Regioselective cope rearrangement and prenyl transfers on indole scaffold mimicking fungal and bacterial dimethylallyltryptophan synthases.

Aromatic prenyltransferases are an actively mined enzymatic class whose biosynthetic repertoire is growing. Indole prenyltransferases catalyze the formation of a diverse set of prenylated tryptophan and diketopiperazines, leading to the formation of fungal toxins with prolific biological activities. At a fundamental level, the mechanism of C4-prenylation of l-tryptophan recently has surfaced to engage a debate between a "direct" electrophilic alkylation mechanism (for wt DMATS and FgaPT2) versus an indole C3-C4 "Cope" rearrangement followed by rearomatization (for mutant FgaPT2). Herein we provide the first series of regioselectively tunable conditions for a Cope rearrangement between C3 and C4 positions. Biomimetic conditions are reported that effect a [3,3]-sigmatropic shift whose two-step process is interrogated for intramolecularity and rate-limiting general base-promoted mechanism. Solvent polarity serves a crucial role in changing the regioselectivity, resulting in sole [1,3]-shifts under decalin. An intermolecular variant is also reported that effectively prenylates the C3 position of l-tryptophan, resulting in products that mimic the structures accessed by bacterial indole prenyltransferases. We report an elaborate investigation that includes screening various substituents and measuring steric and electronic effects and stereoselectivity with synthetically useful transformations.

Synthesis and characterization of oriented glyco-capturing macroligand.

An oriented glyco-capturing macroligand was synthesized by site-specific immobilization of an O-cyanate chain-end-functionalized boronic acid containing polymer (boropolymer) onto an amine surface. The O-cyanate chain-end-functionalized boropolymer was synthesized by arylamine-initiated cyanoxyl-mediated free-radical polymerization in a one-pot fashion. The chain-end O-cyanate was confirmed by (13)C NMR spectroscopy. The specific carbohydrate-binding capacity of the boropolymer was evaluated by an alizarin red S assay. Oriented and covalent immobilization of the O-cyanate chain-end-functionalized boropolymer onto the amine-modified solid surfaces and its specific glyco-capturing capacity were confirmed by the quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques. The oriented multivalent glyco-capturing ligand can be used for efficient carbohydrate and glycoconjugate purification and identification, and thus is expected to constitute a core strategy of glycomics and glycoproteomics and carbohydrate-sensing applications.

Anhydrides of real and hypothetical [hydroxy(R-O)iodo]benzenes.

The anhydrides of [hydroxy(methanesulfonato-O)]iodobenzene (HMIB) and [hydroxy(toluenesulfonato-O)]iodobenzene (HTIB) were prepared by drying acetonitrile solutions of the compounds. The anhydrides of the hypothetical compounds [hydroxy(chloroacetato)-O]iodobenzene and [hydroxy(iodoacetato)-O]iodobenzene were obtained from aqueous solutions. Crystallographic structures were obtained for the anhydrides, except that of HTIB. The electron-domain geometries of the I atoms vis-a-vis secondary I...O bonds were explored. The presence of delocalized bonding in groupings of O and I atoms was suggested. A linear relationship between the C-I-O angles and the I-O bond orders was observed.

Evidence-based emergency medicine: clinical synopsis. No clear winner among dressings for partial-thickness burns.

DATA SOURCES: The authors searched the Cochrane Central Register of Controlled Trials (CENTRAL) and the Cochrane Wounds Group Specialized Register (May 29, 2008), MEDLINE (1950 to May 2008), EMBASE (1980 to May 2008), and CINAHL (1982 to May 2008). STUDY SELECTION: The review included randomized controlled trials examining various burn dressings, frequently using silver sulfadiazine as a control. Dressing types included the following: Studies addressing topical skin agents, full-thickness burns, hand burns, and biological skin replacements were excluded. The primary outcomes included time to complete wound healing and change in wound surface area over time. Secondary outcomes included number of dressing changes, pain, patient satisfaction, infection rate, need for surgery, cost, and hospital length of stay. DATA EXTRACTION AND SYNTHESIS: Studies were reviewed by 2 authors independently and data were abstracted using standardized forms. The authors abstracted and pooled data from eligible studies by using appropriate analytical methods according to the Cochrane Handbook, version 5.0.0. Studies were assessed for the adequacy of randomization and allocation concealment, blinding of providers and participants, potential selection bias after allocation, and completeness of follow-up.

