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Notes: Ischemia results in increased phosphorylation of NMDA receptors. To investigate the possible role of lipid rafts in this increase, lipid rafts and post-synaptic densities (PSDs) were isolated by the extraction of rat brain synaptosomes with Triton X-100 followed by sucrose density gradient centrifugation. Lipid rafts accounted for the majority of PSD-95, whereas SAP102 was predominantly located in PSDs. Between 50 and 60% of NMDA receptors were associated with lipid rafts. Greater than 85–90% of Src and Fyn were present in lipid rafts, whereas Pyk2 was mainly associated with PSDs. Lipid rafts and PSDs were isolated from animals subjected to 15 min of global ischemia followed by 6 h of recovery. Ischemia did not affect the yield, density, flotillin-1 or cholesterol content of lipid rafts. Following ischemia, the phosphorylation of NR1 by protein kinase C and tyrosine phosphorylation of NR2A and NR2B was increased in both lipid rafts and PSDs, with a greater increase in tyrosine phosphorylation occurring in the raft fraction. Following ischemia, NR1, NR2A and NR2B levels were elevated in PSDs and reduced in lipid rafts. The findings are consistent with a model involving close interaction between lipid rafts and PSDs and a role for lipid rafts in ischemia-induced signaling pathways.

Notes: The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia-induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.

Notes: Abstract: The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD-95 binds to NMDA receptor subunits NR2A and NR2B and to signaling molecules such as neuronal nitric oxide synthase and p135synGAP. We investigated the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. Ischemia followed by reperfusion resulted in a decrease in the solubility of the NMDA receptor and PSD-95 in 1% sodium deoxycholate, the decrease being greater in the vulnerable CA1 hippocampal subfield than in the less sensitive CA3/dentate gyrus regions. Solubilization of the kainic acid receptor GluR6/7 and the PSD-95 binding proteins, neuronal nitric oxide synthase and p135synGAP, also decreased following ischemia. The association between PSD-95 and NR2A and NR2B, as indicated by coimmunoprecipitation, was less in postischemic samples than in sham-operated controls. Ischemia also resulted in a decrease in the size of protein complexes containing PSD-95, but had only a small effect on the size distribution of complexes containing the NMDA receptor. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge.

Notes: Abstract: Activation of the N-methyl-d-aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.

Notes: Abstract: We investigated the gene expression levels, the immunoreactive protein prevalence, and the functional activity of N-methyl-d-aspartate (NMDA) receptor complexes at early times after severe global ischemia challenge in rats. The mRNA expression levels for the NR2A and NR2B subunits of NMDA receptors changed to different degrees within different subregions of the hippocampus after reperfusion with respect to sham-operated control. No significant change in expression was observed in the vulnerable CA1 subfield at or before 6 h after challenge for either receptor subunit, although changes in expression in other hippocampal subfields were observed. At 12 and 24 h after challenge, significant decreases in expression for both subunits were found in the vulnerable CA1 subfield, as well as in other hippocampal regions. At the protein level, a significant decrease in the amount of NR2A/NR2B immunoreactivity in the total hippocampus was observed at both 6 and 24 h after reperfusion compared with sham control. Electrophysiological assessment of single-channel NMDA receptor activity in the CA1 subfield indicates that the main conductance state of NMDA receptor channels is maintained 6 h after challenge, although by 18–24 h after challenge, this main conductance state is rarely observed. The NMDA receptor component of the excitatory postsynaptic field potential was found to be significantly diminished from sham control 24 h after challenge, such that only ∼10% of the sham response remained, but was not significantly altered from sham control at 6 h after challenge. These results indicate that decreases in the expression levels, the immunoreactive protein prevalence, and that alterations in the functionality of NMDA receptors occur in the hippocampus at early times after severe transient global ischemia.

