Feasible and effective use of a simulation-based curriculum for post-graduate emergency medicine trainees in India to improve learner self-efficacy, knowledge, and skills.

BACKGROUND: Pediatric emergency medicine training is in its infancy in India. Simulation provides an educational avenue to equip trainees with the skills to improve pediatric care. We hypothesized that a simulation-based curriculum can improve Indian post-graduate emergency medicine (EM) trainees' self-efficacy, knowledge, and skills in pediatric care. METHODS: We designed a simulation-based curriculum for management of common pediatric emergencies including sepsis, trauma, and respiratory illness and pediatric-specific procedures including vascular access and airway skills. Training included didactics, procedural skill stations, and simulation. Measures included a self-efficacy survey, knowledge test, skills checklist, and follow-up survey. Results were analyzed using the Wilcoxon signed-rank test and paired-samples t test. A 6-month follow-up survey was done to evaluate lasting effects of the intervention. RESULTS: Seventy residents from four academic hospitals in India participated. Trainees reported feeling significantly more confident, after training, in performing procedures, and managing pediatric emergencies (p < 0.001). After the simulation-based curriculum, trainees demonstrated an increase in medical knowledge of 19% (p < 0.01) and improvement in procedural skills from baseline to mastery of 18%, 20%, 16%, and 19% for intubation, bag-valve mask ventilation, intravenous access, and intraosseous access respectively (p < 0.01). At 6-month follow-up, self-efficacy in procedural skills and management of pediatric emergencies improved from baseline. CONCLUSIONS: A simulation-based curriculum is an effective and sustainable way to improve Indian post-graduate EM trainees' self-efficacy, knowledge, and skills in pediatric emergency care.

Increased p53 transcription prior to DNA synthesis is regulated through a novel regulatory element within the p53 promoter.

p53 mRNA levels are tightly regulated during the cell cycle with its transcription being induced prior to DNA synthesis. However, the mechanism controlling this regulation is not well defined. Through characterizing an additional 1000 bp of upstream DNA sequences of the murine p53 gene, we identified new positive and negative regulatory elements. Furthermore, we found a trans-acting factor(s) that binds within a positive cis-acting element (-972/-953) in a manner indicative of regulation during the cell cycle. When Swiss3T3 cells are arrested by serum depletion p53 mRNA levels decrease and binding of this regulatory factor(s) to the promoter is reduced. Upon serum stimulation, the regulatory factor(s) binds the promoter and p53 mRNA levels increase prior to the cells entering S phase. When the factors are experimentally sequestered from the promoter or when the regulatory element is deleted from the promoter, p53 promoter activity is reduced. There is no further reduction in p53 promoter activity upon serum depletion and the kinetics of induction upon serum stimulation is delayed by approximately 5 h. These findings indicate that a factor(s) binding within the -972/-953 regulatory element on the p53 promoter is important for the proper regulation of p53 mRNA expression in response to mitogen stimulation. Our initial findings indicate that a member of the C/EBP family of transcription factors may play a role in this regulation.

Cloning and characterization of murine p53 upstream sequences reveals additional positive transcriptional regulatory elements.

Transcriptional regulation of the p53 gene plays an important role leading to elevated expression of mutant p53 alleles in tumor cells. In addition, alterations in p53 transcription levels occur in response to changes in the cell cycle. Previous work had identified a number of regulatory sites at the 5'-end of the murine p53 promoter. During the characterization of the 5'-end of the cloned murine p53 promoter, we identified a 28 bp positive regulatory element that participates in three distinct DNA-protein complexes. The binding by nuclear factors to each one of these sites contributes to the overall activity of the p53 promoter. One site is a potential recognition sequence for members of the ETS family of transcription factors, which are known regulators of the human p53 promoter. Since six nucleotides in the middle of this required element were not present in the previously published sequence of the murine promoter, we recloned this region from C57/BL6 cells and confirmed their presence in the genome. The removal of this regulatory element completely abolishes p53 promoter activity.

The antiproliferative effect of hexadecylphosphocholine toward HL60 cells is prevented by exogenous lysophosphatidylcholine.

