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Biomedical subjects

W A Weigand

Publications and source records attributed to W A Weigand.

13 recordsLinked to original sources
PubMed1999

Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor.

Synthesis of an operon fusion protein was investigated in batch and fed-batch cultures at high cell densities of recombinant Escherichia coli JM105 [pBAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L(-1) DCW) to be attained. This was believed to be the highest reported value for dry cell mass of E. coli strain JM105 expressing two recombinant proteins. Transcription of the two reporter genes, green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT), was under the control of the p(BAD) promoter of the araBAD (arabinose) operon. Each protein was independently translated via separate ribosome binding sites. CAT served as a model recombinant protein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monitored on-line using an intensity-based optical sensor. A linear correlation between the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product under conditions of high biomass concentration and high productivity.

Arabinose↗
PubMed1996

Reactor comparisons for the biodegradation of thiodiglycol, a product of mustard gas hydrolysis.

An environmentally benign method for the mineralization of sulfur mustard has been proposed involving chemical hydrolysis of sulfur mustard to thiodiglycol, and then the biological degradation of thiodiglycol to generate biomass and gaseous carbon dioxide. Alcaligenes xylosoxidans (SH91) was isolated based on its ability to utilize thiodiglycol as a sole carbon source. This article compares different biological reactor designs and experimentally assesses their relative effectiveness in degrading thiodiglycol using pure cultures of SH91. The reactor configurations studied are batch, continuous stirred-tank reactor (CSTR), and CSTR with cell recycle. From the results, it is clear that the CSTR with cell recycle offers superior performance for a given residence time or volume. These pure culture data are necessary for accurate design of a pilot-scale system where mixed cultures will be employed because of a possible incomplete chemical hydrolysis step.

Biodegradation, Environmental↗
PubMed1995

The effect of cellular energetics on foreign protein production.

Escherichia coli strain F-122 was used to determine if there are additional physiological effects, other than decreasing energetic efficiency accompanied by the excretion of the acetate, on foreign protein production. This organism was the host for expressing HIV582-beta-galactosidase fusion protein under the control of the trp promoter, with ampicillin resistance. By comparing parallel batch cultures with and without acetate addition, it was found that the presence of acetate in the media did not influence beta-galactosidase activity. In these experiments, it appears that the low protein productivity often observed during acetate formation is the result of inefficient cell metabolism, rather than acetate acting as a specific inhibitor of protein production.

Acetates↗
PubMed1995

Optimization for a recombinant E. coli fed-batch fermentation.

The operating strategy that produces the maximum foreign protein expression for a fed-batch process is desired. This is achieved by using a feasible quadratic programming (FSQP) algorithm with a structured model that describes cell growth and product formation for recombinant E. coli. Optimization calculations for a fed-batch culture have not been performed with a model of this complexity up to this point. A constraint on the maximum cell concentration was included. For a fixed value of batch time, the results show that the optimal time profile of feed flow rate can increase the yield of foreign protein by 12-29% over a constant feed rate policy. Also, it was found that the computation time for the FSQP algorithm can be reduced significantly by considering suboptimal profiles of the feed rate, with a minor effect on calculated protein yield.

Algorithms↗
PubMed1987

Effects of recombinant plasmid size on cellular processes in Escherichia coli.

The effects of recombinant plasmid size on cell growth and viability, plasmid copy number, and synthesis of plasmid-encoded protein were investigated in Escherichia coli using plasmid pUC8 and four recombinant derivatives containing inserts of Drosophila melanogaster DNA of 1.7-6.0 kb. Growth in log phase was unaffected by plasmid size, but as plasmid size increased, maximum cell density decreased and, with the largest plasmid, cell death was accelerated after the stationary phase was reached. There was also a correlation between increasing plasmid size and decreased viability at high ampicillin concentrations, resistance to which is conferred by the plasmids. These effects were shown not to be due to transcription or translation of Drosophila sequences carried on the recombinant plasmids. Cells harboring the largest plasmid, pBS5 (8.7 kb), fared poorly in competition with plasmid-free cells in mixed cultures, compared with cells harboring pUC8 (2.7 kb). In addition, pBS5 was harbored at significantly fewer copies per cell than pUC8 at all phases of growth and supported much less production of the plasmid-encoded protein, beta-lactamase, than did pUC8. The results suggest that recombinant plasmid size may be an important parameter in the optimization of large-scale production of plasmid-encoded proteins.