Notes: Abstract : We have examined the effects of transient global ischemiaon both the gene expression levels and the functionality of GABABreceptors in rat brain, using antisense in situ hybridization andelectrophysiological evaluations. At the level of gene expression, nosignificant change in GABAB receptor expression was observed in anyhippocampal subfield at either 6 or 12 h after challenge. At 24 hpostchallenge, however, a significant decrease in GABAB receptorexpression was observed in both the CA1 and CA3 subfields, whereas no changewas observed in the dentate granule cell layer. Although expression in boththe vulnerable CA1 and less vulnerable CA3 subfields was diminished at thistime postchallenge, there was no significant difference in the degree of thediminished expression between these subfields. At the functional level, thedose-dependent ability of baclofen (1-100 μM) to inhibit an evokedexcitatory postsynaptic potential (f-EPSP) in the CA1 subfield was evaluatedat 24 h postischemia, in comparison with the dose-response observed insham-operated subjects. No significant differences were observed in theefficacy of GABAB receptor-mediated inhibition of the elicitedf-EPSP at any of the baclofen concentrations examined. These data demonstratethat although the mRNA expression levels for the GABAB receptor arediminished in both vulnerable and less vulnerable neurons of Ammon's horn at24 h following transient global ischemia, the functionality of theGABAB receptor system is maintained at this time postchallenge.

Notes: SYNOPSIS. Observation of the longitudinal flagellum of Ceratium tripos and C. furca with stroboscopic light revealed that this flagellum beats with a planar wave very close in form to a sine wave. The transverse flagellum, however, was seen to beat with a circular or elliptical helical wave, the amplitude of which was such that about half the wave was out of the girdle. With dark-field illumination, suspensions of polystyrene spheres (1.18 μ in diameter) made possible the observation and photography of currents set up by the organism's flagella. This technique revealed an unsuspected component of the force generated by the transverse flagellum, which drives the organism forward.A wire helix, rotating under water, was found to generate a current in one direction along the axis of the helix. When a “girdle” formed from modeling clay was placed around the helix, thus simulating the transverse flagellum, the current was found to flow across the axis of the helix. The girdle in both the model and the dinoflagellate is assumed to cancel those components of force generated by the portion of the helical wave within it which would tend to cause the water to rotate about the helix. The only effective components of this force are therefore those generated by that side of the helix not enclosed in the girdle, and their action is to drive water across the girdle.

Notes: Introduction : Barrett's oesophagus is the most important risk factor in the increase in incidence of oesophageal adenocarcinoma. Photodynamic therapy using porfimer sodium is the only approved endoscopic treatment for use in patients with Barrett's high-grade dysplasia.Aim : To determine clinical characteristics, endoscopic findings and treatment complications in Barrett's high-grade dysplasia patients undergoing photodynamic therapy.Methods : We reviewed our experience using porfimer sodium photodynamic therapy to treat patients with Barrett's oesophagus and high-grade dysplasia or mucosal carcinoma. Data collected included patients characteristics, presentation symptoms, endoscopic findings, subsequent use of surveillance endoscopy and outcome after photodynamic therapy.Results : Since 1997, 102 patients with Barrett's high-grade dysplasia (69 patients) or mucosal adenocarcinoma (33 patients) have been treated with photodynamic therapy using porfimer sodium as an alternative to oesophagectomy (median series follow-up time = 1.6 years). Almost half (46%) of patients had high-grade dysplasia or carcinoma detected on their first endoscopy and the remainder (54%) were found during surveillance of known Barrett's oesophagus. Symptoms typically associated with oesophageal disease were only found in 29 of 47 (62%) patients in whom dysplasia/carcinoma was detected on the initial endoscopy – chest pain in 13 patients, dysphagia in nine patients and chronic gastro-oesophageal disease in seven patients. Comparison of endoscopic characteristics found the median Barrett's glandular segment length was significantly shorter in adenocarcinoma patients (median 3 cm; range: 1–12) vs. Barrett's high-grade dysplasia patients (median 5 cm; range: 1–16, P 〈 0.001). Overall treatment results found complete ablation of glandular epithelium with one course of photodynamic therapy in most patients (56%). Stricture requiring dilation occurred in 20 patients (20%) was the most common serious adverse event. Photodynamic therapy failed to ablate dysplasia or carcinoma in four patients and subsequent oesophagectomy was curative in three of these patients.Conclusions : Approximately 40% of newly diagnosed patients with Barrett's associated dysplasia or carcinoma had no oesophageal symptoms and had carcinoma associated with short segment (3 cm or less). Photodynamic therapy is a highly effective, safe and minimally invasive first-line treatment for patients with Barrett's dysplasia and mucosal adenocarcinoma.

Notes: The role of protein kinase C (PKC) in tyrosine phosphorylation of the N-methyl-d-aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four-vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src-family kinases (SFKs) and Pyk2 were increased in post-synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post-ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia-induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia-induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia-induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src-family kinases.