The mechanisms that account for the anti-proliferative properties of the biologically active lysophospholipid analog hexadecylphosphocholine (HexPC) were investigated in HL60 cells. HexPC inhibited the incorporation of choline into phosphatidylcholine and the pattern of accumulation of soluble choline-derived metabolites pinpointed CTP:phosphocholine cytidylyltransferase (CT) as the inhibited step in vivo. HexPC also inhibited recombinant CT in vitro. HexPC treatment led to accumulation of cells in G2/M phase, triggered DNA fragmentation and caused morphological changes associated with apoptosis. The supplementation of HexPC-treated cells with exogenous lysophosphatidylcholine (LPC) completely reversed the cytotoxic effects of HexPC and restored HL60 cell proliferation in the presence of the drug. LPC provided an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC. This result contrasted with the response of HL60 cells to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) where LPC overcame the cytotoxic effects but did not support continued cell proliferation. Morphological integrity, DNA stability and cell viability were maintained in cells treated with LPC plus either antineoplastic agent. Thus the inhibition of phosphatidylcholine biosynthesis at the CT step accounts for the cytotoxicity of both HexPC and ET-18-OCH3 which is overridden by providing an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC.

Efficacy Expectations and Vocational Interests as Mediators between Sex and Choice of Math/Science College Majors: A Longitudinal Study

A longitudinal study was conducted to test the mediational role of efficacy expectations in relation to sex differences in the choice of a math/science college major. Data on 101 students were gathered prior to their entering college and then again after they had declared a major 3 years later. Path analytic results support the importance of both math self-efficacy beliefs and vocational interest in mathematics in predicting entry into math/science majors and mediating sex differences in these decisions. Also, students who described themselves as more extroverted were less likely to take additional math classes in high school. Students with stronger artistic vocational interests chose majors less related to math and science. School personnel are strongly encouraged to develop programs that challenge the crystallization of efficacy beliefs and vocational interest patterns before students enter college.

The association of lipid activators with the amphipathic helical domain of CTP:phosphocholine cytidylyltransferase accelerates catalysis by increasing the affinity of the enzyme for CTP.

The biochemical mechanism for the regulation of enzyme activity by lipid modulators and the role of the amphipathic alpha-helical domain of CTP:phosphocholine cytidylyltransferase (CT) was investigated by analyzing the kinetic properties of the wild-type protein and two truncation mutants isolated from a baculovirus expression system. The CT[delta 312-367] mutant protein lacked the carboxyl-terminal phosphorylation domain and retained high catalytic activity along with both positive and negative regulation by lipid modulators. The CT[delta 257-367] deletion removed in addition the region containing three consecutive amphipathic alpha-helical repeats. The CT[delta 257-367] mutant protein exhibited a significantly lower specific activity compared to CT or CT[delta 312-367] when expressed in either insect or mammalian cells; however, CT[delta 257-367] activity was refractory to either stimulation or inhibition by lipid regulators. Lipid activators accelerated CT activity by decreasing the Km for CTP from 24.7 mM in their absence to 0.7 mM in their presence. The Km for phosphocholine was not affected by lipid activators. The activity of CT[delta 257-367] was comparable to the activity of wild-type CT in the absence of lipid activators and the CTP Km for CT[delta 257-367] was 13.9 mM. The enzymatic properties of the CT[delta 231-367] mutant were comparable to those exhibited by the CT[257-367] mutant indicating that removal of residues 231 through 257 did not have any additional influence on the lipid regulation of the enzyme. Thus, the region between residues 257 and 312 was required to confer lipid regulation on CT, and the association of activating lipids with this region of the protein stimulated catalysis by increasing the affinity of the enzyme for CTP.

Lysophosphatidylcholine attenuates the cytotoxic effects of the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3- phosphocholine.

A colony-stimulating factor 1-dependent cell line was used to determine the relationship between the inhibition of phospholipid synthesis and the cytotoxic activity of the antineoplastic ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). ET-18-OCH3 inhibited choline incorporation into phosphatidylcholine as well as total phospholipid synthesis. Exposure to ET-18-OCH3 at the G1/S boundary led to the accumulation of cells in G2, whereas the addition of ET-18-OCH3 in the G1 phase of the cell cycle prevented entry into the S phase. In both cases, ET-18-OCH3 treatment triggered DNA fragmentation and morphological changes associated with apoptosis within 10 h. The addition of lysophosphatidylcholine provided an exogenous source of cellular phospholipid and prevented ET-18-OCH3-dependent accumulation of cells in G2 and apoptosis. However, lysophosphatidylcholine did not overcome the ET-18-OCH3-dependent G1 block, although the growth-arrested cells remained viable. These data indicate that restoring phosphatidylcholine synthesis by supplementation with lysophosphatidylcholine overrides the cytotoxic but not the cytostatic activity of ET-18-OCH3.

Lysophosphatidylcholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine inhibit the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase step.

The regulation of the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step by lysophosphatidylcholine (LPC) and the nonhydrolyzable LPC analog, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), was investigated in a colony-stimulating factor 1-dependent murine macrophage cell line. LPC inhibited phosphatidylcholine synthesis in vivo and led to the accumulation of choline and phosphocholine coupled to the disappearance of CDP-choline pointing to CT as the intracellular target. LPC neither inhibited cell growth nor decreased the cellular content of CT or altered the distribution of CT between soluble and particulate subcellular fractions. The inhibition of phosphatidylcholine synthesis was specific for LPC since lysophospholipids lacking the choline headgroup were not inhibitors. ET-18-OCH3 was a more potent inhibitor of phosphatidylcholine synthesis than LPC and caused the translocation of CT from the soluble compartment to the particulate compartment. Both LPC and ET-18-OCH3 were inhibitors of CT activity in vitro and kinetic analysis showed competitive inhibition with respect to the lipid activator. These data point to LPC as a negative regulator of de novo phosphatidylcholine synthesis that acts at the CT step and establish the mechanism for the inhibition of phosphatidylcholine biosynthesis by antineoplastic phospholipids.

Effect of insulin on SN-1,2-diacylglycerol species and de novo synthesis in rat skeletal muscle.

Insulin treatment increases the SN-1,2-diacylglycerol (DAG) concentration in skeletal muscle. Because DAG may participate in transmission or modulation of the insulin receptor signal, we examined the effect of insulin on total DAG and on different DAG species in isolated rat hemidiaphragms incubated with 5 mmol/L glucose. Five DAG species (16:0-18:1 omega 9, 16:0-18:1 omega 7, 18:0-18:1 omega 9, 18:0-18:2 omega 6, and 18:1-18:2) were identified and quantified. After a 5-minute incubation with 60 nmol/L insulin, neither total DAG nor a DAG species increased; exposure to insulin for 10 or 20 minutes increased the concentration of total DAG and of several DAG species. Insulin did not increase DAG in muscles incubated without glucose. Two sources for the insulin-mediated DAG increase were considered: phosphatidylcholine (PC) hydrolysis and de novo DAG synthesis from glucose. Concentrations of choline and phosphocholine in muscle were not increased after 10-minute incubations with insulin. However, insulin increased 14C incorporation from [U-14C]glucose into DAG, triacylglycerol (TAG), and total lipids approximately threefold. Okadaic acid (OKA), an inhibitor of phosphoprotein phosphatases 1 and 2A, increased muscle DAG content and synthesis from glucose, similar to the effect of insulin. Doses of OKA or insulin that increased DAG mass greatly exceeded those required for stimulation of glucose transport. The insulin-mediated, relatively slow increase in muscle DAG observed here likely reflects primarily de novo synthesis from glucose. This effect would be downstream of insulin stimulation of glucose transport. However, a possible insulin-mediated, rapid transient increase in muscle DAG content and PC hydrolysis cannot be ruled out by our studies.

Mail by modem: the BITNET connection.

Nursing faculty in university settings are now able to communicate with colleagues around the country or in other parts of the world through electronic networks such as BITNET. This article describes how members of the Southern Nursing Research Society's (SNRS) Governing Board used the BITNET system for accomplishing the society's work. Essential hardware and software requirements for using the network are described. Guidelines for logging on, sending messages, and e-mail courtesy are explained. Examples are included for using the network for 1) research and manuscript collaboration; 2) organizational communication and planning; and 3) teaching activities.

Okadaic acid, insulin, and denervation effects on glucose and amino acid transport and glycogen synthesis in muscle.

