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Biomedical subjects

C R Lowe

Publications and source records attributed to C R Lowe.

At least 37 records · Page 2Linked to original sources
PubMed1998

Design, synthesis, and application of a protein A mimetic.

Low-molecular-weight synthetic molecules that mimic the activity of native biological macromolecules have therapeutic potential, utility in large-scale production of biopharmaceuticals, and the capacity to act as probes to study molecular recognition events. We have developed a nonpeptidyl mimic for Staphylococcus aureus Protein A (SpA). The specific recognition and complexation elements between the B domain (Fb) of SpA and the Fc fragment of IgG were identified from the x-ray crystallographic structure. Computer-aided molecular modeling was used to design a series of biomimetic molecules around the Phe132-Tyr133 dipeptide involved in its binding to IgG. One of the ligands binds IgG competitively with SpA in solution and when immobilized on agarose beads, with an affinity constant of 10(5)-10(6) M-1. The immobilized artificial Protein A was used to purify IgG from human plasma and murine IgG from ascites fluid, and to remove bovine IgG from fetal calf serum.

Animals↗
PubMed1997

Antibody binding to a functionalized supported lipid layer: a direct acoustic immunosensor.

A direct immunosensor has been developed using an acoustic wave device as a transducer. The device is based on an acoustic waveguide geometry that supports a Love wave. The biorecognition surface, formed on a gold layer, consisted of a biotinylated supported lipid layer which specifically bound streptavidin and, subsequently, biotinylated goat IgG. The modified surface was used as a model immunosensor and successfully detected rabbit anti-goat IgG in the concentration range 3 x 10(-8) - 10(-6) M. Using the anti-goat IgG binding isotherm and the time-resolved measurements of antibody binding, both the binding and rate constants of the reaction were determined. The specificity of each binding step was studied with the acoustic wave device, and it was concluded that the phospholipid bilayer showed a good suppression of nonspecific binding. Comparative measurements using surface plasmon resonance allowed the response of the immunosensor to be quantitatively correlated with mass binding to the surface.

Acoustic Stimulation↗
PubMed1997

Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli.

The structural gene for heroin esterase was cloned from Rhodococcus sp. strain H1 and expressed in Escherichia coli BL21(DE3). The purified enzyme was found to be a tetramer with an M(r) of 137,000 and an apparent K(m) of 0.88 mM for 6-acetylmorphine. The G-x-S-x-G motif was observed in the deduced amino acid sequence, suggesting that the enzyme is a serin esterase.

Acetylesterase↗
PubMed1996

Improved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354.

We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replacement of Glu-354 with all possible amino acid residues was achieved using directed mutagenesis, and produced mutant enzymes with a range of thermostabilities. The mutations E354K and E354R conferred the largest increases in thermostability, suggesting that side-chain size and hydrophobicity, as well as charge, may also be important contributors to the overall thermostability of the polypeptide chain at this position. Unusually for such mutations, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.

Animals↗
PubMed1996

Bioluminescent assay for heroin and its metabolites.

Illicit heroin is trafficked as a solid particulate drug, while heroin abuse is monitored by testing urine samples for its principal metabolites, morphine and morphine-3-glucuronide. Two novel bacterial enzymes were used in the development of a linked-enzyme assay for heroin and its metabolites: heroin esterase, which converts heroin to morphine, and morphine dehydrogenase, which oxidizes morphine to morphinone with the concomitant reduction of NADP+. A bioluminescent assay involving heroin esterase, morphine dehydrogenase, and the bacterial luciferase from Vibrio harveyi was developed and shown to be sensitive to 89 ng/mL heroin and 2.0 ng/mL morphine. Excellent correlation with the results from 83 authentic samples submitted for urine drug screening at a hospital laboratory was obtained. The bioluminescent assay exhibited greater specificity and speed than current immunological screening methods. A novel format of the bioluminescent assay involving immobilized enzymes was sensitive to 101 ng (250 pmol) of heroin and responded well to particulate heroin. This form of the test was sensitive enough to respond to one or two typical particles of illicit heroin.

Acetylesterase↗
PubMed1996

Immunosensors.

Immunosensors are important analytical tools for monitoring antibody-antigen reactions in real time. Recent developments in immunosensors have produced systems that allow rapid and continuous analysis of the binding event without the requirement for added reagents or separation/washing steps. As a result, great interest has focused on commercializing immunosensors for applications in areas such as clinical, environmental and food analysis.

