Identification of a phosphorylation site for calcium/calmodulindependent protein kinase II in the NR2B subunit of the N-methyl-D-aspartate receptor.

The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L. -Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was approximately 50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons.

Dynamic MR digital subtraction angiography using contrast enhancement, fast data acquisition, and complex subtraction.

A dynamic MR angiography technique, MR digital subtraction angiography (MR DSA), is proposed using fast acquisition, contrast enhancement, and complex subtraction. When a bolus of contrast is injected into a patient, data acquisition begins, dynamically acquiring a thick slab using a fast gradient echo sequence for 10-100 s. Similar to x-ray DSA, a mask is selected from the images without contrast enhancement, and later images are subtracted from the mask to generate angiograms. Complex subtraction is used to overcome the partial volume effects related to the phase difference between the flowing and stationary magnetization in a voxel. Vessel signal is the enhancement of flow magnetization resulting from the contrast bolus. MR DSA was performed in 28 patients, including vessels in the lungs, brains, legs, abdomen, and pelvis. All targeted vessels were well depicted with MR DSA. Corresponding dynamic information (contrast arrival time ta and duration of the arterial phase tav) was measured: ta/tav = 3.4/4.7 s for the lung, 10.3/4.9 s for the brain, 12.8/19.3 for the aorta, 15.2/12.6 s for the leg. MR DSA can provide dynamic angiographic images using a very short acquisition time.

Hilar biliary strictures after liver transplantation: cholangiography and percutaneous treatment.

Nonanastomotic hilar bile duct strictures developed in 16 of 152 patients who underwent liver transplantation. The type of pretransplantation liver disease did not significantly affect the likelihood of hilar stricture formation. Possible causes of hilar biliary strictures include hepatic artery occlusion, ductopenic arteriopathic rejection, and cytomegalovirus infection; however, five of the 16 patients had hilar strictures without these complications. Hilar strictures developed within 3 months after transplantation in 11 of the 16 patients. Strictures began as a slight common hepatic duct irregularity and progressed to mucosal cast formation and later to firm strictures. Fifteen of the 16 patients underwent percutaneous stricture dilation. Of 12 patients who no longer have stents, four have had no stricture recurrence for 12-30 months. Eight patients have had to undergo retransplantation or have died. Percutaneous dilations were most likely to result in patient bile ducts if strictures developed within 3 months after transplantation and in the absence of pretransplantation primary sclerosing cholangitis, ductopenic arteriopathic rejection, cytomegalovirus infection, or hepatic artery thrombosis.