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S W Drew

Publications and source records attributed to S W Drew.

9 recordsLinked to original sources
PubMed2003

Measurement of strain-dependent toxicity in the indene bioconversion using multiparameter flow cytometry.

The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains. This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection. The toxic concentrations of the substrate, product, and by-products for each strain have been determined. The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested. The Gram-negative P. putida (421-5 and GM 730) and E. coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms. P. putida GM 730 and the E. coli TDO 123 strains responded similarly to toxic effects, and the E. coli TDO 123 strain was more resistant than the P. putida 421-5 strain. In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches.

Bioreactors↗
PubMed2002

Application of multi-parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene bioconversion.

The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.

Adenosine Triphosphate↗
PubMed1999

Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN.

Indene is oxidized to mixtures of cis- and trans-indandiols and related metabolites by Pseudomonas putida and Rhodococcus sp. isolates. Indene metabolism is consistent with monooxygenase and dioxygenase activity. P. putida resolves enantiomeric mixtures of cis-1,2-indandiol by further selective oxidation of the 1R, 2S-enantiomer yielding high enantiomeric purity of cis-(1S, 2R)-indandiol, a potential intermediate in the synthesis of indinavir sulfate (CRIXIVAN), a protease inhibitor used in the treatment of AIDS. Molecular cloning of P. putida toluene dioxygenase in Escherichia coli confirmed the requirement for the dihydrodiol dehydrogenase in resolving racemic mixtures of cis-indandiol. Rhodococcus sp. isolates convert indene to cis-(1S, 2R)-indandiol at high initial enantiomeric excess and one isolate also produces trans-(1R, 2R)-indandiol, suggesting the presence of monooxygenase activity. Scale up and optimization of the bioconversions to these key synthons for chiral synthesis of potential intermediates for commercial manufacture of indinavir sulfate are described.

Drug Design↗
PubMed1984

Tissue culture as a diagnostic technique for soft-tissue sarcoma of childhood.

A case of childhood small cell undifferentiated sarcoma was grown in vitro in a serum-free culture medium. In vivo the tumor showed no evidence of differentiation by light microscopy, electron microscopy, and immunoperoxidase staining for myoglobin or skeletal muscle myosin. After growth in vitro for two weeks in a defined culture medium, the tumor cells formed myotubes and demonstrated ultrastructural myogenic differentiation. This case demonstrates the use of the tissue culture as a diagnostic aid in the recognition of childhood small cell undifferentiated sarcomas. It also raises questions regarding the origin of these tumors and the ability of in vitro growth to induce differentiation.

Adolescent↗
PubMed1975

Production of cephalosporin C by single and double sulfur auxotrophic mutants of Cephalosporium acremonium.

An early blocked sulfur amino acid auxotroph, Cephalosporium acremonium mutant 274-1 (which could be satisfied by methionine or cysteine), utilized organic sulfur compounds for cephalosporin C production in the following order of decreasing effectiveness; methionine > cystathionine > cysteine, despite the fact that cysteine is considered to be the immediate precursor of the antibiotic. When a genetic block was added to mutant 274-1 in the transsulfuration pathway from cysteine to methionine, the double mutant 11-8 (which grows on methonine but not cysteine) failed to produce cephalosporin C from cysteine even though enough methionine was added to support normal growth. Addition of the non-sulfur analogue, norleucine, resulted in antibiotic production from cysteine in the double mutant. These facts support the hypothesis that methionine stimulation of cephalosporin C production is due to a role of methionine other than that of sulfur donation.

Acremonium↗
PubMed1975

Stimulation of cephalosporin production by methionine peptides in a mutant blocked in reverse transsulfuration.

The previously reported inability of methionine to stimulate cephalosporin C production in a cysteine auxotroph is due to cysteine interference with methionine uptake. In such a cse, "illicit transport" of alanylmethionine can be used to demonstrate the efficacy of methionine in such mutants blocked in the path from methionine to cysteine. This result supports the hypothesis that the stimulatory activity of methionine is not due to its ability to donate sulfur to the caphalosporin C molecule.

Acremonium↗