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Biomedical subjects

B A Andrews

Publications and source records attributed to B A Andrews.

27 records · Page 2Linked to original sources
PubMed1990

Enzymatic cell lysis for product release.

The development of expression systems for recombinant proteins and recombinant protein particles which cannot be secreted and are located in specific cell locations necessitates the development of novel, more selective techniques for cell lysis and or product release. Mechanical cell disruption methods do not discriminate the release of the desired product from among a host of other contaminating molecules and cell debris, and they may also damage the protein product. In contrast, the use of lytic enzyme systems, which can provide biological specificity to the process of cell lysis and product release, shows an interesting potential for controlled lysis. In this chapter we have reviewed the enzymes presently available for cell lysis of bacteria and yeast and have described mathematical models developed to describe the process of lysis, particularly of yeast cells, including simple, structured, and population balance models. Factors affecting the kinetics of lytic enzyme reactions, enzyme recovery, and the effect on downstream operations as well as conditions for use have been discussed. Sources of lytic enzymes and latest developments on enzyme production have been reviewed. Finally, the potential for large-scale use of lytic enzyme technology has been discussed. The design and use of lytic enzyme systems for differential product release from microbial cells have been reviewed. Lytic enzyme systems are usually specific either for yeast or for different types of bacteria. The activity profile of a lytic system will have an effect on the product distribution. This profile can be manipulated at the genetic, physiological, production reactor, enzyme purification, and lysis reactor levels. Alternative process designs that will allow the sequential release of products from different cell locations are discussed. Alternatives are explored by process modeling, process simulation, and optimization techniques. These studies show that the use of lytic enzyme systems has tremendous promise as a method of controlled lysis and differential product release.

Bacteria↗
PubMed1989

Two-dimensional and Doppler echocardiographic assessment of neonatal arterial repair for transposition of the great arteries.

The arterial switch procedure has become an accepted reparative technique for transposition of the great arteries with or without ventricular septal defect. In this study the accuracy of prospective noninvasive imaging in detecting arterial tract obstruction and the prevalence and severity of arterial valvular regurgitation (as assessed by Doppler ultrasound) were evaluated in survivors of arterial repair. All 53 study patients underwent two-dimensional echocardiographic examination 2 days to 20 months (median 7 months) postoperatively; 43 patients also had pulsed and continuous wave Doppler studies. The accuracy of the noninvasive evaluation of arterial tract obstruction was determined by comparison of Doppler maximal instantaneous gradients with peak to peak gradients at nonsimultaneous catheterization in 26 patients. Twenty-one (81%) of the 26 patients underwent catheterization and successful pulsed and continuous wave Doppler examination of the right heart; 17 (81%) of these 21 had a maximal pressure gradient within 20 mm Hg of the peak to peak gradient obtained at catheterization. Echocardiographic identification of the stenotic site was correct in all eight of the patients in this group requiring reoperation. Twenty-three (88%) of the 26 patients who underwent catheterization had successful Doppler interrogation of the aortic tract; 22 (96%) of these 23 had a maximal instantaneous gradient within 20 mm Hg of the peak to peak catheterization gradient. Fourteen (32%) of 43 patients had mild or moderate pulmonary regurgitation by Doppler study. Three (7%) of the 43 had mild aortic regurgitation.

Aortic Valve Insufficiency↗
PubMed1988

Design of enzyme systems for selective product release from microbial cells; isolation of a recombinant protein from yeast.

The development of expression systems for recombinant proteins and recombinant protein particles that cannot be secreted and that are located in specific cell locations necessitates the development of novel, more selective, techniques for cell disruption. Mechanical cell disruption methods do not discriminate the release of the desired product from among a host of other contaminating molecules and cell debris, and they also may damage the protein product. In contrast, the use of lytic enzyme systems, which can provide biological specificity to the process of cell lysis and disruption, shows an interesting potential for controlled lysis. In this report, the design and the use of lytic enzyme systems for differential product release from microbial cells have been reviewed. Lytic enzyme systems are usually specific either for yeast or for different types of bacteria. Moreover, the activity profile of a lytic system will have an effect on the product distribution. This profile can be manipulated at the genetic, physiological, production reactor, enzyme purification, and lysis reactor levels. Alternative process designs that will allow the sequential release of products from different cell locations have been reviewed and discussed. Alternatives have been explored by process modeling, process simulation, and optimization techniques. These studies show that the use of lytic enzyme systems has tremendous promise as a method of controlled lysis and differential product release. Finally, the release of a specific recombinant protein, human serum albumin (HSA) from yeast cells, has been investigated. The low levels of wall-lytic protease present in the Oerskovia lytic enzyme system have no deleterious effect on the protein product, and the level of HSA extracted from two positive yeast clones using lytic enzymes is similar to that extracted after bead breakage.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

The ontogeny of serum GH binding protein in man: a possible indicator of hepatic GH receptor development.

