EMS triage and transport of intoxicated individuals to a detoxification facility instead of an emergency department.

Study Objective: We evaluate the effectiveness and safety of emergency medical services (EMS) provider use of a checklist to triage alcohol-inebriated patients directly to a detoxification facility, rather than an emergency department (ED).

Methods: A retrospective cohort study was conducted of all patients evaluated during a 2-year period, from 2003 to 2005, by EMS providers who used a detoxification evaluation checklist to aid in triage decisionmaking. Patients who did not meet detoxification evaluation checklist criteria were transported to an ED.

Factors associated with the interfacility transfer of the pediatric trauma patient: implications for prehospital triage.

Objective: The goal of this study was to identify prehospital factors associated with increased likelihood of interfacility transfer of pediatric trauma patients. Such factors might serve as a basis for improvements in future field pediatric trauma triage guidelines.

Methods: This was a retrospective cohort study of children aged 12 years or younger with blunt, penetrating, or thermal injuries who were transported by ground emergency medical services from the scene to the emergency department of a Level I, II, or III trauma center within the Denver metropolitan area from January 1, 2000, to December 31, 2008.

The discovery of biaryl acids and amides exhibiting antibacterial activity against Gram-positive bacteria.

Solid-phase synthetic methods for biaryl-based compounds were developed resulting in the construction of two 1000-member libraries. Numerous compounds were identified by high-throughput screening using whole cell screens to exhibit anti-microbial activity against Gram-positive bacteria. A series of biaryl compounds containing natural and unnatural amino acids were made to explore the SAR of the amino acid functionality.

A reconstituted dilute blood clot lysis assay for the medium throughput screening of thrombolytic compounds.

Antithrombotic and clotting factors have long been targets for drug discovery, necessitating the development of blood assays to determine the efficacy of lead compounds prior to animal testing. We have developed a reconstituted blood clot lysis assay which eliminates the need for on-site donors. The assay uses whole blood stored at 4 degrees C obtained from a local blood bank, diluted 1:10 in phosphate buffer.

Exploring structure-activity relationships around the phosphomannose isomerase inhibitor AF14049 via combinatorial synthesis.

Phosphomannose Isomerase (PMI) has been shown by genetic methods to be an essential enzyme in fungal cell wall biosynthesis. The PMI inhibitor AF14049 was discovered as an unanticipated side product from high-throughput library screening against the enzyme from C, albicans. Solid-phase synthetic methods were developed and a series of libraries and discrete analogs synthesized to explore SAR around AF14049.

Encoded combinatorial chemistry: synthesis and screening of a library of highly functionalized pyrrolidines.

The application of a new encoding technology for drug discovery is described. A combinatorial library of mercaptoacyl pyrrolidines has been prepared on a beaded polymeric support. Each polymer bead carries one library constituent in association with an oligomeric "tag," the structure of which is a record of the specific reagents from which that library member was prepared.

A high-density screening format for encoded combinatorial libraries: assay miniaturization and its application to enzymatic reactions.

A novel, miniaturized high-throughput screening format is described for assay of combinatorial libraries generated on beads. This approach, which is ideally suited to encoded libraries synthesized on beads, utilizes the photolytic cleavage of individual compounds into a high-density well array (>6500 wells within a standard 96-well microtiter plate footprint) with well volumes as low as 0.37 microl.

Tissue Factor residue Asp44 regulates catalytic function of the bound proteinase Factor VIIa.

The coagulation pathways are initiated by the cell-surface receptor Tissue Factor (TF), which binds the serine proteinase coagulation Factor VIIa (VIIa), resulting in enhanced catalytic function, both amidolytic, towards small pseudo-substrates, and proteolytic, towards macromolecular substrates. Here we implicate Asp44 in TF as a ligand-interactive residue that, in contrast with previously characterized binding residues, is involved in the enhancement of VIIa catalytic function. Whereas charge neutralization by replacement of Asp44 with Asn did not reduce function of human TF, the exchange by Ala resulted in mutants with 8-fold reduced affinity for binding of VIIa.

Energetic contributions and topographical organization of ligand binding residues of tissue factor.

Tissue factor is the cellular receptor and macromolecular enzymatic cofactor for the serine protease coagulation factor VIIa. The ligand binding extracellular domain of tissue factor consists of two structural modules which fold similar to fibronectin type III modules, consistent with the classification of tissue factor as a member of the class 2 cytokine receptor family. On the basis of the three-dimensional structure, we here analyze the importance of tissue factor residues for binding of ligand by scanning alanine mutagenesis.

