Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin

Ca(2+) influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor leads to activation and postsynaptic accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and ultimately to long term potentiation, which is thought to be the physiological correlate of learning and memory. The NMDA receptor also serves as a CaMKII docking site in dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of Thr(286). This autophosphorylation reduces the off rate to a level (t(12) = approximately 23 min) that is similar to that observed for dissociation of the T286D mutant CaMKII (t(12) = approximately 30 min) from spines after its glutamate-induced accumulation (Shen, K., Teruel, M. N., Connor, J. H., Shenolikar, S., and Meyer, T. (2000) Nat. Neurosci. 3, 881-886). CaMKII as well as the previously identified NR1 binding partners calmodulin and alpha-actinin bind to the short C-terminal portion of the C0 region of NR1. Like Ca(2+)/calmodulin, autophosphorylated CaMKII competes with alpha-actinin-2 for binding to NR1. We conclude that the NR1 C0 region is a key site for recruiting CaMKII to the postsynaptic site, where it may act in concert with calmodulin to modulate the stimulatory role of alpha-actinin interaction with the NMDA receptor.     

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.     

Selectivity and promiscuity of the first and second PDZ domains of PSD-95 and synapse-associated protein 102

PDZ domains typically interact with the very carboxyl terminus of their binding partners. Type 1 PDZ domains usually require valine, leucine, or isoleucine at the very COOH-terminal (P(0)) position, and serine or threonine 2 residues upstream at P(-2). We quantitatively defined the contributions of carboxyl-terminal residues to binding selectivity of the prototypic interactions of the PDZ domains of postsynaptic density protein 95 (PSD-95) and its homolog synapse-associated protein 90 (SAP102) with the NR2b subunit of the N-methyl-d-aspartate-type glutamate receptor. Our studies indicate that all of the last five residues of NR2b contribute to the binding selectivity. Prominent were a requirement for glutamate or glutamine at P(-3) and for valine at P(0) for high affinity binding and a preference for threonine over serine at P(-2), in the context of the last 11 residues of the NR2b COOH terminus. This analysis predicts a COOH-terminal (E/Q)(S/T)XV consensus sequence for the strongest binding to the first two PDZ domains of PSD-95 and SAP102. A search of the human genome sequences for proteins with a COOH-terminal (E/Q)(S/T)XV motif yielded 50 proteins, many of which have not been previously identified as PSD-95 or SAP102 binding partners. Two of these proteins, brain-specific angiogenesis inhibitor 1 and protein kinase Calpha, co-immunoprecipitated with PSD-95 and SAP102 from rat brain extracts.     

TIP,a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.     

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs

Oxidant stress, in vivo or in vitro, isknown to induce oxidative changes in human red blood cells (RBCs). Ourobjective was to examine the effect of augmenting RBC glutathione(GSH) synthesis on 1) degenerative protein loss and2) RBC chemokine- and free radical-scavenging functions inthe oxidatively stressed human RBCs by using banked RBCs as a model.Packed RBCs were stored up to 84 days at 1-6°C in Adsol or inthe experimental additive solution (Adsol fortified with glutamine,glycine, and N-acetyl-L-cysteine). Supplementingthe conventional additive with GSH precursor amino acids improved RBCGSH synthesis and maintenance. The rise in RBC -glutamylcysteineligase activity was directly proportional to the GSH content andinversely proportional to extracellular homocysteine concentration,methemoglobin formation, and losses of the RBC proteins band 3, band4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffyantigen (P < 0.01). Reduced loss of Duffy antigencorrelated well with a decrease in chemokine RANTES (regulated uponactivation, normal T-cell expressed, and secreted) concentration. Weconclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSHsynthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, inwhich RBCs are subjected to chronic/acute oxidant stresses.