Survival impact of post-operative immunotherapy in resected stage III cutaneous melanomas in the checkpoint era.

BACKGROUND: Checkpoint inhibitors have shown improvement in recurrence-free survival in the post-operative setting for node-positive melanoma and were first approved in late 2015. However, single-agent checkpoint therapies have yet to show benefit to overall survival (OS) for lower-risk stage III cancers. We evaluated the OS benefit of post-operative immunotherapy in the National Cancer Database (NCDB). PATIENTS AND METHODS: Patient cases were selected from the NCDB 2020 Participant Use File. Patients diagnosed with stage III cutaneous melanoma between 2016 and 2019 who underwent definitive resection for their melanoma were included. OS between those who received post-operative immunotherapy within 84 days of surgery and those who did not was analyzed by the Kaplan-Meier method. Demographic and clinical characteristics between the two groups were compared via Cox proportional hazard models. RESULTS: 14 978 patients with stage III melanoma were included. Of those, 34.9% (n = 5234) received post-operative immunotherapy and 65.1% (n = 9744) did not. Using the American Joint Committee on Cancer version 8 (AJCCv8) staging, 36-month survival was significantly higher in patients who received post-operative immunotherapy compared to no post-operative systemic therapy in those diagnosed with stage IIIB (88.0% versus 84.7%, P = 0.011), IIIC (75.6% versus 68.1%, P < 0.001), or IIID (59.2% versus 48.4%, P = 0.002). No significant improvement in 36-month survival was seen in patients who received post-operative immunotherapy in patients with stage IIIA disease (93.0% versus 92.2%, P = 0.218). CONCLUSIONS: Post-operative immunotherapy had an OS benefit in patients with AJCCv8 stage IIIB, IIIC, and IIID disease, but had no significant survival benefit for patients with stage IIIA melanomas.

The World Federation of Societies of Anaesthesiologists: supporting education in the developing world.

The World Federation of Societies of Anaesthesiologists (WFSA) was formed in 1955 and is currently composed of 120 national societies. The aims of WFSA are to improve the standards of anaesthesia worldwide, with a particular emphasis in developing countries. This article details the structure of the WFSA, the various activities carried out by the different committees, and our achievements in education and training.

Ultrasound-guided thoracentesis: is it a safer method?

STUDY OBJECTIVES: The objectives of this study are as follows: (1) to determine the incidence of complications from thoracentesis performed under ultrasound guidance by interventional radiologists in a tertiary referral teaching hospital; (2) to evaluate the incidence of vasovagal events without the use of atropine prior to thoracentesis; and (3) to evaluate patient or radiographic factors that may contribute to, or be predictive of, the development of re-expansion pulmonary edema after ultrasound-guided thoracentesis. DESIGN: Prospective descriptive study. SETTING: Saint Thomas Hospital, a tertiary referral teaching hospital in Nashville, TN. PATIENTS: All patients referred to interventional radiology for diagnostic and/or therapeutic ultrasound-guided thoracentesis between August 1997 and September 2000. RESULTS: A total of 941 thoracenteses in 605 patients were performed during the study period. The following complications were recorded: pain (n = 25; 2.7%), pneumothorax (n = 24; 2.5%), shortness of breath (n = 9; 1.0%), cough (n = 8; 0.8%), vasovagal reaction (n = 6; 0.6%), bleeding (n = 2; 0.2%), hematoma (n = 2; 0.2%), and re-expansion pulmonary edema (n = 2; 0.2%). Eight patients with pneumothorax received tube thoracostomies (0.8%). When > 1,100 mL of fluid were removed, the incidence of pneumothorax requiring tube thoracostomy and pain was increased (p < 0.05). Fifty-seven percent of patients with shortness of breath during the procedure were noted to have pneumothorax on postprocedure radiographs, while 16% of patients with pain were noted to have pneumothorax on postprocedure radiographs. Vasovagal reactions occurred in 0.6% despite no administration of prophylactic atropine. Re-expansion pulmonary edema complicated 2 of 373 thoracenteses (0.5%) in which > 1,000 mL of pleural fluid were removed. CONCLUSIONS: The complication rate with thoracentesis performed by interventional radiologists under ultrasound guidance is lower than that reported for non-image-guided thoracentesis. Premedication with atropine is unnecessary given the low incidence of vasovagal reactions. Re-expansion pulmonary edema is uncommon even when > 1,000 mL of pleural fluid are removed, as long as the procedure is stopped when symptoms develop.