The alkylating properties of chlorambucil.

Previous work has indicated an aziridinium ion mechanism in the hydrolysis of chlorambucil, and the present work on the alkylation of nucleophiles fully supports this mechanism. This mechanism forms the basis for understanding the kinetics of alkylation reactions because their rates are limited by the rate of formation of the aziridinium ion and the alkylation reaction competes with the hydrolytic reaction. We have measured alpha N, where alpha N(N) is the rate of reaction of the aziridinium ion with a nucleophile N relative to its reaction with water for several nucleophiles that are related to those found in proteins.

Key ligand interface residues in tissue factor contribute independently to factor VIIa binding.

Scanning alanine mutagenesis of the cell surface protease receptor tissue factor suggested importance of residues Lys20, Ile22, Asp58, Arg135, and Phe140 for binding of ligand, the serine protease coagulation factor VIIa. Ligand binding by single alanine replacement mutants was characterized by functional assays which concordantly demonstrated a calculated 1-2.5 kcal/mol reduction in free energy of binding as a result of each of the mutations.

Mutational mapping of functional residues in tissue factor: identification of factor VII recognition determinants in both structural modules of the predicted cytokine receptor homology domain.

Alanine scanning mutagenesis of tissue factor, the initiating receptor and cofactor molecule for the coagulation pathways, was used to define residue side chains with functional contributions. Approximately half of the residues were exchanged, and several stretches of functional residues throughout the entire extracellular domain were identified which contributed to overall coagulant function. Mutants were further characterized with respect to their affinity for binding of ligand, providing evidence that identified functional sequence spans are involved in ligand interaction.

Angiotensin converting enzyme: substrate inhibition.

Phosphate, borate, and Tris inhibit angiotensin converting enzyme (ACE), but HEPES buffer is inert. Measurements of substrate inhibition were made in HEPES buffer at pH 7.0 and 25 degrees C and 37 degrees C.

Purification of bovine angiotensin converting enzyme.

A change has been made in the commonly used lisinopril affinity gel procedure for purifying angiotensin converting enzyme. The new method greatly decreases the time required and greatly increases the yield of pure enzyme. All of the enzyme in various bovine tissues was extracted with 0.

The binding of zinc to angiotensin-converting enzyme.

The equilibrium constant for the dissociation of zinc ion from angiotensin-converting enzyme (ACE) was measured using zinc ion buffers of zinc chloride and nitrilotriacetic acid (NTA). The dissociation constant is 6.4 X 10(-10) M.

Hybridization and partial cDNA sequence analyses of bovine lung angiotensin I-converting enzyme.

The mRNA encoding angiotensin I-converting enzyme, a zinc-metallo dipeptidyl carboxyhydrolase, has been identified in extracts prepared from bovine lung tissue. Bovine lung poly(A) + mRNAs were subjected to electrophoresis and northern blot hybridization analysis using a radiolabeled synthetic 24-deoxyoligonucleotide probe complementary to eight codons for amino acids at the active-site of the enzyme (Harris, R.B.

Cleavage of human von Willebrand factor by platelet calcium-activated protease.

In human platelet lysates prepared by addition of nonionic detergent (Triton X-100) or by sonication, the multimer composition and electrophoretic mobility of platelet von Willebrand factor (vWF) were consistently modified under conditions that would favor activation of the endogenous calcium-activated, sulfhydryl-dependent neutral protease (CAP). By sodium dodecylsulfate-agarose gel electrophoresis, native platelet vWF contained some multimers that were larger than those characteristic of plasma vWF. Modified platelet vWF contained a multimer population equivalent to or smaller than that of plasma vWF plus an additional fast-migrating band.

Interaction of von Willebrand factor with human platelets in the plasma milieu.

The binding of von Willebrand factor (vWf) to stimulated platelets in the plasma milieu was performed using a radiolabeled monoclonal antibody to vWf. Plasma proteins specifically inhibited the thrombin- and ADP/epinephrine-induced vWf binding to activated platelets but did not inhibit the ristocetin-induced vWf binding. When normal plasma was heat defibrinated, monoclonal-labeled vWf was bound to platelets following thrombin or ADP/epinephrine stimulation.