Effects of okadaic acid (OKA) and calyculin A, cell-permeating specific inhibitors of phosphoprotein phosphatases-1 and -2A, were studied in isolated rat hemidiaphragms. OKA stimulated glucose transport (half-maximum = approximately 0.1 microM; maximum = approximately 1 microM) but was less effective than 6 nM insulin. Insulin and OKA effects were not additive. OKA diminished or abolished glucose transport-stimulation by insulin. System A amino acid transport was also stimulated by OKA, insulin was more effective, and preexposure to OKA inhibited insulin stimulation. Calyculin A affected both transport systems similarly to OKA. OKA did not affect basal glycogen synthesis but abolished its stimulation by insulin. Denervated muscles develop post-receptor insulin resistance. Glucose transport and glycogen synthesis were essentially unresponsive to insulin 3 days postdenervation; however, glucose transport was stimulated by OKA similarly to controls. OKA did not affect glycogen synthesis in denervated muscle except for abolishing a small insulin effect. The data suggest similar acute regulation of glucose and system A amino acid transport in muscle. Enhanced Ser/Thr phosphorylation of unidentified protein(s) stimulates both processes but inhibits their full stimulation by insulin. Postdenervation insulin resistance likely reflects impaired signal transduction.

Extracorporeal membrane oxygenation treatment. One-year outcomes for North Carolina infants.

Initial survival rates for the first 45 North Carolina ECMO neonates in the study group are somewhat better than national figures. Developmental functioning as assessed at one year of age is comparable to that reported in other studies. A congenital diaphragmatic hernia diagnosis was significantly related to poorer outcome. Measures of family functioning approximate those reported for families of non-ECMO neonates treated in intensive care. Support provided by ECMO staff was rated at a notably high level. With nearly three thousand survivors and multiple numbers of ECMO centers now operating in this country, ECMO has made the transition from an experimental treatment to an established procedure for treatment of neonates in pulmonary failure. A few beginning attempts to utilize ECMO with older patients are being made. Neonatologists are now directing efforts toward refining techniques and selection criteria. Further evaluation of longer-term effects of this type of treatment is essential. Effective protocols for follow-up care need to be designed, particularly for those at greatest risk for developmental disability.

Purification from human pregnancy serum of a low molecular weight mitogen similar to placental lactogen and growth hormone.

Recently, we isolated from the serum of pregnant women a factor that induced rapid proliferation of a lactogen-dependent rat lymphoma cell line (Nb2). This mitogenic factor is reasonably specific to pregnancy, since it was present in serum samples from second trimester as well as term-pregnant women, but not in those of adult men or cycling females. It is unlikely that this mitogenic activity (referred to as pregnancy mitogen [PM]) is due to contamination by classical lactogens, since acetone fractionation of serum yielded a preparation devoid of placental lactogen and prolactin, as determined by radioimmunoassays. Further purification of acetone precipitates from term-pregnant serum by ion exchange chromatography and gel filtration yielded a mitogenic activity with a relative mol wt of approximately 10,000. PM activity in the NB2 cell bioassay was not affected by the presence of prolactin antiserum. However, its activity was immunoneutralized by coincubation with anti-placental lactogen serum and, to a lesser extent, anti-growth hormone serum. It appears that PM was not generated by our extraction procedure, since gel filtration of whole serum also yielded a bioactive fraction of approximately 10 kDa. PM was further purified to homogeneity by high-performance liquid chromatography. Examination of the preliminary amino acid composition of PM revealed differences from that of a bioactive fragment of growth hormone and a corresponding portion of placental lactogen, suggesting that PM could be either a molecular variant of these hormones or a novel protein.

Effects of insulin and phospholipase C in control and denervated rat skeletal muscle.

Phospholipase C (PLC), an enzyme that increases endogenous 1,2-diacylglycerol (DAG), caused dose-dependent stimulation of 2-deoxy-D-glucose (2-DG) uptake in rat soleus muscles; the maximal effect was less than that of insulin. In denervated muscles the effect of insulin on 2-DG uptake was markedly reduced, whereas the response to PLC was identical to that of control muscles. Both PLC and insulin stimulated glucose incorporation into glycogen in control but not in denervated solei. Amino acid transport was unaffected by PLC; however, the enzyme completely inhibited the stimulation of amino acid transport by insulin. PLC did not activate the insulin receptor tyrosine kinase but decreased activation of the receptor by insulin in vivo. Basal muscle DAG content increased after denervation. Incubation with PLC markedly increased DAG in control and in denervated muscle. Insulin increased total DAG mass less than PLC in control muscles and did not affect DAG in denervated muscles. In media without added Ca2+, PLC stimulation of DAG production was impaired, and 2-DG uptake was unresponsive to PLC. The data are consistent with, but do not prove, that a subpopulation of DAGs may participate in insulin-mediated stimulation of glucose transport. They also suggest that the denervation-induced insulin resistance of glucose transport may reflect impaired generation of certain DAGs involved in the signaling cascade.