Prevalence and clinical course of pleural effusions at 30 days after coronary artery and cardiac surgery.

The present prospective study was designed to determine the prevalence of pleural effusion at approximately 28 days after cardiac surgery and their subsequent course. This consecutive case study included 389 patients; 312 had only coronary artery bypass graft surgery (CABG) surgery, 37 had both valve and CABG surgery, and 40 had only valve surgery. Chest radiographs were obtained approximately 28 days postoperatively. Patients were subsequently contacted by telephone 3, 6, and 12 months postoperatively and questioned about the presence of fluid in their chest and related symptoms. The prevalence of pleural effusions in the patients undergoing only CABG surgery (63%) or CABG surgery plus valve surgery (62%) was significantly (p = 0.05) higher than that in the patients undergoing valve surgery only (45%). The prevalence of effusions occupying more than 25% of the hemithorax was 9.7%. The primary symptom associated with these larger effusions was dyspnea. Chest pain and fever were uncommon. Over the 12-month follow-up, the effusions tended to resolve. In conclusion, the prevalence of pleural effusions occupying more than 25% of the hemithorax is approximately 10%, 28 days postoperatively. These larger pleural effusions produce dyspnea but not chest pain or fever, and most of the effusions disappear gradually over the subsequent months.

Regulation of growth and tumorigenicity of breast cancer cells by the low molecular weight GTPase Rad and nm23.

Rad is the prototypic member of a family of novel Ras-related GTPases that is normally expressed in heart, skeletal muscle, and lung and that has been shown to exhibit a novel form of bi-directional interaction with the nm23 metastasis suppressor. In the present study, we have investigated the expression of Rad in normal and neoplastic breast tissues by Western blot and immunohistochemistry and the functional effect of altered Rad expression in breast cancer cell lines. We found that, although Rad is frequently expressed in normal breast tissue (23/30 Rad+ve), expression is usually lost in adjacent invasive carcinoma (8/30 Rad+ve; P < 0.0001). However, where Rad expression persists in a small proportion of tumors, it is associated with higher grade, larger size, and extensive axillary nodal involvement (n = 48; P = 0.035, P = 0.016, P = 0.022, respectively). Furthermore, Rad is also highly expressed in a breast cancer cell line with high tumorigenic and metastatic potential (MDA-MB231). To further examine the role of Rad in breast cancer, we stably transfected a Rad-ve breast cancer cell line (MDA-MB435). We observed an increase in growth and marked increased colony formation in soft agar in vitro (P < 0.05) and an increase in tumor growth rate in nude mice (P < 0.05). Moreover, coexpression of nm23 with wild-type Rad inhibited the effect of Rad on growth of these cells in culture and markedly inhibited tumor growth in vivo. Additional transfection studies with mutated Rad cDNAs revealed that the growth-promoting effects of Rad appeared to be mediated through its NH2- and COOH-terminal regions, rather than its GTPase domain, and might involve acceleration of cell cycle transition. These findings suggest that Rad may act as an oncogenic protein in breast tissues and demonstrate a potential mechanism by which interaction between Rad and nm23 may regulate growth and tumorigenicity of breast cancer.

Routine measurement of pleural fluid amylase is not indicated.

BACKGROUND: The routine measurement of pleural fluid amylase is frequently recommended, but the cost-effectiveness of this procedure is unknown. METHODS: To assess the utility of routine measurement of pleural fluid amylase in evaluating pleural effusions, we measured amylase, glucose, lactate dehydrogenase, and protein levels and blood cell counts in 379 patients undergoing thoracentesis during a 22-month period from 1997 to 1999. Of these, 199 had effusions after cardiac surgery; 61, malignant; 48, transudative; 28, parapneumonic; 2, chylous; 2, rheumatoid; 1, tuberculous; and 1, from chronic pleuritis. There were 37 exudates of unknown origin. RESULTS: Measurement of pleural fluid amylase levels did not assist in determining the origin of the effusion in any of the patients. Amylase levels greater than 100 U/L (normal serum level in our laboratory is 30-110 U/L) were found in 5 (1.3%) of 379 patients: 1 patient with congestive heart failure (amylase, 173 U/L), 2 with post-cardiac surgery effusions (144 U/L and 130 U/L), 1 with pneumonia (109 U/L), and 1 with lung cancer (105 U/L). CONCLUSIONS: The routine measurement of pleural fluid amylase levels is neither clinically indicated nor cost-effective. We suggest that pleural fluid serum amylase levels be measured only if there is a pretest suspicion of acute pancreatitis, chronic pancreatic disease, or esophageal rupture.

