Deletions within the amino-terminal half of the c-src gene product that alter the functional activity of the protein.

To examine how amino acid sequences outside of the catalytic domain of pp60c-src influence the functional activity of this protein, we have introduced deletion mutations within the amino-terminal half of pp60c-src. These mutations caused distinct changes in the biochemical properties of the c-src gene products and in the properties of cells infected with retroviruses carrying these mutant c-src genes. Cells expressing the c-srcNX protein, which contains a deletion of amino acids 15 to 89, displayed a refractile, spindle-shaped morphology, formed intermediate-sized, tightly packed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Thus, deletion of amino acids 15 to 89 can activate the kinase activity and transforming potential of the c-src gene product. Deletion of amino acids 112 to 225, however, did not increase the kinase activity or transforming ability of pp60c-src; indeed, deletion of these sequences in c-srcHP suppressed phenotypic alterations induced by pp60c-src. Cells expressing the c-srcNP or c-srcBS gene products (containing deletions of amino acids 15 to 225 and 55 to 169, respectively) displayed a fusiform, refractile morphology and formed diffuse colonies in soft agar; the mutant proteins displayed an increased in vitro protein-tyrosine kinase activity. However, only a few cellular proteins contained elevated levels of phosphotyrosine in vivo. Thus, deletions downstream of amino acid 89 severely restricted the ability of c-src to phosphorylate cellular substrates in vivo without affecting the intrinsic tyrosine kinase activity of the c-src gene product. These results suggest the existence of at least two modulatory regions within the amino-terminal half of pp60c-src that are important for the regulation of tyrosine kinase activity and for the interaction of pp60c-src with cellular substrates.

Identification of the mismatch negativity in the responses of individual listeners.

The mismatch negativity (MMN) was recorded from normal adults in three stimulus conditions: two contrast conditions and a control condition in which standard and deviant stimuli were identical. Averaged waveforms were analyzed by examiners blind to the evoking stimulus condition. Hit rates, a false alarm rate, and d' values were determined based on the number of MMNs identified in each condition. Hit rates were low and the false alarm rate was relatively high, resulting in unacceptably small d' values. The relationship between MMN findings and corresponding behavioral discrimination data for individual listeners was not systematic. Factors that may contribute to ambiguity and error in MMN data analysis are discussed.

Reliability of the mismatch negativity in the responses of individual listeners.

The mismatch negativity (MMN) was recorded from 12 normal adults during four biweekly sessions. Responses were elicited by a synthetically generated speech contrast (/dalpha/-/galpha/) that all listeners discriminated with at least 90 percent accuracy. Standard and deviant waveforms were replicable across sessions for all listeners; however, replicability of the derived difference waveforms was poor. Of greater importance, the MMN identification rate was too low (29%) to allow reliability to be evaluated. The implications that these findings may have on clinical applicability are discussed.

Hemodynamic changes during laparoscopic cholecystectomy in patients with severe cardiac disease.

STUDY OBJECTIVE: To evaluate the hemodynamic changes and need for pharmacologic interventions during laparoscopic cholecystectomy in patients with severe cardiac dysfunction. DESIGN: Prospective open study. SETTING: University hospital. PATIENTS: 17 ASA physical status III and IV patients with severe cardiac dysfunction undergoing elective laparoscopic cholecystectomy. INTERVENTIONS: A standardized general anesthetic and surgical technique was used for all patients. In addition to routine monitoring, invasive hemodynamic monitoring included radial and pulmonary artery (PA) cannulation. MEASUREMENTS AND MAIN RESULTS: Hemodynamic parameters were recorded prior to induction of anesthesia, 5 minutes after induction of anesthesia but prior to incision, 5 minutes after carbon dioxide (CO2) insufflation and head-up tilt, every 10 minutes after change of position, after deflation of the abdomen and return to supine position, and 10 minutes after attaining supine position. Need for any pharmacologic interventions [to maintain mean arterial pressure (MAP) < 100 mmHg and/or systemic vascular resistance (SVR) < 2,000 dynes sec/cm-5, and/or cardiac index (CI) > 1.5 L/min/m2] and the incidence of any myocardial morbidity and mortality was noted. CI decreased significantly (p < 0.05) following insufflation and remained low until exsufflation. MAP, SVR, and PA occlusion pressure increased significantly (p < 0.05) after CO2 insufflation. Three of the 17 patients required administration of nitroglycerin to maintain the MAP and SVR within the accepted limits, one of whom also required administration of dobutamine to maintain CI. There was no myocardial morbidity or mortality in the perioperative period. CONCLUSION: Laparoscopic cholecystectomy in patients with severe cardiac dysfunction results in significant hemodynamic changes.

Differential release of mast cell interleukin-6 via c-kit.

Mast cells represent a potential source of interleukin-6 (IL-6) and other cytokines that have been implicated in host defense, tissue maintenance/remodeling, immunoregulation, and many other biologic responses. In acquired immune responses to parasites or allergens, the extensive IgE-dependent activation of mast cells via Fc epsilonRI can result in the release of large quantities of biogenic amines that are stored in the cells' cytoplasmic granules as well as the production of lipid mediators and many cytokines; these products together can orchestrate an intense inflammatory response. We now report that activation of mouse mast cells via c-kit, the receptor for the pleiotropic survival/growth factor, stem cell factor (SCF), can induce the release of IL-6. Upon challenge with SCF, bone marrow-derived cultured mouse mast cells (BMCMCs) released amounts of IL-6 that were greater than 100-fold more than those produced by unstimulated cells, but that were substantially less than those produced in response to IgE and specific antigen. Moreover, BMCMCs released IL-6 upon challenge with concentrations of SCF that resulted in little or no detectable release of tumor necrosis factor-alpha, leukotriene C4, histamine, or serotonin. These findings indicate that SCF, a widely expressed protein that is critical for mast cell development and survival, can also regulate the differential release of mast cell mediators.

Increased activation of L-type voltage-dependent calcium channels is associated with glycine enhancement of N-methyl-D-aspartate-stimulated dopamine release in global cerebral ischemia/reperfusion.

We investigated the relationships among N-methyl-D-aspartate, glycine, L-type voltage-dependent calcium channels, and [3H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [3H]PN200-110 ([3H]isradipine) to L-type voltage-dependent calcium channels, that open as a consequence of N-methyl-D-aspartate-induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage-sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N-methyl-D-aspartate-stimulated [3H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N-methyl-D-aspartate-stimulated [3H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N-methyl-D-aspartate-stimulated release in all treatment groups. The enhancement of N-methyl-D-aspartate-stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30-min reperfused animals. These data suggest that glycine may facilitate opening of the voltage-dependent calcium channels activated by N-methyl-D-aspartate and that this facilitation is blocked by the antagonist nitrendipine.