Experiments in rabbits suggest that a serum GH binding protein (GH-BP) probably is derived largely from hepatic membrane bound GH receptors. Human serum contains a specific GH binding protein which can be easily measured by incubation with [125I] hGH and separation of [125I] hGH-BP complexes from free [125I] hGH by gel filtration through an Ultrogel AcA 44 minicolumn. In each assay GH-BP activity of a reference (normal young adult) serum is similarly run and the results are expressed as a percentage of the activity of the unknown serum divided by the GH-BP activity of the reference serum after correction for the expected inhibition of GH binding resulting from the GH content of the unknown serum. The mean relative specific GH binding protein (RSGH-BP) of cord serum from 11 premature infants was only 3.2 +/- 1.4% (SE) and of cord serum from 17 full term infants was 14.9 +/- 2.5%. During the first two decades of life there was a progressive rise of RSGH-BP with considerable individual variation. The mean serum RSGH-BP of 13 such subjects was 54.5 +/- 6.2%. More uniform RSGH-BP results were obtained in serum from 15 young adults, 91.7 +/- 7.4%. Lower RSGH-BP 77.2 +/- 5.4% was found in serum from 12 healthy older adults (age 60 to 70 years). The low levels of RSGH-BP in fetal serum are consistent with the reported low concentrations of GH receptors in sheep and rat fetal liver membranes. We suggest the measurement of GH-BP activity provides a simple, noninvasive measure of the ontogeny of GH receptors of human beings.

Adolescent↗
PubMed1982

Absence of the booster phenomenon in serial tuberculin skin testing.

The Center for Disease has recently recommended that tuberculin skin testing of new hospital employees be done in two steps because of the so-called "booster phenomenon." To assess the feasibility of the two-step method in our hospital, we performed a pilot study using this method on all our new hospital employees during a 6-month period. From July 1, 1980, to December 31, 1980, all newly hired employees of the Strong Memorial Hospital were given a two-step tuberculin skin test as part of their preemployment health evaluation. During this time, 571 new employees were tested, 416 (73%) of whom completed both steps. The mean age of the 416 employees was 26 yr. Fourteen of 448 employees (3.1%) had positive reactions on the first test. None of the 416 employees had positive reactions on the second test. Additional visits for skin test administration, costs of informational materials, administrative expenses, and supplies added approximately $2,600 to the costs of our tuberculin skin testing program during the study period. The absence of the booster phenomenon in our employee population may be due to (1) the relatively young age of our hospital employee population who may be less likely to have remote infection with Mycobacterium tuberculosis and/or (2) the relatively uncommon occurrence of nontuberculous mycobacterial infection in our area of the country. Depending on the age of employees of an institution and its geographic location, the two-step method may not detect any additional reactors. We recommend that hospitals contemplating adoption of this method first perform a similar pilot study in order to assess the frequency of the booster phenomenon in their employee populations.

Adolescent↗
PubMedDate not supplied

Is there a rational method to purify proteins? From expert systems to proteomics.

The purification of recombinant proteins for therapeutic or analytical applications requires the use of several chromatographic steps in order to achieve a high level of purity. A range of techniques is available such as anion and cation exchange chromatography, which can be carried out at different pHs, and hence used at different steps, hydrophobic interaction chromatography, gel filtration and affinity chromatography. Evidently when confronted with a complex mixture of partially unknown proteins or a clarified cell extract there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98, 99.5 or 99.9%). In this review we will show how an initial "proteomic" characterization of the complex initial mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a maximum level of purity with a minimum number of steps. The chosen methodology was implemented in a computer based expert system. The first algorithm developed was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants. The second algorithm developed was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The successful application of the expert system approach, based on an initial proteomic characterization, to the practical cases of protein mixtures and clarified fermentation supernatant is presented and discussed. The purification strategy proposed was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with an "unknown" supernatant of Bacillus subtilis producing a recombinant beta-1,3-glucanase. The system was robust to errors <10% which is the range that can be found in the experimental determination of the properties in the database of product and contaminants. On the other hand, the system was sensitive both to larger variations (>20%) in the properties of the contaminant database and the protein product and to variations in one protein property (e.g. hydrophobicity).

Animals↗
PubMedDate not supplied

Metabolic control analysis of monoclonal antibody synthesis.

A general route for protein synthesis in eukaryotic cells has been proposed and applied to monoclonal antibody (MAb) synthesis. It takes into account transcription of the gene, binding of ribosomes to mRNA, and polypeptide elongation including binding to SRP (signal recognition particles) and SRP-receptor, competing translocation, folding and glycosylation, assembly of the heavy and light chains in a tetrameric protein and Golgi processing and secretion. A comprehensive model was built on the basis of the proposed pathway. The model takes into account the mechanism of each step. Metabolic control analysis (MCA) principles were applied to the general pathway using the proposed model, and control coefficients were calculated. The results show a shared flux control (of both pathway flux and flux ratio at the branch) among different steps, i.e., transcription, folding, glycosylation, translocation and building blocks synthesis. The steps sharing the control depend on the concentration of building blocks, pathway flux and levels of OST (oligosacharyl transferase), BiP (heavy chain binding protein) and PDI (protein disulfide isomerase). Model predictions compare well with experimental data for MAb synthesis, explaining the control structure of the route and the heterogeneity of the product and also addressing future targets for improvement of the production rate of MAbs.

Antibodies, Monoclonal↗