DNA, Recombinant↗
PubMed1976

Pharmacokinetics of chlorpromazine-induced miotic response in rabbits.

The time variation of changes in the chlorpromazine-induced pupil diameter decrease was studied following varying bolus and slowly infused intravenous doses administered to rabbits. The observed pharmacological response data were coverted, via, the use of a dose-effect curve, to values theoretically corresponding to relative biophasic drug levels. These values were, in turn, used to construct a linear pharmacokinetic model of the drug bioavailability input equilibrium pharmacological response output dynamics of the system. The use of a time domain, MULTIFIT, computerized method of fitting the data to obtain a pharmacokinetic model was compared to the use of a frequency response, PLTEST, approach. The fidelity of the model in quantitatively relating the time course of systemic drug bioavailability to observed pupil response was verified by the satisfactory agreement obtained by directly comparing experimentally known amounts of drug intravenously infused with corresponding values computed from observed changes in pupil size. The applicability of using pharmacological data for quantitative bioavailability and pharmacokinetic analysis of chlorpromazine is demonstrated. This finding is particularly significant because no suitable chemical or radiological direct assay technique exists for determining levels of chlorpromazine, except for high doses, in body fluids.

Animals↗
PubMed1976

Bioavailability and pharmacokinetic analysis of chlorpromazine-induced rectal temperature depression in rabbits.

Chlorpromazine-induced depression of rectal temperatures in rabbits kept at 20 degrees was used to determine relative biophasic drug levels corresponding to observed hypothermic response intensities, which then served to establish a triexponential linear mathematical model describing drug transference and drug action in this system. Comparisons of various experimentally known, slow intravenous infusion drug inputs of chlorpromazine with drug inputs computed by deconvolution, using the derived model and observed temperature depressions, served to verify the accuracy of the model for the 0.50-4.0 mg/kg dosage range.

Animals↗
PubMedDate not supplied

On the mechanism of growth of cells (Bacillus amyloliquefaciens) in the mixed aqueous two-phase system.

The growth of Bacillus amyloliquefaciens in the aqueous two-phase system, made up of polyethylene glycol, dextran, and water, was investigated. Generally, Bacillus partitions in the dextran phase, but the magnitude of the separation depends largely on the overall composition of polymers in the phase system. The kinetics of growth of Bacillus amyloliquefaciens was studied in the polyethylene glycol-rich continuous phase, dextran-rich dispersed phase, and in the mixed phase. From the kinetic data it appears that increasing the overall polymer composition causes the cells to adsorp at the interface. On the other hand, partition measurements indicate that increasing polymer concentrations make the cell partitioning more one-sided. This anomaly is explained by studying the interfacial adsorption of cells via dynamic surface tension measurements.

Adsorption↗
PubMedDate not supplied

Bioreactor strategies for the treatment of growth-inhibitory waste: an analysis of thiodiglycol degradation, the main hydrolysis product of sulfur mustard.

The microbial degradation of thiodiglycol, the primary hydrolysis product of sulfur mustard, by a pure culture of Alcaligenes xylosoxydans ssp. xylosoxydans (SH91) was accomplished in laboratory scale stirred tank reactors. This is a major component of the overall biodegradation process proposed for the complete mineralization of sulfur mustard. Several configurations were evaluated for degradation efficiency including batch, repeated batch, continuous stirred tank reactor (CSTR), and two-stage series CSTR. The repeated batch reactor provided the highest degradation rate of thiodiglycol. Further, this method degraded thiodiglycol in the liquid broth to below the detection limits (0.03 mM). Both batch and repeated batch experiments were simulated by an unstructured mathematical model. Simulation results were in agreement with the experimental data, particularly at low TDG concentration (around 30 mM). This study demonstrates the degradation of thiodiglycol using bioreactors and, more generally, is an experimental study of bioreactor designs for the degradation of growth-inhibitory substances.

Alcaligenes↗