Vascular endothelial growth factor level correlates with transforming growth factor-beta isoform levels in pleural effusions.

BACKGROUND: Recent studies have demonstrated high levels of vascular endothelial growth factor (VEGF) in exudative pleural effusions and a possible etiologic role. The factors regulating VEGF accumulation in the pleural space are unknown. Transforming growth factor (TGF)-beta is a potent stimulator of VEGF expression in vitro. We hypothesized that TGF-beta induces VEGF production in pleural tissues, and, hence, the pleural fluid VEGF levels should correlate with the levels of TGF-beta in pleural fluid of different etiologies. METHODS: Seventy pleural fluid samples were analyzed. These included 20 malignant, 13 post-coronary artery bypass grafting (CABG), 8 parapneumonic, 11 miscellaneous exudative, and 18 congestive heart failure (CHF) pleural effusions. RESULTS: Pleural fluid VEGF levels showed good correlation with those of TGF-beta(1) (r = 0.58; p < 0. 0001), TGF-beta(2) (r = 0.43; p < 0.001), and lactate dehydrogenase (r = 0.65; p < 0.001). The levels of TGF-beta(1) and TGF-beta(2) also were correlated (r = 0.60; p < 0.0001). The median levels of TGF-beta(1) (2,480 pg/mL) and TGF-beta(2) (266 pg/mL) in the CHF group were significantly lower than those in the malignant (TGF-beta(1), 4,902 pg/mL; TGF-beta(2), 428 pg/mL), post-CABG (TGF-beta(1), 5,456 pg/mL; TGF-beta(2), 377 pg/mL), parapneumonic (TGF-beta(1), 5,024 pg/mL; TGF-beta(2), 464 pg/mL), and miscellaneous exudate groups (TGF-beta(1), 7,690 pg/mL; TGF-beta(2), 369 pg/mL). There was no significant difference in TGF-beta(1) and TGF-beta(2) levels among the four exudate groups. CONCLUSIONS: VEGF levels in pleural effusions are significantly correlated with the levels of TGF-beta(1) and beta(2) isoforms. VEGF, TGF-beta(1), and TGF-beta(2) levels were all higher in exudative effusions than in effusions secondary to CHF.

Interaction of the Ras-related protein associated with diabetes rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation.

Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP.Rad was also reconverted to GTP.Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

Lysophosphatidic acid and apoptosis of nerve growth factor-differentiated PC12 cells.

The lipid biomediator lysophosphatidic acid (LPA) elicits a unique response in hippocampal neurons, LPA induces neuronal apoptosis. This study explores the effects of LPA on cells with neuronal properties, nerve growth factor-differentiated PC6 cells, a clone of PC12 cells. LPA induced apoptosis in these cells as assessed by chromatin condensation, terminal dUTP nick end-labeling of DNA, protection against these nuclear alterations by a general caspase inhibitor and the lack of release of lactic dehydrogenase. LPA caused oxidative stress, namely a decreased reduction of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. This oxidative stress appears to be of functional significance, since cells were protected by pretreatment with the antioxidant propyl gallate and by stable transfection with cDNA encoding the antioxidant enzyme, manganese superoxide dismutase. Mitochondrial and nitric oxide participation in LPA-induced apoptosis are suggested by the protection afforded by pretreatment with either cyclosporin A, an inhibitor of mitochondrial permeability transition, or nitric oxide synthase inhibitors. The nitric oxide synthase inhibitor findings are novel, since to our knowledge, LPA has not heretofore been associated with an increase in nitric oxide. In addition, as observed for many neurotoxic agents, insulin-like growth factor I protected against LPA-induced apoptosis of PC6 cells.

The ras-related protein rad associates with the cytoskeleton in a non-lipid-dependent manner.