Insulin administration in vivo increases 1,2-diacylglycerol in rat skeletal muscle.

Based on in vitro studies, an insulin-mediated increase in muscle 1,2-diacylglycerol (DAG) content has been proposed as a signal for the insulin induced stimulation of glucose transport. A recent study [Turinsky, J., Bayly, B.P. and O'Sullivan, D.M. (1990) J. Biol. Chem. 265, 7933-7938] challenged this hypothesis because no increase in muscle 1,2-diacylglycerol was observed after in vivo infusions of insulin at doses which markedly stimulated muscle glucose transport. We observed a 30-45% increase in DAG in rat gastrocnemius and diaphragm muscles, 5-15 min after intramuscular or intravenous injections of 1-3 U of insulin per rat, doses which would be expected to activate insulin receptors more fully. The effects on DAG were similar whether or not hypoglycemia was prevented by co-injection of glucose.

Synergy of monocytes and lymphocytes from idiopathic minimal lesion nephrotic patients in relapse in the production of the supernatant factor that increases rat glomerular basement membrane sulfate uptake.

We have previously shown a significant increase in 35sulfate uptake in rat glomerular basement membrane (GBM) when glomeruli were cocultured with peripheral blood mononuclear cells (PBMC) from idiopathic minimal lesion nephrotic syndrome (IMLNS) patients in relapse or with the supernatants of the same PBMC cultures. The purpose of this study was to determine the cell source of the supernatant factor. Rat glomeruli were cocultured with PBMC, with monocytes or with lymphocytes from 11 patients with IMLNS in relapse. Monocytes and lymphocytes were separated using adherence to plastic technique. Rat glomeruli cultured without PBMC served as controls. There was a significant increase in 35sulfate uptake in the GBM when glomeruli were cocultured with PBMC (geometric mean [GM]: 794 cpm/mg dry glomerular weight) as compared to glomeruli cultured with monocytes (GM: 316) (p less than 0.0005), glomeruli cultured with lymphocytes (GM: 309) (p less than 0.05), and glomeruli cultured alone (GM: 302) (p less than 0.00005). No significant differences in 35sulfate uptake were seen between monocytes, lymphocytes and glomeruli alone. These data showed that monocytes and lymphocytes are needed for the production of the supernatant factor that increases rat GBM 35SO4 uptake. Based on these and previous data (Concanavalin A stimulation of IMLNS PBMC results in an increased GBM uptake of 35sulfate, Pediatric Research 20: 321, 1986), we postulate that monocytes could trigger or amplify the production of the supernatant factor by lymphocytes.

Effect of concanavalin A on nephrotic peripheral blood mononuclear cells mediated increased 35sulfate uptake in rat glomerular basement membrane.

We have previously shown a significant increase in 35sulfate uptake in rat glomerular basement membrane (GBM) when glomeruli were cocultured with peripheral blood mononuclear cells (PBMC) from patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) in relapse, but an uptake not different than normal controls if glomeruli were incubated with PBMC of patients in remission. In the present study we examined 35sulfate uptake by GBM after PBMC from 12 IMLNS patients in remission were stimulated with Concanavalin A (Con A) (10 micrograms/ml of culture media). There was a significant increase in 35sulfate GBM uptake when glomeruli were cocultured with Con A-stimulated IMLNS PBMC (geometric mean), 331 cpm/mg dry glomerular weight) as compared to glomeruli cocultured with IMLNS PBMC (geometric mean, 200) (p = 0.048); glomeruli alone stimulated with Con A (geometric mean, 182) (p = 0.008) or glomeruli alone (geometric mean, 146) (p = 0.002). No significant differences were seen between the groups when glomeruli were cocultured with PBMC from 12 normal adults. These data show that Con A stimulated PBMC from IMLNS patients in remission alter the sulfate metabolism of rat GBM. The stimulation of PBMC with Con A reproduces the increase in 35sulfate uptake observed when glomeruli are cocultured with PBMC from IMLNS in relapse. Sulfated compounds in the GBM may play a role in glomerular permeability. Since stimulated nephrotic PBMC alter the metabolism of GBM sulfated compounds, these findings may have pathogenic significance.