Protein NMR-based screening in drug discovery.

Protein NMR as a screening tool in drug discovery has become prominent within the past ten years. Advances in protein manipulation, whether by biological means (labeling) or physical means (NMR), have created a powerful method that is able to observe ligand-target interactions in solution.

Simulation based teamwork training for emergency department staff: does it improve clinical team performance when added to an existing didactic teamwork curriculum?

OBJECTIVE: To determine if high fidelity simulation based team training can improve clinical team performance when added to an existing didactic teamwork curriculum. SETTING: Level 1 trauma center and academic emergency medicine training program. PARTICIPANTS: Emergency department (ED) staff including nurses, technicians, emergency medicine residents, and attending physicians. INTERVENTION: ED staff who had recently received didactic training in the Emergency Team Coordination Course (ETCC) also received an 8 hour intensive experience in an ED simulator in which three scenarios of graduated difficulty were encountered. A comparison group, also ETCC trained, was assigned to work together in the ED for one 8 hour shift. Experimental and comparison teams were observed in the ED before and after the intervention. DESIGN: Single, crossover, prospective, blinded and controlled observational study. Teamwork ratings using previously validated behaviorally anchored rating scales (BARS) were completed by outside trained observers in the ED. Observers were blinded to the identification of the teams. RESULTS: There were no significant differences between experimental and comparison groups at baseline. The experimental team showed a trend towards improvement in the quality of team behavior (p = 0.07); the comparison group showed no change in team behavior during the two observation periods (p = 0.55). Members of the experimental team rated simulation based training as a useful educational method. CONCLUSION: High fidelity medical simulation appears to be a promising method for enhancing didactic teamwork training. This approach, using a number of patients, is more representative of clinical care and is therefore the proper paradigm in which to perform teamwork training. It is, however, unclear how much simulator based training must augment didactic teamwork training for clinically meaningful differences to become apparent.

Pharmacokinetics and metabolism of nateglinide in humans.

The pharmacokinetics and metabolism of nateglinide were studied in six healthy male subjects receiving a single oral (120 mg) and intravenous (60 mg) dose of [14C]nateglinide in randomized order. Serial blood and complete urine and feces were collected for 120 h post dose. Nateglinide was rapidly (approximately 90%) absorbed, with peak blood and plasma concentrations at approximately 1 h post dose. The maximal plasma concentrations of radioactivity (6360 ngEq/ml) and nateglinide (5690 ng/ml) were comparable, and plasma radioactivity concentrations were about twice those of blood at all times. Oral bioavailability was 72%, indicating only a modest first-pass effect. After either dose, plasma nateglinide concentrations declined rapidly with elimination half-lives of 1.5 to 1.7 h and plasma clearance of 7.4 l/h. Plasma radioactivity was eliminated more slowly with half-lives of 52 and 35 h in plasma and blood, respectively, after the oral dose. The contribution of this more slowly eliminated component to the AUC(0-infinity) was minor. Nateglinide was extensively metabolized, with excretion predominantly (84-87%) in urine. Only approximately 16% of the dose was excreted unchanged in urine after either dosing route. The major metabolites were the result of oxidative modifications of the isopropyl group. Three of these were monohydroxylated, two of which appeared to be diastereoisomers. Additionally, one metabolite with an unsaturation in the isopropyl group and two diol-containing isomers were identified. Glucuronic acid conjugates resulting from direct glucuronidation of the carboxylic acid were also present. The major metabolite in plasma and urine was the result of hydroxylation of the methine carbon of the isopropyl group.

Functional decline in independent elders after minor traumatic injury.