Rad is the prototypic member of a new family of Ras-related proteins (Rad, Gem, and Kir) which lack typical C-terminal amino acid motifs for isoprenylation. In mouse C2C12 muscle cell lines about 50% of Rad protein resides in the cytosol and behaves as a hydrophilic protein partitioning away from TX-114. The remainder of Rad is associated with plasma and internal membranes. The association of Rad with the membrane does not occur through the lipid bilayer, but instead depends on the interaction of Rad with the cytoskeleton or membrane skeleton. In contrast to Ras, biosynthetic labeling of cellular proteins in C2Cl2 cells with [3H]palmitic acid demonstrates that Rad is not modified with this fatty acid, and inhibition of isoprenylation with lovastatin treatment has no effect on Rad subcellular distribution. Furthermore, removal of the C-terminal 11 amino acids that are precisely conserved in all three Rad family members has no effect on Rad subcellular distribution. Addition of the 9 amino acids from the C-terminus of H-Ras to the truncated Rad protein results in a redistribution of Rad from the cytosol to the membrane skeleton without the presence of any detectable lipid modification of the chimeric protein. These data suggest that Rad possesses unique cellular localization signals which, in contrast to other Ras-related family members, do not depend on the lipid modification of the C-terminus.

Effects of phosphorylation on function of the Rad GTPase.

Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases.

Rad and Rad-related GTPases interact with calmodulin and calmodulin-dependent protein kinase II.

Members of the Rad family of GTPases (including Rad, Gem, and Kir) possess several unique features of unknown function in comparison to other Ras-like proteins, with major N-terminal and C-terminal extensions, a lack of typical prenylation motifs, and several non-conservative changes in the sequence of the GTP binding domain. Here we show that Rad and Gem bind to calmodulin (CaM)-Sepharose in vitro in a calcium-dependent manner and that Rad can be co-immunoprecipitated with CaM in C2C12 cells. The interaction is influenced by the guanine nucleotide binding state of Rad with the GDP-bound form exhibiting 5-fold better binding to CaM than the GTP-bound protein. In addition, the dominant negative mutant of Rad (S105N) which binds GDP, but not GTP, exhibits enhanced binding to CaM in vivo when expressed in C2C12 cells. Peptide competition studies and expression of deletion mutants of Rad localize the binding site for CaM to residues 278-297 at the C terminus of Rad. This domain contains a motif characteristic of a calmodulin-binding region, consisting of numerous basic and hydrophobic residues. In addition, we have identified a second potential regulatory domain in the extended N terminus of Rad which, when removed, decreases Rad protein expression but increases the binding of Rad to CaM. The ability of Rad mutants to bind CaM correlates with their localization in cytoskeletal fractions of C2C12 cells. Immunoprecipitates of calmodulin-dependent protein kinase II, the cellular effector of Ca2+-calmodulin, also contain Rad, and in vitro both Rad and Gem can serve as substrates for this kinase. Thus, the Rad family of GTP-binding proteins possess unique characteristics of binding CaM and calmodulin-dependent protein kinase II, suggesting a role for Rad-like GTPases in calcium activation of serine/threonine kinase cascades.

Comparison of a modified double-lumen endotracheal tube with a single-lumen tube with enclosed bronchial blocker.

This study compared the modified BronchoCath double-lumen endotracheal tube with the Univent bronchial blocker to determine whether there were objective advantages of one over the other during anesthesia with one-lung ventilation (OLV). Forty patients having either thoracic or esophageal procedures were randomly assigned to one of two groups. Twenty patients received a left-side modified BronchoCath double-lumen tube (DLT), and 20 received a Univent tube with a bronchial blocker. The following were studied: 1) time required to position each tube until satisfactory placement was achieved; 2) number of times that the fiberoptic bronchoscope was required; 3) frequency of malpositions after initial placement with fiberoptic bronchoscopy; 4) time required until lung collapse; 5) surgical exposure ranked by surgeons blinded to type of tube used; and 6) cost of tubes per case. No differences were found in: 1) time required to position each tube (DLT 6.2 +/- 3.1 versus Univent 5.4 +/- 4.5 min [mean +/- SD]); 2) number of bronchoscopies per patient (DLT median 2, range 1-3 versus Univent median 3, range 2-5); or 3) time to lung collapse (DLT 7.1 +/- 5.4 versus Univent 12.3 +/- 10.5 min). The frequency of malposition was significantly lower for the DLT (5) compared to the Univent (15) (P < 0.003). Blinded evaluations by surgeons indicated that 18/20 DLT provided excellent exposure compared to 15/20 for the Univent group (P = not significant). We conclude that in spite of the greater frequency of malposition seen with the Univent, once position was corrected adequate surgical exposure was provided. In the Univent group the incidence of malposition and cost involved were both sufficiently greater that we cannot find cost/ efficacy justification for routine use of this device.