Animals↗
PubMed1996

Degradation of cocaine by a mixed culture of Pseudomonas fluorescens MBER and Comamonas acidovorans MBLF.

A mixed culture that could utilize cocaine as the sole source of carbon and energy for growth was isolated by selective enrichment. The individual microorganisms within this mixed culture were identified as Pseudomonas fluorescens (termed MBER) and Comamonas acidovorans (termed MBLF). Each microorganism was shown to be unable to grow to any appreciable extent on 10 mM cocaine in the absence of the other. C. acidovorans MBLF was found to possess an inducible cocaine esterase which catalyzed the hydrolysis of cocaine to ecgonine methyl ester and benzoate. C. acidovorans was capable of growth on benzoate at concentrations below 5 mM but was unable to metabolize ecgonine methyl ester. P. fluorescens MBER was capable of growth on either benzoate as the sole source of carbon or ecgonine methyl ester as the sole source of carbon and nitrogen. P. fluorescens MBER was found to initiate the degradation of ecgonine methyl ester via ecgonine, pseudoecgonine, and pseudoecgonyl-coenzyme A. Subcellular studies resulted in the identification of an ecgonine methyl esterase, an ecgonine epimerase, and a pseudoecgonyl-coenzyme A synthetase which were induced by growth on ecgonine methyl ester or ecgonine. Further metabolism of the ecgonine moiety is postulated to involve nitrogen debridging, with the production of carbonyl-containing intermediates.

Benzoates↗
PubMed1995

Affinity labeling of recombinant ricin A chain with Procion blue MX-R.

Recombinant ricin A chain was irreversibly modified by Procion blue MX-R, a dichlorotriazinyl analogue of Cibacron blue F3G-A, at pH 7.5 and 4 degrees C in 90 h with over 95% loss of activity in an in vitro translation assay. The presence of total yeast RNA reduced the covalent attachment of Procion blue MX-R to ricin A chain. Quantitatively modified ricin A chain contained 2 mol Procion blue MX-R/mol 29-kDa subunit. Tryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a blue peptide corresponding to Gln5-Arg26 of ricin A chain. Thus, a likely dye-binding site on recombinant ricin A was identified. This region is removed from the active-site cleft of recombinant ricin A but may be involved in its substrate binding.

Affinity Labels↗
PubMed1995

An amperometric opiate assay.

Current techniques for the detection and measurement of diacetylmorphine (heroin), morphine and their principal metabolite morphine-3-glucuronide (M3G) are based mainly on chromatography or immunoassay. No enzymatic method for the detection of these compounds has yet been reported. Two novel microbial enzymes have been isolated and characterized in this laboratory: an acetylmorphine carboxyesterase (heroin esterase) and a morphine dehydrogenase (MDH). These highly specific enzymes have been incorporated in an amperometric assay for heroin and morphine using phenazine methosulphate as a mediator. The assay gives a rapid and sensitive response to heroin and morphine, with a detection limit for morphine of 6.8 micrograms ml-1 (23.7 microM).

Acetylesterase↗
PubMed1995

A scanning tunnelling microscopic study of site-specifically immobilized immunoglobulin G on gold.

A convenient and efficient method for the site-specific incorporation of foreign cysteine residues at the C-termini of immunoglobulin G (IgG) using carboxypeptidase-Y-catalyzed transpeptidation is explored as a means of ensuring oriented immobilization of IgG on gold. A scanning tunnelling microscopic study of the immobilization of the modified IgG molecules on gold surfaces is reported. The results show not only that some globular features are observed to form striking surface patterns with a geometric size close to that of the fragments of IgG but also that the conformation of the bound IgG molecules appears more stable when adsorbed on gold. The effect of the immobilization method on these topographic features is discussed.

Animals↗
PubMed1994

Optical biosensor for monitoring microbial cells.

The potential of a new optical biosensor, the resonant mirror, for detecting whole cells is demonstrated. Staphylococcus aureus (Cowan-1) cells, which express protein-A at their surface, were detected by binding to human immunoglobulin G (IgG) immobilized on an aminosilane-derivatized sensor surface at concentrations in the range 8 x 10(6)-8 x 10(7) cells/mL. A control S. aureus strain (Wood-46), which does not express protein-A, gave no significant response. Immobilization of the capture ligand on aminosilane surfaces with and without a hydrogel coating of carboxymethyl-dextran was compared. The greatest binding response was observed with non-dextran-coated surfaces. The sensitivity of the technique was increased a 1000-fold by using a human IgG-colloidal gold conjugate (30 nm) in a sandwich assay format. S. aureus (Cowan-1) cells were detected in spiked milk samples at cell concentrations from 4 x 10(3)-1.6 x 10(6) cells/mL using the sandwich assay.