OBJECTIVES: To describe injury types, patterns, and health status in independently functioning elder patients presenting to the emergency department (ED) after a minor traumatic injury; and 2) to assess short-term functional decline in this population at three-month follow-up. METHODS: This was a prospective observational study of elder patients (age > 65 years) discharged home from the ED after evaluation and treatment for an acute traumatic injury. Patients were excluded if they were not independently functioning or had an acute delirium. Type and mechanism of injury sustained during the ED visit were recorded. Functional status was assessed during the visit and three months later using activities of daily living (ADL) and instrumental activities of daily living (IADL) scores. RESULTS: One hundred six subjects were enrolled in the study. Mean age was 74.8 years. The most common injuries observed were contusion (n = 35, 33%, 95% CI = 24% to 42%), fractures (n = 28, 26%, 95% CI = 18% to 36%), lacerations (n = 20, 19%, 95% CI = 12% to 28%), and sprains (n = 12, 11%, 95% CI = 6% to 19%), which represented more than 90% of the injuries. Eighty-eight (83%) patients completed three-month follow up. Of these, 6 of 88 (6.82%, 95% CI = 3% to 14%) declined in their ADL scores and 20 of 88 (22.73%, 95% CI = 14% to 33%) declined in their IADL scores at three months. Primary injury type, specifically contusion, was more prevalent in patients who had a decline in ADL score, as compared with those who did not have a decline in ADL score (chi-square p<0.001). In addition, anatomic locations of injury were different between those patients with and without a decline in IADL scores (chi-square p = 0.008). Gender differences were also found; females were more likely to be injured by a slip, trip, or fall indoors (36 of 58, 62%) than outdoors (22 of 58, 38%); males injured by this mechanism were more likely to be injured outdoors (14 of 20, 70%) as opposed to indoors (6 of 20, 30%), chi-square p = 0.013. CONCLUSIONS: A significant proportion of functional elder patients with minor traumatic injury are at risk for short-term functional decline. Decline in ADL is related to injury type, while IADL decline is related to anatomic location of injury. Emergency physicians should consider initiating follow-up evaluation and possible intervention in highly functioning elders after minor traumatic injury.

NMR spectroscopy in beta cell engineering and islet transplantation.

Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to beta cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c-myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl-2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy. (2) Overnight incubation of islets and beta cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR-detectable markers using conventional 13C and 31P NMR spectroscopy of extracts. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high-resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused beta cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy.

Abdominal compartment syndrome.

Intra-abdominal hypertension (IAH) associated with organ dysfunction defines the abdominal compartment syndrome (ACS). Elevated intra-abdominal pressure (IAP) adversely impacts pulmonary, cardiovascular, renal, splanchnic, musculoskeletal/integumentary, and central nervous system physiology. The combination of IAH and disordered physiology results in a clinical syndrome with significant morbidity and mortality. The onset of the ACS requires prompt recognition and appropriately timed and staged intervention in order to optimize outcome. The history, pathophysiology, clinical presentation, and management of this disorder is outlined.

Clostridium difficile toxins influence hepatocyte protein synthesis through the interleukin 1 receptor.

HYPOTHESIS: Clostridium difficile toxins require interleukin 1 (IL-1) production or a functioning IL-1 receptor to elicit acute-phase protein production by murine hepatocytes. DESIGN: Experimental study. SETTING: Research laboratory at the DVA Medical Center, St Louis, Mo. CELLS STUDIED: Hepatocytes prepared from normal mice, from knockout mice deficient in IL-1 production due to loss of IL-1 converting enzyme, or from knockout mice deficient in the IL-1 p80 receptor. INTERVENTIONS: Cells were treated with lipopolysaccharide, a crude C difficile toxin extract, or purified C difficile toxins A or B for 24 hours in vitro, then radiolabeled with (35)S methionine. Newly synthesized acute-phase proteins were identified by electrophoresis and autoradiography. MAIN OUTCOME MEASURES: Synthesis of a 23-kd acute-phase protein in response to the various stimuli. RESULTS: Lipopolysaccharide, C difficile culture extract, and purified toxins A and B stimulated the synthesis of the 23-kd acute-phase protein by hepatocytes from normal mice and by hepatocytes from knockout mice deficient in the IL-1 converting enzyme. However, hepatocytes from knockout mice deficient in the IL-1 p80 receptor failed to produce this acute-phase protein when treated with the C difficile toxins, although they responded fully to lipopolysaccharide. CONCLUSIONS: Stimulation of acute-phase protein synthesis by C difficile toxins does not require IL-1 production, but does require a functioning IL-1 p80 receptor. This suggests that some of the actions of these toxins are mediated by this receptor.

Calcium-stimulated insulin secretion in diffuse and focal forms of congenital hyperinsulinism.

