Diffusive and electrostatic effects with insolubilized enzymes subject to substrate inhibition.
Explore the source record for details and available documents.
Bacteria↗
Biomedical subjects
M L Shuler
Publications and source records attributed to M L Shuler.
Diffusive and electrostatic effects with insolubilized enzymes.
Explore the source record for details and available documents.
Catalysis↗
Hydrodynamic focusing and electronic cell-sizing techniques.
The technique of hydrodynamic focusing, used to improve the resolution of the Coulter counter for the sizing of bacteria, was examined. Latex particles of 0.26 mum to 6.7 mum volume were used to examine the characteristics of the system with and without hydrodynamic focusing. The system then was evaluated for sizing mixed bacterial populations as well as single populations. Possible applications are also discussed.
Journal Article↗
A preliminary physiologically based pharmacokinetic model for naphthalene and naphthalene oxide in mice and rats.
Naphthalene is a toxicant with unusual species and tissue specificity that has been the subject of in vitro studies. We describe a preliminary physiologically based pharmacokinetic (PBPK) model for naphthalene constructed solely from in vitro data for comparison to animal data without the use of adjustable parameters. The prototypical PBPK model containing five lumped tissue compartments was developed to describe the uptake and metabolism of naphthalene by mice and rats dosed intraperitoneally (i.p.) and orally (po). The model incorporates circulation and biotransformation of the semistable reactive intermediate, naphthalene oxide, as well as the parent compound naphthalene. Circulation is included because the toxic action of naphthalene has been proposed to be caused by the formation of a reactive metabolite in one organ (liver) and its circulation to another organ (lung) being adversely affected by the metabolite. The model allows conversion of naphthalene oxide into dihydrodiol, glutathione (GSH) conjugates, 1-naphthol (non-enzymatically) and covalently bound adducts with proteins. Model simulations are compared with previously reported in vivo measurements of glutathione depletion, mercapturic acid formation, and covalently bound protein formation. The mouse model predicts accurately the amount of mercapturates excreted, the effect of various pretreatments, and the extent of covalent binding in the lung and liver resulting from ip administration, including the sharp increase in binding between 200 and 400 mg/kg.
Absorption↗
Process options in hepatitis B surface antigen extraction from transgenic potato.
The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined. The effects of temperature, the reducing agent beta-mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined. Sedimentation profiles were performed to characterize HBsAg assembly into virus-like particles. Increasing the temperature of the sample for about 1 min increased the measured HBsAg antigenic activity. The optimum temperature was around 50 degrees C. A 3-fold enhancement of the antigenic activity was obtained in extract from transgenic potato expressing HBsAg, when monoclonal antibodies were used to assay for HBsAg. When antigenic activity was determined by polyclonal antibodies, no enhancement in the antigenic activity was obtained. Temperature may affect the conformation of the a epitope to which the monoclonal antibodies bind or alter the fluidity of surface lipid regions. BME increased the antigenic activity of HBsAg up to 4-fold when monoclonal antibodies directed against the a determinant were used, but there was no increase with polyclonal antibodies. This observation suggests that BME affects the structure or presentation of the a epitope. In the presence of BME and leupeptin, a proteinase inhibitor, higher antigenic activity was obtained. Leupeptin might protect the antigen, which might become more susceptible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases. Although both temperature and BME increased the antigenic activity of HBsAg individually, when combined their interaction was antagonistic, resulting in reduced antigenic activity. Different proteinase inhibitors, including leupeptin, aprotinin, E-64, pefabloc, and pepstatin, had no significant effect on HBsAg from potato extract in a 2 h period in the absence of BME. The sedimentation profile of potato-produced HBsAg was determined in 5-30% sucrose gradients. Yeast-derived recombinant HBsAg was used as a positive control. The HBsAg from transgenic potato showed sedimentation and density properties that are very similar to the yeast-produced antigen, indicating assembly into virus-like particles. BME treatment did not change the sedimentation profile.
Hepatitis B Surface Antigens↗
Characterization of a perfusion reactor utilizing mammalian cells on microcarrier beads.