Biosensing Techniques↗
PubMed1994

Physical adsorption of immunoglobulin G on gold studied by scanning tunnelling microscopy.

This paper describes a preliminary scanning tunnelling microscope (STM) study of the physical adsorption of a mouse monoclonal anti-hCG (human chorionic gonadotrophin) immunoglobulin G (IgG) on vacuum-evaporated gold surfaces in air. Ellipsoid-like protrusions of various geometric sizes assembled in different surface patterns were observed on scanning adsorbed IgG molecules. It is believed that both the adsorption process per se and the tip-molecule interactions are key factors which determine the topographical features of adsorbed IgG molecules.

Adsorption↗
PubMed1994

Identification of a heroin esterase in Rhodococcus sp. strain H1.

A strain of a Rhodococcus sp. (termed H1) capable of utilizing heroin as its sole carbon and energy source was isolated by selective enrichment. An inducible heroin esterase was partially purified and shown to catalyze the hydrolysis of both of the acetylester groups of heroin. The enzyme displays optimum activity at pH 8.5 and appears to be a trimer of identical subunits with an M(r) or 39,000 and a native M(r) of 120,000.

Acetylesterase↗
PubMed1993

Nucleotide sequence and over-expression of morphine dehydrogenase, a plasmid-encoded gene from Pseudomonas putida M10.

Pseudomonas putida M10 was originally isolated from factory waste liquors by selection for growth on morphine. The NADP(+)-dependent morphine dehydrogenase that initiates morphine catabolism is encoded by a large plasmid of 165 kb. Treatment of P. putida M10 with ethidium bromide led to the isolation of a putative plasmid-free strain that was incapable of growth on morphine. The structural gene for morphine dehydrogenase, morA, has been located on the plasmid by oligonucleotide hybridization, by coupled transcription-translation of cloned restriction fragments and by nucleotide sequence analysis and is contained within a 1.7 kb SphI fragment that has been cloned into Escherichia coli. The cloned dehydrogenase enzyme is expressed at high levels in E. coli resulting in a 65-fold increase in morphine dehydrogenase activity in cell-free extracts compared with P. putida M10. Morphine dehydrogenase was rapidly purified to homogeneity, as judged by SDS/PAGE, by a one-step affinity chromatography procedure on Mimetic Orange 3 A6XL. The properties of the purified enzyme were identical with those previously reported for P. putida M10 morphine dehydrogenase. The morA gene was sequenced and the deduced amino acid sequence confirmed by N-terminal amino acid sequencing of the over-expressed protein. The predicted amino acid sequence of morA, deduced from the nucleotide sequence, indicated that morphine dehydrogenase did not belong to the non-metal-requiring short-chain class of dehydrogenases, but was more closely related to the aldo-ketoreductases.

Alcohol Oxidoreductases↗
PubMed1993

Novel affinity separations based on perfluorocarbon emulsions. Development of a perfluorocarbon emulsion reactor for continuous affinity separations and its application in the purification of human serum albumin from blood plasma.

Perfluorocarbon affinity emulsions are generated by the homogenisation of a perfluorocarbon oil with a polymeric fluorosurfactant previously derivatised with an affinity ligand and subsequently cross-linked in situ. This procedure gives rise to a novel liquid affinity adsorbent that can be used for continuous protein purification. Discrete emulsion droplets were found to be unstable when pumped for prolonged periods; however, when flocculated, the emulsion floccules with diameters of around 125 microns, were very stable and sedimented faster. A four-stage reactor unit (perfluorocarbon emulsion reactor for continuous affinity separations, PERCAS) was designed and constructed to carry out continuous separations, and exploited the unusual properties of the absorbent, i.e. liquid nature and high density. Each of the four stages of PERCAS consisted of a mixing tank, for contacting between emulsion phase and aqueous phase, adjacent to a settling tank for the subsequent separation of emulsion from the aqueous phase. Using PERCAS adsorption, washing, elution and re-equilibration of the emulsion could be carried out continuously with emulsion recycle. Using single-component adsorption of human serum albumin to a perfluorocarbon affinity emulsion derivatised with the triazine dye C.I. Reactive Blue 2, PERCAS was optimised with respect to flow-rates and input concentrations. The work was then extended to the continuous purification of essentially homogeneous human serum albumin from blood plasma.

Adsorption↗