The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.     

Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B

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.     

Depolarization-Dependent Tyrosine Phosphorylation in Rat Brain Synaptosomes

Synaptosomes from rat forebrain were analyzed for the presence of phosphotyrosine-containing proteins by immunoblotting with antiphosphotyrosine antibodies. Using this technique, 10-11 phosphotyrosine-containing proteins were detected. Depolarization of synaptosomes by transfer to a high (41 mM) K+ medium resulted in increases in the phosphotyrosine content of several synaptosomal proteins, the most pronounced increase being associated with a membrane protein of M(r) 117,000 (ptp117). Additional proteins exhibiting depolarization-dependent increases in phosphotyrosine content had molecular weights of 39,000, 104,000, 135,000, and 160,000. The depolarization-dependent increase in the phosphotyrosine content of ptp117 was apparent within 30 s of the onset of depolarization, reached a maximum between 3 and 5 min, and then decreased to near control values by 30 min. The increase in tyrosine phosphorylation of ptp117 was dependent on the concentration of K+ in the depolarizing medium and was maximal with [K+] in excess of 50 mM. It was also calcium dependent and did not occur in the absence of extracellular calcium. The addition of veratridine to the incubation medium also resulted in an increase in the tyrosine phosphorylation of ptp117. The results suggest that the phosphorylation of synaptic proteins on tyrosine residues may be involved in the regulation or modulation of synaptic activity.     

Changes in phosphorylation of the NMDA receptor in the rat hippocampus induced by status epilepticus

Systemic administration of pilocarpine preceded by lithium induces status epilepticus (SE) that results in neurodegeneration and may lead to the development of spontaneous recurrent seizures. We investigated the effect of Li/pilocarpine-induced SE on phosphorylation of the NMDA receptor in rat hippocampus. Phosphorylation of NR1 by PKC on Ser890 was decreased to 45% of control values immediately following 1 h of SE. During the first 3 h following the termination of SE, phosphorylation of Ser890 increased 4-fold before declining to control values by 24 h. Phosphorylation of NR1 by PKA was also depressed relative to controls immediately following SE and transiently increased above control values upon the termination of SE. SE was accompanied by a general increase in tyrosine phosphorylation of hippocampal proteins that lasted for several hours following the termination of seizures. Tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDAR increased 3-4-fold over control values during SE, continued to increase during the first hour following SE and then declined to control levels by 24 h. SE resulted in the activation of Src and Pyk2 associated with the postsynaptic apparatus, suggesting a role for these enzymes in the SE-induced increase in tyrosine phosphorylation. Changes in phosphorylation of the NMDA receptor may play a role in the pathophysiological consequences of SE.     

Xylanase-induced reduction of chlorine dioxide consumption during elemental chlorine-free bleaching of different pulp types

The potential of crude xylanase from Thermomyces lanuginosus and Xylanase P (a commercial xylanase) was evaluated in bleaching of various paper pulp types. Xylanases released chromophores and reducing sugars and decreased kappa number of pulps. Chlorine-bleached, alkali-extracted bagasse and post-oxygen kraft pulps, pretreated with enzymes, gained over 5 brightness points over controls. Biobleaching of soda-aq pulp with Xylanase P produced chlorine dioxide savings of up to 30% or 4.5 kg chlorine dioxide t^–1 pulp.     

Synaptic Protein Tyrosine Kinase: Partial Characterization and Identification of Endogenous Substrates