Prevalence of hepatitis C, hepatitis B, and human immunodeficiency virus in a Grand Rapids, Michigan emergency department.

OBJECTIVE: The objective of this study is to provide updated prevalence information on hepatitis C, hepatitis B, and human immunodeficiency virus (HIV) among patients in a high-volume emergency department (ED) located in a medium-sized, Midwestern city. BACKGROUND: This study provides updated information regarding the prevalence of the blood-borne pathogens hepatitis C, hepatitis B, and HIV among ED patients. Prior studies of this type have focused on large inner-city populations with high incidence rates of blood-borne diseases. These studies have limited applicability to other common ED settings. METHODS: A convenience sample of 404 patients was selected using blood previously drawn independent of the study. Patient-identifying information was unlinked from study results, which allowed waiver of informed consent from the Institutional Review Board. This blood was then tested for hepatitis C, hepatitis B, and HIV. RESULTS: Prevalence of hepatitis C antibody was 4.0%, relative to the overall US population prevalence of 1.8%. Hepatitis B(s)Ag was present in 0.7% and HIV prevalence was 0.8%. There were no coinfections; therefore, there was a combined prevalence of blood-borne pathogens of 5.5%. CONCLUSIONS: The combined prevalence of blood-borne pathogens of 5.5% supports previous recommendations of universal precautions, particularly in settings where the overall prevalence may be underestimated.

Catabolism of 4-hydroxyacids and 4-hydroxynonenal via 4-hydroxy-4-phosphoacyl-CoAs.

4-Hydroxyacids are products of ubiquitously occurring lipid peroxidation (C(9), C(6)) or drugs of abuse (C(4), C(5)). We investigated the catabolism of these compounds using a combination of metabolomics and mass isotopomer analysis. Livers were perfused with various concentrations of unlabeled and labeled saturated 4-hydroxyacids (C(4) to C(11)) or 4-hydroxynonenal. All the compounds tested form a new class of acyl-CoA esters, 4-hydroxy-4-phosphoacyl-CoAs, characterized by liquid chromatography-tandem mass spectrometry, accurate mass spectrometry, and (31)P-NMR. All 4-hydroxyacids with five or more carbons are metabolized by two new pathways. The first and major pathway, which involves 4-hydroxy-4-phosphoacyl-CoAs, leads in six steps to the isomerization of 4-hydroxyacyl-CoA to 3-hydroxyacyl-CoAs. The latter are intermediates of physiological beta-oxidation. The second and minor pathway involves a sequence of beta-oxidation, alpha-oxidation, and beta-oxidation steps. In mice deficient in succinic semialdehyde dehydrogenase, high plasma concentrations of 4-hydroxybutyrate result in high concentrations of 4-hydroxy-4-phospho-butyryl-CoA in brain and liver. The high concentration of 4-hydroxy-4-phospho-butyryl-CoA may be related to the cerebral dysfunction of subjects ingesting 4-hydroxybutyrate and to the mental retardation of patients with 4-hydroxybutyric aciduria. Our data illustrate the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.

Competence of new emergency medicine residents in the performance of lumbar punctures.

BACKGROUND: Medical students are taught some procedural skills during medical school, but there is no uniform set of procedures that all students learn before residency. OBJECTIVE: To determine the level of competence in the performance of a lumbar puncture (LP) by new postgraduate year 1 (PGY1) emergency medicine (EM) residents. METHODS: An observational study was conducted at three EM residencies with 42 PGY1 residents who recently graduated from 26 various medical schools. The LP procedure was divided into 26 major and 44 minor steps to create a scoring protocol. The model, procedure, and scoring protocol were validated by experienced emergency physicians. Subjects performed the procedure without interruption or feedback on an LP training model using a standard LP kit. A step was scored as "performed correctly" if two of the three evaluators concurred. Pre- and poststudy questionnaires assessed subjects' prior instruction and clinical experience with LP, self-confidence, sense of relevance, motivation, and fatigue. RESULTS: Subjects completed an average of 14.8 (57%; 95% confidence interval [95% CI] = 53% to 61%) of the major steps (range: 4-26) and 19.1 (43%; 95% CI = 42% to 45%) of the minor steps (range: 7-28) in 14.3 minutes (range: 3-22). Sixty-nine percent failed to obtain cerebrospinal fluid from the model. Subjects' levels of confidence changed slightly on a five-point scale from 2.8 ("little-to-some") before the test to 2.5 after the test. Eighty-three percent of the subjects previously performed LPs on patients during medical school (average attempts = 2.2; range: 0-10), but only 40% of those who did so were supervised by an attending during their first attempt. CONCLUSIONS: In the cohort studied, new PGY1 EM residents had not attained competence in performing LPs from training in medical school. Most new PGY1 residents probably require training, practice, and close, direct supervision of this procedure by attending physicians until the residents demonstrate competent performance.

