Alteration of Ca2+ dependence of neurotransmitter release by disruption of Ca2+ channel/syntaxin interaction

Presynaptic N-type calcium channels interact with syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) through a binding site in the intracellular loop connecting domains II and III of the alpha1 subunit. This binding region was loaded into embryonic spinal neurons of Xenopus by early blastomere injection. After culturing, synaptic transmission of peptide-loaded and control cells was compared by measuring postsynaptic responses under different external Ca2+ concentrations. The relative transmitter release of injected neurons was reduced by approximately 25% at physiological Ca2+ concentration, whereas injection of the corresponding region of the L-type Ca2+ channel had virtually no effect. When applied to a theoretical model, these results imply that 70% of the formerly linked vesicles have been uncoupled after action of the peptide. Our data suggest that severing the physical interaction between presynaptic calcium channels and synaptic proteins will not prevent synaptic transmission at this synapse but will make it less efficient by shifting its Ca2+ dependence to higher values.     

Nonopioid analgesics shorten the duration of postoperative ileus

Morphine inhibits propagating and stimulates nonpropagating colon contractions in monkeys and humans. The use of morphine or other opioids that inhibit propulsive contractions prolongs postoperative ileus. In contrast, ketorolac tromethamine, a nonsteroidal analgesic, has no effect on colon contractions in monkeys. In 14 patients having elective abdominal operations, bipolar electrodes were implanted on the right (n = 13) and left (n = 10) colon. Group A (n = 8) received ketorolac, 30 mg IM q6h, for pain relief. Group B (n = 6) needed supplemental morphine, 2-10 mg IV or IM, plus ketorolac to control their pain. Myoelectric activity was recorded from each subject on postop Days 1-5 and analyzed by computer for electrical control activity (ECA), short and long electrical response activity (ERA), and propagation of long ERA. There was a difference between the two groups in return of propagated long ERA bursts that correlated with clinical recovery from postoperative ileus. Postoperative analgesia with ketorolac resulted in faster resolution of ileus compared to morphine plus ketorolac because opioid-induced motor abnormalities in the colon were avoided.     

The propeptide of the vitamin K-dependent carboxylase substrate accelerates formation of the gamma-glutamyl carbanion intermediate

Vitamin K-dependent carboxylase catalyzes the post-translational gamma-carboxylation of 9-12 glutamyl residues of several blood coagulation proteins. Carboxylase purified from Chinese hamster ovary (CHO) cells as a recombinant FLAG-carboxylase fusion protein [Sugiura, I., et al. (1996) J. Biol. Chem. 271, 17837-17844] was utilized with pentapeptide substrate FL[3H-R,S]EAL with high specific radioactivity to probe the timing of glutamyl Cgamma-3H cleavage relative to Cgamma-COO- bond formation by 14CO2 incorporation rates. Studies were conducted over a range of NaH14CO3 concentrations to assess uncoupling of gamma-glutamyl carbanion formation and over a range of concentrations of ProPT18, the 18-residue peptide corresponding to the -18 to -1 propeptide region of prothrombin known to affect the catalytic efficiency of carboxylase. At saturation, ProPT18 accelerates Cgamma-3H cleavage 11-13-fold and Cgamma-14CO2- formation 6-7-fold, converting a Cgamma-3H cleavage/Cgamma-14CO2- formation ratio of 1.2-1.4 in the absence of ProPT18 to 2.3-2.8 in its presence, a relative increase in and uncoupling of Cgamma-3H cleavage from C-C bond formation. When the HCO3- concentration was varied, the V/K3H+/V/K14CO2 ratios rose as HCO3- fractional saturation dropped to a ratio of 9.3-10.8/l at low bicarbonate, indicating an uncoupling of nine out of ten gamma-glutamyl carbanion formations from carboxylative capture, consistent with prior reports on microsomal enzyme [Larson, A. E., et al. (1981) J. Biol. Chem. 256, 11032-11035]. These results with pentapeptide substrate FLEAL validate reversible gamma-glutamyl carbanion formation by pure carboxylase and indicate the ProPT18 increase in catalytic efficiency is in selective lowering of an energy barrier preceding the gamma-glutamyl carbanion intermediate.     