The subcellular distribution of protein tyrosine kinase in rat forebrain was determined using [Val5]-angiotensin II as exogenous substrate. Enzyme activity was present in each of the fractions analyzed and was enriched in synaptic membranes (SMs) and the synaptosomal soluble fraction (2.2- and 2.5-fold over the homogenate, respectively). SMs also phosphorylated polyglutamyltyrosine (pGT; molar ratio of 4:1), the Vmax for angiotensin and pGT phosphorylation being 26.3 +/- 1.6 and 142 +/- 4 pmol/min/mg, respectively. Extraction of SMs with several different detergents resulted in enhanced enzyme activity and the solubilization of 33-37% of the angiotensin and 43-70% of the pGT-phosphorylating activity. Isolated postsynaptic densities (PSDs) contained tyrosine kinase and phosphorylated angiotensin and pGT. The Vmax values for angiotensin and pGT phosphorylation by PSDs were 17 +/- 5 and 23 +/- 1 pmol/min/mg, respectively. Six putative endogenous substrates for SM tyrosine kinase, with molecular weights of 205K, 180K, 76K, 60K, 50K, and 45K, were identified. Each of these proteins, except p76, was phosphorylated in the detergent-insoluble residue obtained following the extraction of SMs with Triton X-100 as well as in PSDs, indicating that the postsynaptic apparatus is an active site of tyrosine phosphorylation. The phosphorylation of p76 was localized to the Triton X-100 extract and also occurred in the synaptosomal soluble fraction. The results indicate that tyrosine kinase and its substrates are located in both pre- and postsynaptic compartments and suggest a role for this enzyme in synaptic function.     

Identification of Lectin-Purified Neural Glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA Receptor Subunits: Conservation of Glycosylation at the Synapse

Abstract: The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N -glycosidase F and endo -β- N -acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for ∼50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N -glycosidase F and endo -β- N -acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.     

Evaluation of a Well-Established Task-Shifting Initiative: The Lay Counselor Cadre in Botswana

Background

Evidence supports the implementation of task shifting to address health worker shortages that are common in resource-limited settings. However, there is need to learn from established programs to identify ways to achieve the strongest, most sustainable impact. This study examined the Botswana lay counselor cadre, a task shifting initiative, to explore effectiveness and contribution to the health workforce.

Methods

This evaluation used multiple methods, including a desk review, a national lay counselor survey (n = 385; response = 94%), in-depth interviews (n = 79), lay counselors focus group discussions (n = 7), lay counselors observations (n = 25), and client exit interviews (n = 47).

Results

Interview and focus group data indicate that lay counselors contribute to essentially all HIV-related programs in Botswana and they conduct the majority of HIV tests and related counseling at public health facilities throughout the country. Interviews showed that the lay counselor cadre is making the workload of more skilled health workers more manageable and increasing HIV acceptance in communities. The average score on a work-related knowledge test was 74.5%. However for 3 questions, less than half answered correctly. During observations, lay counselors demonstrated average competence for most skills assessed and clients (97.9%) were satisfied with services received. From the survey, lay counselors generally reported being comfortable with their duties; however, some reported clinical duties that extended beyond their training and mandate. Multiple factors affecting the performance of the lay counselors were identified, including insufficient resources, such as private counseling space and HIV test kits; and technical, administrative, and supervisory support.

Conclusion

Lay counselors are fulfilling an important role in Botswana''s healthcare system, serving as the entry point into HIV care, support, and treatment services.

Recommendation

For this and other similar task shifting initiatives, it is important that lay counselors'' responsibilities are clear and that training and support are adequate to optimize their effectiveness.     

Differential effects of hypoxia-ischemia on subunit expression and tyrosine phosphorylation of the NMDA receptor in 7- and 21-day-old rats

The effect of cerebral hypoxia-ischemia (HI) on levels and tyrosine phosphorylation of the NMDA receptor was examined in 7- (P7) and 21 (P21)-day-old rats. Unilateral HI was administered by ligation of the right common carotid artery and exposure to an atmosphere of 8% O2/92% N2 for 2 (P7) or 1.5 (P21) h. This duration of HI produces significant infarction in nearly all of the survivors with damage being largely restricted to the cortex, striatum, and hippocampus of the hemisphere ipsilateral to the carotid artery ligation. NR2A levels in the right hemisphere of P7 pups were markedly reduced after 24 h of recovery, while NR1 and NR2B remained unchanged. In contrast, NR2B, but not NR2A, was reduced after HI at P21. At both ages, HI resulted in a transient increase in tyrosine phosphorylation of a number of forebrain proteins that peaked between 1 and 6 h of recovery. At both P7 and P21, tyrosine phosphorylation of NR2B was enhanced 1 h after HI and had returned to basal levels by 24 h. HI induced an increase in tyrosine phosphorylation of NR2A in 21 day, but not in 7-day-old animals. The differential effects of HI on the NMDA receptor at different post-natal ages may contribute to changing sensitivity to hypoxia-ischemia.