Overexpression of Rad inhibits glucose uptake in cultured muscle and fat cells.

Rad is a Ras-like GTPase that was isolated by subtraction cloning of human muscle and shown to have increased expression in some individuals with Type II diabetes. To ascertain the potential role of Rad in insulin-mediated signaling, we have overexpressed Rad in myocyte and adipocyte cell lines. Expression of Rad resulted in a 50-90% reduction in insulin-stimulated 2-deoxyglucose glucose uptake in C2C12 murine myotubes, L6 rat myotubes, and 3T3-L1 adipocytes and a 25% reduction in 3-O-methylglucose uptake in 3T3-L1 adipocytes. This occurred despite unaltered levels of glucose transporter expression, with no detectable change in Glut4 translocation and with no alteration in insulin receptor or substrate phosphorylation or phosphatidylinositol 3-kinase activity. These data indicate that Rad is a negative regulator of glucose uptake and that this effect may be due to a decrease in the intrinsic activity of the transporter molecules, rather than an effect on the translocation of Glut4.

Molecular cloning of the human rad gene: gene structure and complete nucleotide sequence.

We have isolated and sequenced human genomic DNA clones encoding the Ras-related GTP-binding protein, Rad. The gene spans 3.75 kb and consists of five exons and four introns. Translation initiates from the first of two in-frame methionine residues in the second exon. Several potential transcription cis-elements were revealed throughout the 1.7 kb 5'-flanking region, including 'E box' and CArG binding sites for regulators of transcription in muscle.

Rad, a novel Ras-related GTPase, interacts with skeletal muscle beta-tropomyosin.

Rad, a prototypic member of a subfamily of Ras-related GTPases, is overexpressed in skeletal muscle of type II diabetic humans. By expression screening of mouse embryo and human skeletal muscle cDNA libraries, we found that Rad interacted with skeletal muscle beta-tropomyosin. In the mouse skeletal muscle cell line C2C12, this interaction was significantly increased by the calcium ionophore A23187. A23187 also caused a time- and concentration-dependent decrease in total cellular Rad with increased interaction between tropomyosin and Rad in the detergent-soluble fraction and the appearance of Rad in the cytoskeleton. In C2C12 cells stably overexpressing a putative dominant negative mutant of Rad (S105N), there was an increase in the amount of tropomyosin in Rad immunoprecipitates. In cells overexpressing wild type Rad, much of Rad was associated with the cytoskeleton and was no longer responsive to A23187. In far-Western blotting and guanine nucleotide saturation studies, GDP-Rad bound to tropomyosin far better than GTP-Rad. We conclude that Rad interacts with skeletal muscle beta-tropomyosin and the cytoskeleton in a guanine nucleotide-dependent manner. These data suggest that Rad may be involved in skeletal muscle motor function and cytoskeletal organization.

Can atherosclerotic coronary arteries vasodilate? An intraoperative high-frequency epicardial echocardiographic study.

Our purpose was to evaluate the vasodilating responses of atherosclerotic coronary arteries using intraoperative high-frequency (12 MHz) epicardial echocardiography. We obtained continuous high-frequency epicardial echocardiographic recordings during surgery, and determined cross-sectional lumen area from 17 coronary arterial segments (12 patients). Nitroglycerin (100 to 400 micrograms/min) was administered intravenously to reduce mean (+/- SEM) arterial pressure 14 +/- 1.8 mm Hg. The cross-sectional arterial images were classified using 3 different parameters: arterial lumen area, percentage of the arterial wall circumference that was atherosclerotic (wall thickness > 0.7 mm), and presence of an eccentrically shaped arterial lumen (maximal/minimal luminal diameter > 1.5). Nine arterial segments had small (< 5.0 mm2) arterial lumens (1.7 +/- 0.40 mm2 [+/- SEM; range 0.6 to 3.9]). With nitroglycerin, the luminal area increased 0.8 +/- 0.28 mm2 (range 0 to 2.5), and 39 +/- 12.1% (range 0 to 117). The remaining 8 segments had larger (> 5.0 mm2) lumens (8.7 +/- 0.91 mm2 [range 5.0 to 11.9]). With nitroglycerin the luminal area increased 4.3 +/- 1.11 mm2 (range 1.4 to 11.4), and 51 +/- 10.2% (range 16 to 96). Seven arterial segments had eccentric lumens; mean maximal/minimal ratio was 1.8 +/- 0.08 (range 1.6 to 2.0). The area increased 39 +/- 7.3% (range 16 to 71) with nitroglycerin. In the 10 concentrically shaped lumens (maximal/minimal lumen diameters 1.3 +/- 0.04 [range 1.1 to 1.5]), nitroglycerin increased luminal area by 48 +/- 12.6% (range 0 to 117) (p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the in vitro phosphorylation sites on Gs alpha mediated by pp60c-src.