Our overall objective is to develop a cell culture analogue bioreactor (CCA) that can be used together with a corresponding physiologically based pharmacokinetic model (PBPK) to evaluate molecular mechanisms of toxicity. The PBPK is a mathematical model that divides the body into compartments representing organs, integrating the kinetic, thermodynamic, and anatomical parameters of the animal. The CCA bioreactor is a physical replica of the PBPK; where the PBPK specifies organs, the CCA bioreactor contains compartments with a corresponding cell type that mimics some of the characteristic metabolism of that organ. The device is a continuous, dynamic system composed of multiple cell types that interact through a common circulating cell culture medium. The CCA bioreactor and the model can be coupled to evaluate the plausibility of the molecular mechanism that is input into the model. This paper focuses on the design, development, and characterization of a CCA bioreactor to be used in naphthalene dose response studies. A CCA bioreactor prototype developed previously is improved upon by culturing the cells on microcarrier beads. Microcarrier beads with cells attached can form packed beds with cell culture medium perfusing the beds. In this study, two packed beds of cells, one with L2 cells (rat lung) and one with H4IIE cells (rat hepatoma), are linked in a physiologically relevant arrangement by a common recirculating cell culture medium. Studies of this CCA bioreactor presented here include mixing profiles, effect of reactor environment on cell viability and intracellular glutathione, naphthalene distribution profile, and initial naphthalene dosing studies. Unlike the prototype system there is no detectable response to naphthalene addition; in a companion paper we show that this discrepancy can be explained by differences in liquid residence times in the organ compartments. The perfusion reactor design is shown to have significant operating improvements over prototype designs.
Animals↗
Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein.
The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.
Alkaline Phosphatase↗
Escherichia coli host cell modifications in continuous culture affecting heterologous protein overproduction: a population dynamics study.
There are many published studies of plasmid segregational instability in Escherichia coli in the literature. However, the formation of plasmid-free segregants can be controlled by the addition of selective chemical agents like antibiotics. This solution has become commonplace in both the laboratory and industry. On the other hand, host cell modifications, which result in low production of plasmid-encoded protein and lead to loss of culture productivity, have not been adequately addressed. Continuous culture of an inducible (ptac) Escherichia coli vector containing strain, RB791(pKN), was characterized by strong dynamic changes in the cell population and product (beta-lactamase) expression. Long-term cultivation resulted in the loss of high-level production of beta-lactamase. Loss of productivity was not due to the formation of plasmid-free cells or structural modifications to the plasmid; instead, continuous operation resulted in a culture dominated by irreversibly altered, low-producing cells. Two distinct classes of lac- mutants which inhibited induction were identified (Y- and I(s)).
Chromosomes, Bacterial↗
Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system.
Eight cell lines derived from the insects Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, and Estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. Cell density was found to be an important factor in comparing the cell lines due to a density-dependent inhibition of specific protein and virus production that appeared to result from cell-cell contact. After infection of cells at low-density specific beta-galactosidase production per cell would drop between 3- and 6-fold in five of the eight cell lines when plated on tissue culture plates at near-confluent and confluent cell densities. The cell lines Sf 21 and Sf 9 were least sensitive to cell density. After accounting for cell density effects and differences in cell size, two cell lines, BTI Tn 5B1-4 and BTI TnM, were identified that were superior to the other cell lines, including Sf 21 and Sf 9, in beta-galactosidase production. Optimal volumetric and specific beta-galactosidase production from Tn 5B1-4 and TnM cells was 2-fold and 5-fold higher, respectively, in both cell lines than the optimal production from Sf 9 or Sf 21 cells. The Tn 5B1-4 cell line also had the highest viability of all the cell lines at 3 days postinfection and could be adapted to serum-free media.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of plasmid copy number and runaway plasmid replication on overproduction and excretion of beta-lactamase from Escherichia coli.
Runaway plasmid replication can be used to increase target gene dosage and thereby overproduce proteins within the bacterium Escherichia coli. However, the presence of excessive plasmid DNA often alters normal cell functions. High copy number plasmids with strong promoters place a severe metabolic burden on the cell, causing a decreased specific growth rate and changes in cell physiology. Induction of beta-lactamase synthesis from the tac promoter on plasmid pKN causes runaway plasmid replication and excretion of beta-lactamase. Runaway plasmid replication results from readthrough of tac promoter transcripts into the replication region of the plasmid. Both high plasmid copy numbers and a strong promoter (tac) are necessary to achieve the level of overproduction necessary for excretion of beta-lactamase, but high-level target protein synthesis is detrimental to the cell. A derivative of pKN which is more easily regulated was constructed by adding the lacI gene to the plasmid.