OBJECTIVES: To identify infants with hyperinsulinism caused by defects of the beta-cell adenosine triphosphate-dependent potassium channel complex and to distinguish focal and diffuse forms of hyperinsulinism caused by these mutations. STUDY DESIGN: The acute insulin response to intravenous calcium stimulation (CaAIR) was determined in 9 patients <20 years with diffuse hyperinsulinism caused by defective beta-cell sulfonylurea receptor (SUR1(-/-)), 3 patients with focal congenital hyperinsulinism (6 weeks to 18 months), a 10-year-old with insulinoma, 5 with hyperinsulinism/hyperammonemia syndrome caused by defective glutamate dehydrogenase (6 months to 28 years), 4 SUR1(+/-) heterozygotes with no symptoms, and 9 normal adults. Three infants with congenital focal disease, 1 with diffuse hyperinsulinism, and the child with insulinoma underwent selective pancreatic intra-arterial calcium stimulation with hepatic venous sampling. RESULTS: Children with diffuse SUR1(-/-) disease and infants with congenital focal hyperinsulinism responded to CaAIR, whereas the normal control group, patients with hyperinsulinism/hyperammonemia syndrome, and SUR1(+/-) carriers did not. Selective arterial calcium stimulation of the pancreas with hepatic venous sampling revealed selective, significant step-ups in insulin secretion that correlated anatomically with the location of solitary lesions confirmed surgically in 2 of 3 infants with congenital focal disease and in the child with insulinoma. Selective arterial calcium stimulation of the pancreas with hepatic venous sampling demonstrated markedly elevated baseline insulin levels throughout the pancreas of the infant with diffuse hyperinsulinism. CONCLUSIONS: The intravenous CaAIR is a safe and simple test for identifying infants with diffuse SUR1(-/-) hyperinsulinism or with focal congenital hyperinsulinism. Preoperative selective arterial calcium stimulation of the pancreas with hepatic venous sampling can localize focal lesions causing hyperinsulinism in children. The combination of these calcium stimulation tests may help distinguish focal lesions suitable for cure by local surgical resection.

Protease-activated receptors 1 and 4 are shut off with distinct kinetics after activation by thrombin.

Protease-activated receptors 1 and 4 (PAR1 and PAR4) mediate thrombin signaling in human platelets. Whether these receptors are redundant, interact, or serve only partially overlapping functions is unknown. We report that PAR1 and PAR4 signal with distinct tempos. In transfected fibroblasts, PAR4 triggered substantially more phosphoinositide hydrolysis per activated receptor than PAR1 and was shut off more slowly than PAR1. Shutoff and internalization of PAR1 depends upon phosphorylation of its carboxyl tail upon receptor activation. In contrast to PAR1, phosphorylation of PAR4 was undetectable, and activation-dependent internalization of PAR4 was much slower than that seen for PAR1. Mutation of potential phosphorylation sites in the carboxyl tail of PAR1 enhanced PAR1 signaling, whereas analogous mutations in PAR4 had no effect. Thus PAR4 signaling is shut off less rapidly than PAR1, probably due to differences in receptor phosphorylation. PAR1 and PAR4 also signaled with distinct tempos in platelets. PAR1 triggered a rapid and transient increase in intracellular calcium, whereas PAR4 triggered a more prolonged response. Together, the tempo of these responses accounted for that triggered by thrombin. Thus differences in the rates at which PAR1 and PAR4 are shut off allow thrombin to trigger intracellular signaling with distinct temporal characteristics.

Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function.

Thrombin activates protease-activated receptors (PARs) by specific cleavage of their amino-terminal exodomains to unmask a tethered ligand that binds intramolecularly to the body of the receptor to effect transmembrane signaling. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGKF, lacks potency and is of limited utility. In a structure-activity analysis of PAR4 peptides, AYPGKF was approximately 10-fold more potent than GYPGKF and, unlike GYPGKF, elicited PAR4-mediated responses comparable in magnitude to those elicited by thrombin. AYPGKF was relatively specific for PAR4 in part due to the tyrosine at position 2; substitution of phenylalanine or p-fluorophenylalanine at this position produced peptides that activated both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to inhibit both receptors. Identifying a single agonist for both receptors raises the possibility that a single antagonist for both receptors might be developed. The AYPGKF peptide is a useful new tool for probing PAR4 function. For example, AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR1, PAR4 couples to G(q) and not G(i).

Clostridium difficile toxin: cytoskeletal changes and lactate dehydrogenase release in hepatocytes.