Solution NMR studies of the A beta(1-40) and A beta(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation.

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of the 40-residue A beta(1-40) and 42-residue A beta(1-42) peptides into amyloid plaques. The structural changes associated with the conversion of monomeric A beta peptide building blocks into multimeric fibrillar beta-strand aggregates remain unknown. Recently, we established that oxidation of the methionine-35 side chain to the sulfoxide (Met35(red) --> Met35(ox)) significantly impedes the rate of aggregation and fibrillation of the A beta peptide. To explore this effect at greater resolution, we carefully compared the (1)H, (15)N, and (13)C NMR chemical shifts of four A beta peptides that had the Met35 reduced or oxidized (A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox)). With the use of a special disaggregation protocol, the highly aggregation prone A beta peptides could be studied at higher, millimolar concentrations (as required by NMR) in aqueous solution at neutral pH, remaining largely monomeric at 5 degrees C as determined by sedimentation equilibrium studies. The NOE, amide-NH temperature coefficients, and chemical shift indices of the (1)H alpha, (13)C alpha, and (13)C beta established that the four peptides are largely random, extended chain structures, with the Met35(ox) reducing the propensity for beta-strand structure at two hydrophobic regions (Leu17-Ala21 and Ile31-Val36), and turn- or bendlike structures at Asp7-Glu11 and Phe20-Ser26. Additional NMR studies monitoring changes that occur during aging at 37 degrees C established that, along with a gradual loss of signal/noise, the Met35(ox) significantly hindered upfield chemical shift movements of the 2H NMR signals for the His6, His13, and His14 side chains. Taken together, the present NMR studies demonstrate that the Met35(red) --> Met35(ox) conversion prevents aggregation by reducing both hydrophobic and electrostatic association and that the A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox) peptides may associate differently, through specific, sharp changes in structure during the initial stages of aggregation.

Enhancement of solubility and bioavailability of beta-lapachone using cyclodextrin inclusion complexes.

PURPOSE: To explore the use of cyclodextrins (CD) to form inclusion complexes with beta-lapachone (beta-lap) to overcome solubility and bioavailability problems previously noted with this drug. METHODS: Inclusion complexes between beta-lap and four cyclodextrins (alpha-, beta-, gamma-, and HPbeta-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and 1H-NMR spectroscopy. Biologic activity and bioavailability of beta-lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). RESULTS: Phase solubility studies showed that beta-lap solubility increased in a linear fashion as a function of alpha-, beta-, or HPbeta-CD concentrations but not gamma-CD. Maximum solubility of beta-lap was achieved at 16.0 mg/ml or 66.0 mM with HPbeta-CD. Fluorescence and 1H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between beta-CD and HPbeta-CD with beta-lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of beta-lap in beta-CD or HPbeta-CD inclusion complexes (TD50 = 2.1 microM). Animal studies in mice showed that the LD50 value of beta-lap in an HPbeta-CD inclusion complex is between 50 and 60 mg/kg. CONCLUSIONS: Complexation of beta-lap with HPbeta-CD offers a major improvement in drug solubility and bioavailability.

Amphipols can support the activity of a membrane enzyme.

Amphipathic polymers ("amphipols") were introduced several years ago (Tribet, C.; Audebert, R.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047-15050) as an alternative method for solubilizing integral membrane proteins in stable, nativelike conformations. However, direct maintenance of full membrane protein functionality in amphipol solutions has not previously been demonstrated in the absence of added lipid or detergent. In this contribution, the first zwitterionic amphipol "PMAL-B-100" is introduced. PMAL-B-100 not only maintains membrane protein structure and solubility, but also supports the full catalytic activity of an integral membrane enzyme, diacylglycerol kinase, in the complete absence of additional lipid or detergent. All of the roles which a lipid bilayer normally plays in maintaining diacylglycerol kinase's structure and in facilitating catalysis are satisfied by the environment and interactions supplied by PMAL-B-100.