Structural studies of EcoRII methylase: exploring similarities among methylases

EcoRII methyltransferase (M.EcoRII) is a cytosine-C5 DNA methylating enzyme. A model of its three-dimensional structure is proposed on the basis of homology modeling. Crystal structures of two members of the same family of enzymes, HaeIII and HhaI methyltransferases (M.HaeIII and M.HhaI respectively), were used as template molecules. Molecular dynamics was used to ensure sampling of conformationally stable structures. The final model has good geometry. The DNA and cofactor binding residues are in expected positions and form proper interactions. M.EcoRII is 147 amino acids longer than the template molecules, and hence the model contains several loops that are significantly longer than those in M.HaeIII and M.HhaI. The model provides a framework for interpretation and designing site-directed mutants that have a potential to improve crystallization experiments of this enzyme, and possibly other similar enzymes.      

The Utah intracortical Electrode Array: a recording structure for potential brain-computer interfaces

We investigated the potential of the Utah Intracortical Electrode Array (UIEA) to provide signals for a brain-computer interface (BCI). The UIEA records from small populations of neurons which have an average signal-to-noise ratio (SNR) of 6:1. We provide specific examples that show the activities of these populations of neurons contain sufficient information to perform control tasks. Results from a simple stimulus detection task using these signals as inputs confirm that the number of neurons present in a recording is significant in determining task performance. Increasing the number of units in a recording decreases the sensitivity of the response to the stimulus; decreasing the number of units in the recording, however, increases the variability of the response to the stimulus. We conclude that recordings from small populations of neurons, not single units, provide a reliable source of sufficiently stimulus selective signals which should be suitable for a BCI. In addition, the potential for simultaneous and proportional control of a large number of external devices may be realized through the ability of an array of microelectrodes such as the UIEA to record both spatial and temporal patterns of neuronal activation.     

Left atrial appendage flow velocity and spontaneous echo contrast in patients with rheumatic mitral stenosis: a multiplane transesophageal echocardiographic study

BACKGROUND: Left atrial spontaneous echo contrast (LASEC), a putative marker of thrombo-embolic risk, is commonly located in the left atrial appendage (LAA). The aims of this work were to evaluate, using multiplane transesophageal echography, the echocardiographic determinants, specifically LAA outflow Doppler velocity, in the presence of SEC in patients with rheumatic MS. METHODS: Transthoracic and transesophageal echocardiographic tests were performed on 61 patients. The patients were divided into 3 groups based on the presence and type of valvular disease. Patients in group I (n = 28) presented with rheumatic mitral stenosis (MS). Patients in group II (n = 18) presented with valvular heart disease other than MS, and patients in group III (n = 15) had no history of valvular heart disease. The left atrium and appendage were examined for the presence of spontaneous echocontrast and thrombus, using multiplane echo scopy with transducer rotation. Minimal and maximal appendage areas were measured, on a computer-assisted bablet, by tracing a line from the top of the limbus of the left upper pulmonary vein to the appendage endocardial border. The LAA ejection fraction was calculated according to the formula: (maximal area-minimal area)/maximal area. Mitral valvular condition was evaluated with transthoracic and transesophageal echocardiography. Left atrial appendage blood flow velocity profiles were obtained with pulsed-wave Doppler at the orifice of the LAA. RESULTS: LASEC was present in 18 of 28 patients with mitral stenosis (64.3%). Patients with LASEC showed a greater incidence of atrial fibrillation (14/18 vs 12/43, p < 0.005), larger LAD (53.67 +/- 8.74 vs 40.54 +/- 14.85, p < 0.005), smaller LAAEF (38.7 +/- 1.53 vs 69.5 +/- 24.0, p < 0.05), smaller LAAMEV (20.28 +/- 10.07 vs 2.95 +/- 25.11, p < 0.005) and smaller LAAMFV (24.6 +/- 12.23 vs 36.00 +/- 11.01, p < 0.01), when compared with patients without LASEC. For group I, LAAEF, LAAMEV and LAAFV were smaller in patients with SEC than in patients without SEC (p < 0.005, p < 0.05, p < 0.01). However LAD values were similar for patients with and without SEC (53.67 +/- 8.75 vs 54.20 +/- 18.81, p = NS). Both LAAMEV and LAAMFV were related to SEC in patients with atrial fibrillation. However, LAD did not show the same trend. CONCLUSIONS: LASEC is more commonly observed in patients with rheumatic mitral stenosis or atrial fibrillation. Both LAAMEV and LAAMFV are associated with SEC in these patients.     

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.