BACKGROUND: We have found that Clostridium difficile toxins can evoke hepatocyte acute-phase protein synthesis, and that this effect is dependent on a functioning interleukin-1 (IL-1) receptor. The present study was undertaken to determine if C. difficile toxicity, as determined by actin rearrangement and lactate dehydrogenase (LDH) release, also requires a functioning IL-1 receptor. METHODS: Primary hepatocyte cultures were prepared from normal mice, knockout mice deficient in the IL-1-converting enzyme (ICE), and knockout mice deficient in the IL-1 p80 receptor. Hepatocytes were treated for 24 h with C. difficile culture extract, purified C. difficile toxin A, or purified C. difficile toxin B. The actin cytoskeleton was examined using confocal microscopy, and LDH release was measured by spectrophotometric analysis. RESULTS: C. difficile culture extract, toxin A, and toxin B induced collapse of the actin cytoskeleton in hepatocytes from normal mice. Hepatocytes from both the ICE-deficient mice and the IL-1 p80 receptor-deficient mice demonstrated similar responses to both toxins. These toxins also induced significant LDH release in a concentration-dependent fashion in the normal hepatocytes and the ICE-deficient hepatocytes. However, no significant increase in LDH release was observed in hepatocytes from IL-1 p80 receptor-deficient mice. CONCLUSIONS: C. difficile toxins induce actin cytoskeletal collapse independent of IL-1 or the IL-1 receptor. In contrast, toxin-stimulated LDH release was dependent on the presence of the IL-1 receptor. Thus, separate pathways appear to mediate toxic effects as manifested by actin rearrangement and LDH release.

High resolution MAS-NMR in combinatorial chemistry.

High-resolution magic angle spinning (hr-MAS) NMR is a powerful tool for characterizing organic reactions on solid support. Because magic angle spinning reduces the line-broadening due to dipolar coupling and variations in bulk magnetic susceptibility, line widths approaching those obtained in solution-phase NMR can be obtained. The magic angle spinning method is amenable for use in conjunction with a variety of NMR-pulse sequences, making it possible to perform full-structure determinations and conformational analysis on compounds attached to a polymer support. Diffusion-weighted MAS-NMR methods such as SPEEDY (Spin-Echo-Enhanced Diffusion-Filtered Spectroscopy) can be used to remove unwanted signals from the solvent, residual reactants, and the polymer support from the MAS-NMR spectrum, leaving only those signals arising from the resin-bound product. This review will present the applications of high-resolution magic angle spinning NMR for use in combinatorial chemistry research.

Aqueous and vitreous penetration of levofloxacin after oral administration.

OBJECTIVE: To investigate the penetration of levofloxacin, an optical S-(-)isomer of ofloxacin, into the aqueous and vitreous humor after oral administration. DESIGN: Randomized, clinical trial comparing tissue levels of levofloxacin after one or two doses 12 hours apart. PARTICIPANTS: Forty-five patients undergoing initial vitrectomy between February 1997 and June 1997 at the UIC Eye Center. METHODS: Aqueous, vitreous, and serum samples were obtained and later analyzed from 45 patients after oral administration of 1 500-mg tablet (group 1, 22 patients) or 2 500-mg tablets (group 2, 23 patients) 12 hours apart before surgery. MAIN OUTCOME MEASURES: Aqueous, vitreous, and serum concentrations of levofloxacin (micrograms/milliliter). RESULTS: Group 1 achieved mean aqueous, vitreous, and serum levels of 0.59 +/- 0.48 microg/ml, 0.32 +/- 0.34 microg/ml, and 4.34 +/- 3.59 microg/ml, respectively. Group 2 achieved mean aqueous, vitreous, and serum levels of 1.90 +/- 0.97 microg/ml, 2.39 +/- 0.70 microg/ml, and 8.02 +/- 3.14 microg/ml. CONCLUSIONS: Mean inhibitory aqueous and vitreous MIC90 levels were achieved against a majority of ocular pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pneumoniae (vitreous), Bacillus cereus (vitreous), Haemophilus influenzae, Moraxella catarrhalis, and most gram-negative aerobic organisms except Pseudomonas aeruginosa after two doses given 12 hours apart. Mean MIC90 levels were obtained in the vitreous for a majority of pathogens responsible for traumatic, postoperative, or bleb-related endophthalmitis.

