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Biomedical subjects

Kathleen M McKenzie

Publications and source records attributed to Kathleen M McKenzie.

7 recordsLinked to original sources
PubMed2006

Paclitaxel induces calcium oscillations via an inositol 1,4,5-trisphosphate receptor and neuronal calcium sensor 1-dependent mechanism.

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Despite a well documented tubulin-stabilizing effect, many side effects of taxol therapy cannot be explained by cytoskeletal mechanisms. In the present study submicromolar concentrations of taxol, mimicking concentrations found in patients, induced cytosolic calcium (Ca(2+)) oscillations in a human neuronal cell line. These oscillations were independent of extracellular and mitochondrial Ca(2+) but dependent on intact signaling via the phosphoinositide signaling pathway. We identified a taxol binding protein, neuronal Ca(2+) sensor 1 (NCS-1), a Ca(2+) binding protein that interacts with the inositol 1,4,5-trisphosphate receptor from a human brain cDNA phage display library. Taxol increased binding of NCS-1 to the inositol 1,4,5-trisphosphate receptor. Short hairpin RNA-mediated knockdown of NCS-1 in the same cell line abrogated the response to taxol but not to other agonists stimulating the phosphoinositide signaling pathway. These findings are important for studies involving taxol as a research tool in cell biology and may help to devise new strategies for the management of side effects induced by taxol therapy.

Biotin↗
PubMed2006

Identification and characterization of single chain anti-cocaine catalytic antibodies.

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.

Alanine↗
PubMed2006

Complete reaction cycle of a cocaine catalytic antibody at atomic resolution.

Antibody 7A1 hydrolyzes cocaine to produce nonpsychoactive metabolites ecgonine methyl ester and benzoic acid. Crystal structures of 7A1 Fab' and six complexes with substrate cocaine, the transition state analog, products ecgonine methyl ester and benzoic acid together and individually, as well as heptaethylene glycol have been analyzed at 1.5-2.3 angstroms resolution. Here, we present snapshots of the complete cycle of the cocaine hydrolytic reaction at atomic resolution. Significant structural rearrangements occur along the reaction pathway, but they are generally limited to the binding site, including the ligands themselves. Several interacting side chains either change their rotamers or alter their mobility to accommodate the different reaction steps. CDR loop movements (up to 2.3 angstroms) and substantial side chain rearrangements (up to 9 angstroms) alter the shape and size (approximately 320-500 angstroms3) of the antibody active site from "open" to "closed" to "open" for the substrate, transition state, and product states, respectively.

Antibodies, Catalytic↗
PubMed2005

Quorum sensing in Vibrio harveyi: probing the specificity of the LuxP binding site.

Quorum sensing activity was investigated in the bacterium Vibrio harveyi using a series of both natural and nonnatural analogs of DPD, the penultimate precursor to autoinducer AI-2. The progression of molecules that were both synthesized and investigated includes enantiomeric variants, carbon-chain extension, and hydroxyl-functional group addition/deletions of DPD. The compilation of these studies reveals a binding cleft that can accommodate a number of different structural variants of DPD, albeit with invariably lower activities.

Bacterial Proteins↗