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.     

Positive selection driving the evolution of a gene of male reproduction, Acp26Aa, of Drosophila: II. Divergence versus polymorphism

BACKGROUND: Sirolimus, a novel immunosuppressant that inhibits cytokine-driven cell proliferation and maturation, prolongs allograft survival in animal models. After a phase I trial in stable renal transplant recipients documented that cyclosporine and sirolimus have few overlapping toxicities, we conducted an open-label, single-center, phase I/II dose-escalation trial to examine the safety and efficacy of this drug combination. METHODS: Forty mismatched living-donor renal transplant recipients were sequentially assigned to receive escalating initial doses of sirolimus (0.5-7.0 mg/m2/day), in addition to courses of prednisone and a concentration-controlled regimen of cyclosporine. We conducted surveillance for drug-induced side effects among sirolimus-treated patients and compared their incidence of acute rejection episodes as well as mean laboratory values with those of a historical cohort of 65 consecutive, immediately precedent, demographically similar recipients treated with the same concentration-controlled regimen of cyclosporine and tapering doses of prednisone. RESULTS: The addition of sirolimus reduced the overall incidence of acute allograft rejection episodes to 7.5% from 32% in the immediately precedent cyclosporine/prednisone-treated patients. At 18- to 47-month follow-up periods, both treatment groups displayed similar rates of patient and graft survival, as well as morbid complications. Although sirolimus-treated patients displayed comparatively lower platelet and white blood cell counts and higher levels of serum cholesterol and triglycerides, sirolimus did not augment the nephrotoxic or hypertensive proclivities of cyclosporine. The degree of change in the laboratory values was more directly associated with whole blood trough drug concentrations than with doses of sirolimus. CONCLUSIONS: Sirolimus potentiates the immunosuppressive effects of a cyclosporine-based regimen by reducing the rate of acute rejection episodes.     

Can an immunologically, nonreactive potential allograft recipient undergo transplantation without a donor-specific crossmatch?

Performance of the pretransplant crossmatch requires 4 or more hours . Delays in the crossmatch might alter operating room availability and thereby increase donor organ cold ischemia time that might then result in increased risk of delayed graft function. To avoid these problems, recipients could be identified who would be expected to display negative donor crossmatches and who could be transplanted with a concurrent or retrospective rather than a pretransplant crossmatch. We, therefore, evaluated the percent reactive antibodies and donor IgG-antihuman globulin (AHG) crossmatch results of 1165 sera from 220 potential allograft recipients. Twenty-five (11%) of 220 recipients consistently displayed a 0% PRA and, with only one exception, their sera (n= 156) tested IgG-AHG crossmatch-negative against potential cadaveric donors (a 0.6% IgG-AHG positive crossmatch risk). These data suggest that the timing of the pretransplant serum crossmatch could be altered for a highly selected group of immunologically nonreactive recipients.