely to contribute to the atherogenicity of the remnant lipoproteins. (+info)
Nicotinic cholinergic receptors in the rat cerebellum: multiple heteromeric subtypes.
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Nicotinic receptors (nAChRs) in the cerebellum have been implicated in the pathology of autism spectrum disorders (Lee et al., 2002; Martin-Ruiz et al., 2004). The subtypes of nAChRs in the cerebellum are not known in any detail, except that, in addition to the homomeric alpha7 subtype, there appears to be one or more heteromeric subtypes consisting of combinations of alpha and beta subunits. To begin to better understand the potential roles of these heteromeric nAChRs in cerebellar circuitry and their potential as targets for nicotinic drugs, we investigated their subunit composition. Using subunit-selective antibodies in sequential immunoprecipitation assays, we detected six structurally distinct heteromeric nAChR populations in the rat cerebellum. Among these were several subtypes that have not been encountered previously, including alpha3alpha4beta2 and alpha3alpha4beta4 nAChRs. This diversity suggests that nAChRs play multiple roles in cerebellar physiology. (+info)
Novel function for vascular endothelial growth factor receptor-1 on epidermal keratinocytes.
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Vascular endothelial growth factor (VEGF-A), a potent stimulus for angiogenesis, is up-regulated in the skin after wounding. Although studies have shown that VEGF is important for wound repair, it is unclear whether this is based solely on its ability to promote angiogenesis or if VEGF can also promote healing by acting directly on non-endothelial cell types. By immunohistochemistry and reverse transcriptase-polymerase chain reaction, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was detected in murine keratinocytes during wound repair and in normal human epidermal keratinocytes (NHEKs). The presence of VEGF receptors on NHEKs was verified by binding studies with 125I-VEGF. In vitro, VEGF stimulated the proliferation of NHEKs, an effect that could be blocked by treatment with neutralizing VEGFR-1 antibodies. A role for VEGFR-1 in keratinocytes was also shown in vivo because treatment of excisional wounds with neutralizing VEGFR-1 antibodies delayed re-epithelialization. Treatment with anti-VEGFR-1 antibodies also reduced the number of proliferating keratinocytes at the leading edge of the wound, suggesting that VEGF sends a proliferative signal to these cells. Together, these data describe a novel role for VEGFR-1 in keratinocytes and suggest that VEGF may play several roles in cutaneous wound repair. (+info)
131I-anti-CD45 antibody plus busulfan and cyclophosphamide before allogeneic hematopoietic cell transplantation for treatment of acute myeloid leukemia in first remission.
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In an attempt to improve outcomes for patients with acute myeloid leukemia (AML) after allogeneic hematopoietic cell transplantation (HCT), we conducted a phase 1/2 study in which targeted irradiation delivered by 131I-anti-CD45 antibody was combined with targeted busulfan (BU; area-under-curve, 600-900 ng/mL) and cyclophosphamide (CY; 120 mg/kg). Fifty-two (88%) of 59 patients receiving a trace 131I-labeled dose of 0.5 mg/kg anti-CD45 murine antibody had higher estimated absorbed radiation in bone marrow and spleen than in any other organ. Forty-six patients were treated with 102 to 298 mCi (3774-11 026 MBq) 131I, delivering an estimated 5.3 to 19 (mean, 11.3) Gy to marrow, 17-72 (mean, 29.7) Gy to spleen, and 3.5 Gy (n = 4) to 5.25 Gy (n = 42) to the liver. The estimated 3-year nonrelapse mortality and disease-free survival (DFS) were 21% and 61%, respectively. These results were compared with those from 509 similar International Bone Marrow Transplant Registry patients who underwent transplantation using BU/CY alone. After adjusting for differences in age and cytogenetics risk, the hazard of mortality among all antibody-treated patients was 0.65 times that of the Registry patients (95% CI 0.39-1.08; P = .09). The addition of targeted hematopoietic irradiation to conventional BU/CY is feasible and well tolerated, and phase 2 results are sufficiently encouraging to warrant further study. (+info)
Diurnal regulation of the gastrin-releasing peptide receptor in the mouse circadian clock.
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In mammals, circadian rhythms are generated by the suprachiasmatic nuclei (SCN) of the hypothalamus. SCN neurons are heterogeneous and can be classified according to their function, anatomical connections, morphology and/or peptidergic identity. We focus here on gastrin-releasing peptide- (GRP) and on GRP receptor- (GRPr) expressing cells of the SCN. Pharmacological application of GRP in vivo or in vitro can shift the phase of circadian rhythms, and GRPr-deficient mice show blunted photic phase shifting. Given the in vivo and in vitro effects of GRP on circadian behavior and on SCN neuronal activity, we investigated whether the GRPr might be under circadian and/or diurnal control. Using in situ hybridization and autoradiographic receptor binding, we localized the GRPr in the mouse SCN and determined that GRP binding varies with time of day in animals housed in a light-dark cycle but not in conditions of constant darkness. The latter results were confirmed with Western blots of SCN tissue. Together, the present findings reveal that changes in GRPr are light driven and not endogenously organized. Diurnal variation in GRPr activity probably underlies intra-SCN signaling important for entrainment and phase shifting. (+info)
Increased extrajunctional acetylcholine sensitivity produced by chronic acetylcholine sensitivity produced by chronic post-synaptic neuromuscular blockade.
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1. Anaesthetized rats were paralysed for periods of up to 3 days by chronic administration of D-tubocurarine (DTC), succinylcholine or alpha-bungarotoxin. 2. After 3 days of treatment with DTC, the phrenic nerve remained active. Neuromuscular transmission and spontaneous miniature end-plate potentials (m.e.p.p.s) were restored after removal of the DTC. Resting potentials and input resistances of muscle fibres that had been paralysed for 3 days were similar to those in denervated fibers. 3. Chronic neuromuscular blockade increased the binding of [125-I]-alpha-bungarotoxin by extrajunctional regions of muscle. The time course of the increase was similar to that seen after denervation. Binding to muscles from animals that were anaesthetized and respirated, but not paralysed, was not increased. 4. Three days of paralysis increased the sensitivity of the extrajunctional muscle membrane to acetylcholine (ACh) applied by iontophoresis. 5. Approximately the same proportion of muscle fibres from muscles paralysed for 3 days gave overshooting action potentials in the presence of tetrodotoxin 10-minus 6 g/ml. as did fibres form muscles denervated for 3 days. 6. Chronic paralysis did not change the accumulation of acetylcholinesterase above a ligation in the sciatic nerve. 7. These results are consistent with the idea that extrajunctional ACh sensitivity is normally controlled by muscle activity. (+info)