The role of ecotin dimerization in protease inhibition.

Ecotin is a homodimeric protein from Escherichia coli that inhibits many serine proteases of the chymotrypsin fold, often with little effect from the character or extent of enzyme substrate specificity. This pan-specificity of inhibition is believed to derive from formation of a heterotetrameric complex with target proteases involving three types of interface: the dimerization interface, a primary substrate-like interaction, and a smaller secondary interaction between the partner ecotin subunit and the protease. A monomeric ecotin variant (mEcotin) and a single-chain ecotin dimer (scEcotin) were constructed to study the effect of a network of protein interactions on binding affinity and the role of dimerization in broad inhibitor specificity.

Predicting relative binding affinities of non-peptide HIV protease inhibitors with free energy perturbation calculations.

The relative binding free energies in HIV protease of haloperidol thioketal (THK) and three of its derivatives were examined with free energy calculations. THK is a weak inhibitor (IC50 = 15 microM) for which two cocrystal structures with HIV type 1 proteases have been solved [Rutenber, E. et al.

Assessment of cornea viability by confocal laser scanning microscopy and MTT assay.

Purpose: Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness.

Methods: New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature.

Enhanced transepithelial transport of peptides by conjugation to cholic acid.

The potential of the intestinal bile acid transporter to serve as a shuttle for small peptide molecules was investigated. Eleven peptides with a 2-6 amino acid backbone were conjugated to the 24-position of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid (cholic acid) via an amide bond using an automated peptide synthesizer. In a human intestinal cell line (CaCo-2), cholic acid-peptide conjugates were able to inhibit the transepithelial transport of [3H]taurocholic acid, a natural substrate for the bile acid carrier, at a 100:1 conjugate/substrate ratio.

Use of the intestinal bile acid transporter for the uptake of cholic acid conjugates with HIV-1 protease inhibitory activity.

Purpose: To investigate the ability of the human intestinal bile acid transporter to transport cholic acid conjugates with potential HIV-1 protease inhibitory activity.

Methods: Cholic acid was conjugated at the 24 position of the sterol nucleus with various amino acids and amino acid analogs. The CaCo-2 cell line was used as a model to investigate the interaction of these bile acid conjugates with the human intestinal bile acid transporter.

Residues specifically involved in down-regulation but not internalization of the m1 muscarinic acetylcholine receptor.

Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells.

Purification and characterization of proteasomes from Trypanosoma brucei.

Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities, and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. To determine the possible role that proteasomes may play in controlling the life cycle of African trypanosomes, we have isolated proteasomes from the bloodstream and the insect (procyclic) forms of Trypanosoma brucei by DEAE-cellulose chromatography and glycerol gradient fractionation in the presence of ATP.

Delocalizing trypsin specificity with metal activation.

Recognition for proteolysis by trypsin depends almost exclusively on tight binding of arginine or lysine side chains by the primary substrate specificity pocket. Although extended subsite interactions are important for catalysis, the majority of binding energy is localized in the P1 pocket. Analysis of the interactions of trypsin with the P1 residue of the bound inhibitors ecotin and bovine pancreatic trypsin inhibitor suggested that the mutation D189S would improve metal-assisted trypsin N143H, E151H specificity toward peptides that have a Tyr at P1 and a His at P2'.

Cloning by functional complementation in Trypanosoma brucei.

A procyclic Trypanosoma brucei double-knockout mutant lacking the ornithine decarboxylase (ODC) gene was transfected with a T. brucei genomic library in the expression vector pTSO-HYG4, which utilizes the PARP promoter and replicates extrachromosomally by virtue of a minicircle origin of replication. Transfectants which grew in the absence of exogenous putrescine, the product of the ODC-catalyzed reaction, were obtained at a frequency of 1.

Solution structure of the GTPase activating domain of alpha s.

We have used heteronuclear three-dimensional NMR spectroscopy to determine the solution structure of a 141 residue protein containing the GTPase activating domain from the alpha chain of the heterotrimeric G protein Gs. The domain contains six alpha-helices and is stable and structured in solution despite having been excised from the intact Gs protein. The N-terminal ten and C-terminal 11 residues of the protein are unstructured in solution while the core is well determined by the 2483 distance and torsion restraints derived from the NMR spectra.

The electrical characteristics of human skin in vivo.

Purpose: The objectives of this study were to investigate the impedance properties of human skin in vivo and to examine the effect of iontophoresis upon them.

Methods: Having established the intra- and inter-individual variation in basal values of skin impedance, the effect of varying iontophoretic current density, ionic strength and counter-ion on the rate of recovery of skin impedance after iontophoresis was investigated.

Results: Passage of an iontophoretic current caused a significant reduction in the magnitude of the skin impedance.

Synthesis and mass-spectrometric characterization of human serum albumins modified by covalent binding of two non-steroidal anti-inflammatory drugs: tolmetin and zomepirac.

Human serum albumins modified by covalently bound tolmetin or zomepirac were synthesized as models for similar products formed in vivo from acyl glucuronides. Activated esters of both drugs were prepared with 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-imide, and then allowed to react with human serum albumin. Tryptic digests of both protein products were analysed by HPLC to identify peptides containing covalently bound drugs, and binding sites on albumin were identified by high-performance tandem MS.

Investigating the extension of pairwise distance pharmacophore measures to triplet-based descriptors.

Distances between key functional groups have been used for some time as molecular descriptors in 3D database screening and clustering calculations. More recently, a number of groups have explored triplets of molecular centers to describe key ligand features in terms of the properties of triangles. Three-body distances are attractive, since they retain more information than pairwise representations.

Engineered metal regulation of trypsin specificity.

Histidine substrate specificity has been engineered into trypsin by creating metal binding sites for Ni2+ and Zn2+ ions. The sites bridge the substrate and enzyme on the leaving-group side of the scissile bond. Application of simple steric and geometric criteria to a crystallographically derived enzyme-substrate model suggested that histidine specificity at the P2' position might be achieved by a tridentate site involving amino acid residues 143 and 151 of trypsin.

New molecular shape descriptors: application in database screening.

Geometric descriptors are becoming popular tools for encoding molecular shape, for use in database screening and clustering calculations. They provide condensed representations of complex objects and, as a consequence, can usually be compared quite rapidly. Here we present a number of new descriptors and methods for the quantification of molecular shape similarity.

Pharmacokinetic interaction between benzene metabolites, phenol and hydroquinone, in B6C3F1 mice.

There is strong evidence that metabolites are responsible for adverse effects of benzene. Benzene myelotoxicity, reproduced by coadministering phenol (PH) and hydroquinone (HQ) but not when these benzene metabolites were administered alone, has been postulated to be induced by PH stimulating the myeloperoxidase-mediated oxidation of HQ to the toxic 1,4-benzoquinone in bone marrow. A pharmacokinetic interaction between PH and HQ is also hypothesized to contribute to the observation.

Origins of structural diversity within sequentially identical hexapeptides.

Efforts to predict protein secondary structure have been hampered by the apparent structural plasticity of local amino acid sequences. Kabsch and Sander (1984, Proc. Natl.