Outcome of vitreoretinal surgery and penetrating keratoplasty using temporary keratoprosthesis.

PURPOSE: The use of a temporary keratoprosthesis has allowed earlier surgical intervention in eyes with coexisting vitreoretinal and corneal disease. We analyzed our experience with this type of surgery. METHODS: We retrospectively reviewed charts of patients in whom a temporary keratoprosthesis was used between 1987 and 1998. Analysis was focused on ocular history, indications for surgery, visual acuity (VA), intraocular pressure, anatomic results, and complications. RESULTS: A temporary keratoprosthesis was used in 31 eyes, 22 (71.0%) of which were for trauma-related indications. In 6 (19.4%) of the operated eyes, the fellow eye also had severely reduced VA. Retinal detachments were present in 30 (96.8%) eyes; most had evident proliferative vitreoretinopathy. Twelve (38.7%) eyes had vitreous hemorrhage, and 20 (64.5%) had corneal scars. Improvement in VA was seen initially in 45.1% of patients, and 51.6% maintained equal or better VA at their final visit as compared with before surgery. The common documented reasons for poor final VA were recurrent retinal detachments deemed inoperable (32.3%), phthisis (22.6%), and optic atrophy or macular scar (16.1%). Corneal grafts remained clear in 41.9%. Nine patients had further surgery. The most significant complication was one case of sympathetic ophthalmia. CONCLUSIONS: Combined vitreoretinal and corneal surgery using temporary keratoprostheses has been used in our institution to treat eyes with extreme abnormalities. Outcomes were less favorable than some reported in the literature, probably because of the severity of disease for which temporary keratoprostheses were reserved. Although results are probably better than the natural course of the disease, patients should be informed of realistic expectations for improvement and potential complications when offered this option.

Overuse of splenic scoring and computed tomographic scans.

BACKGROUND: As the most commonly injured abdominal organ in blunt trauma, the management of splenic injury has undergone evolution. The risk of blood transfusions administered in an attempt to save the spleen has lowered the threshold for operation and also expanded the limits for nonoperative management. An in-depth analysis was carried out of risk factors on patients requiring immediate surgery and those who fail non-operative management based on organ injury scaling grading by computed tomographic (CT) scan and operation. The application of nonoperative management in the elderly population and the use of follow-up CT scanning and sonography in the outpatient setting was also examined. METHODS: Between January of 1991 and June of 1996, 226 consecutive blunt splenic trauma, injured patients at a Level I trauma center were evaluated. All subsequent CT scans and sonograms in the inpatient and outpatient setting were analyzed. The Student's t test, Pearson chi2 analysis with Yates correction, and analysis of variance were used to compare between and among groups. RESULTS: There were 153 men (67.7%), an average age of 34.8 years, an average Injury Severity Score of 24.4, and 28 deaths (12%). There was a significant difference with respect to Injury Severity Score, Glasgow Coma Scale score, Revised Trauma Score, units of packed red blood cells transfused, length of stay, intensive care unit length of stay, mean splenic injury grade, and cost between patients observed initially and those operated on initially. There was no significant difference in age between the two groups. Of 170 patients, 37 patients (22%) who had an initial CT scan underwent immediate exploratory laparotomy. The remaining 133 patients (78%) had nonoperative management; however, 15 patients (11%) failed the period of observation. Five in this group had a laparotomy secondary to other causes and another six were operated on within 24 hours of their injury for their splenic injury. Thus, only four of the nonoperative management patients (3%) actually failed nonoperative splenic management after 24 hours of injury. There were 100 second CT scans obtained. Three of these patients, who had developed hemodynamic instability, required operation for a bleeding spleen. The subsequent CT scan was confirmatory in these three patients who resided in the intensive care unit. All other CT scans and sonograms for clinically unremarkable patients failed to yield any alteration in care based on the scans. CONCLUSION: Blunt splenic injured patients can be safely observed; however, there are certain risk factors in those requiring immediate surgery and those failing nonoperative management. The CT scan underestimates injury, possibly related to a progression of bleeding found at the time of operation. No outpatient studies altered the course of management. Age also did not influence outcome. Thus, in the dedicated trauma center, nonoperative management of blunt splenic injury patients does not lead to undue morbidity or mortality. Once discharged, follow-up radiographs in asymptomatic patients are not necessary.