Overexpression of pp60c-src in mouse fibroblasts potentiates both agonist-induced signalling through beta-adrenergic receptors and cyclic AMP accumulation in response to cholera toxin [Bushman, Wilson, Luttrell, Moyers and Parsons (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7462-7466; Moyers, Bouton and Parsons (1993) Mol. Cell. Biol. 13, 2391-2400]. In reconstitution experiments in vitro, phosphorylation of Gs alpha by immune-complexed pp60c-src resulted in enhanced rates of receptor-mediated guanosine 5'-[gamma-thio]triphosphate (GTP[S]) binding and GTP hydrolysis [Hausdorff, Pitcher, Luttrell, Linder, Kurose, Parsons, Caron and Lefkowitz (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5720-5724]. These results suggest that one mechanism by which pp60c-src affects signalling through the beta-adrenergic receptor is by phosphorylation and functional alteration of the G protein. To elucidate how phosphorylation of Gs alpha might affect its function, we subjected phosphorylated, recombinant Gs alpha to tryptic phosphopeptide analysis. Phosphotryptic peptides were purified by h.p.l.c. and analysed by Edman degradation to determine the cycle numbers at which radiolabelled phosphotyrosine was released. Candidate peptides that contained Tyr residues at the corresponding positions were synthesized, phosphorylated in vitro by pp60c-src, and their migrations in two-dimensional electrophoresis/t.l.c. were compared with those of tryptic phosphopeptides from intact Gs alpha. We report here that Gs alpha is phosphorylated on two residues by pp60c-src, namely, Tyr-37 and Tyr-377. Tyr-37 lies near the site of beta gamma binding in the N-terminus, within a region postulated to modulate GDP dissociation and activation by GTP [Johnson, Dhanasekaran, Gupta, Lowndes, Vaillancourt and Ruoho (1991) J. Cell Biochem. 47, 136-146], while Tyr-377 is located in the extreme C-terminus, within a region of Gs alpha important for receptor interaction [Sullivan, Miller, Masters, Beiderman, Heideman and Bourne (1987) Nature (London) 334, 712-715]. The location of these residues suggests that phosphorylation may affect the function of both of these regulatory domains.

Assessment of an interactive learning system with "sensorized" manikin head for airway management instruction.

An interactive, self-study learning system for airway management instruction that utilizes a "sensorized" manikin head (Actronics Inc., Pittsburgh, PA) was compared to didactic instruction from anesthesiologists during third-year medical student anesthesia rotations. Before students were allowed to participate in airway management on anesthetized patients, they were randomly separated into two groups. One group received instruction from the learning system, and the other group was given a lecture with guided practice on a standard tracheal intubating manikin. Differences between groups were then assessed using 22 separate variables as all students performed actual airway management on patients undergoing general anesthesia. Anesthesia faculty, residents, and nurse anesthetists, blinded to group, served as assessors. There were 48 and 49 students in the didactic instruction and learning system groups, respectively. Beginning experience level of students with respect to airway management was similar between groups before the anesthesia rotations. There were 185 and 188 evaluation forms completed to assess the didactic instruction and learning system groups, respectively. Demographic data regarding patients were recorded. Patients in the learning system group on whom students performed airway management were older, had a larger average body mass index, and their airways more frequently received higher Mallampati classifications (glottic structures more difficult to visualize). No difference in the quality of airway management efforts or in students' appraisal of their own performances was seen between groups. Neither group demonstrated more rapid development of psychomotor skills. Students were equally satisfied with both methods of instruction.(ABSTRACT TRUNCATED AT 250 WORDS)