DNA Replication↗
A computer model for intracellular pH regulation in Chinese hamster ovary cells.
A single-cell model for Chinese hamster ovary (CHO) cells has been extended to study the regulation of intracellular pH. This model provides for the first time a direct mechanistic linkage between intracellular pH control and cellular metabolism. Among the known mechanisms that regulate and disturb pHi, the Na/H antiport, effect of lactate and ammonia, proton leakage, and external pH have been included in the model. The ability of the model to predict both steady-state and transient pHi has been tested against experimental studies. The "regular model" that has been used to predict cell growth in suspension and batch cultures can predict the steady-state pHi and the transient response of pHi to an alkaline load well, but it had to be modified in terms of the maximum activity of the Na/H antiport in order to simulate the transient response of pHi to an acid load. The modification indicates the possibility that a cell could activate the Na/H antiport under acute acid load but return to its basal level of Na/H antiport activity under steady-state conditions, even when the cell interior is under low-pH conditions (6.7-7.0).
Amino Acids↗
Production of human alkaline phosphatase, a secreted, glycosylated protein, from a baculovirus expression system and the attachment-dependent cell line Trichoplusia ni BTI-Tn 5B1-4 using a split-flow, air-lift bioreactor.
A split-flow, air-lift bioreactor for the cultivation of insect cells to produce recombinant protein is described. It can be used advantageously with attached cell systems. This bioreactor incorporates two sections: a rise and a downcomer. Trichoplusia ni BTI-Tn 5B1-4 cells are grown on a support material of glass beads or microcarriers placed in the downcomer. This cell line is more productive than other commonly used insect cell lines, but it has the disadvantage of being difficult to use at large volumes since it is not easily adaptable to suspension culture. Adequate oxygen demand is supplied by sparging without direct exposure of cells to air bubbles. Nutrients are supplied convectively to the attached cells on support material as the fluid flows through the downcomer. The split-flow, air-lift bioreactor appears to be suitable for insect cell culture and is potentially scalable. It can provide a high surface-to-volume ratio and can be operated in batch or continuous mode. A lab-scale prototype bioreactor has been constructed and tested for the production of a secreted, glycosylated recombinant protein (human alkaline phosphatase) or seAP using an Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) vector. With a ratio of riser cross-sectional area to downcomer cross-sectional area of 1, an aspect ratio of 4.4, an air-flow rate of 54 mL/min in the riser, and a bed of 2400 3-min nonporous glass beads, 10.7 micrograms/mL of seAP was produced using an MOI (multiplicity of infection or the ratio of plaque-forming units to cells) of 10.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkaline Phosphatase↗
A simple model to predict the effectiveness of molecules that block attachment of human rhinoviruses and other viruses.
The binding of viruses to cell surfaces is often mediated by cell surface receptors. The use of soluble receptors, such as intracellular adhesion molecule-1 (ICAM-1) for human rhinovirus (HRV), CD4 for human immunodeficiency virus (HIV), and CR2 for Epstein-Barr virus, for in vivo antiviral therapy is under serious investigation. A number of synthetic compounds that affect HRV attachment and uncoating (termed WIN compounds) are also being studied. However, the mechanism behind the dose-response effect of these agents in preventing infection has not been clearly demonstrated. In addition to simple blocking (by receptors) or inactivation of binding sites (by WIN compounds) on the virus surface, other mechanisms of inhibition have been proposed and demonstrated, including cooperative inactivation of neighboring sites, receptor-induced viral attachment protein (VAP) shedding, virus particle inactivation, and inhibition of multivalent virus binding. We present a simple mathematical model to predict the effect of these molecules on virus infection by incorporating only the blocking or site inactivation step of the blocking molecule and its resulting effect on attachment. The ability of the model to reproduce the response of a virus to a dose of blocking molecules is used to distinguish the role of blockers in inhibiting attachment from the other mechanisms of viral inactivation that have been proposed. The model includes both the reversible attachment of the virus to its cellular receptor and to soluble receptors or synthetic molecules (blockers).(ABSTRACT TRUNCATED AT 250 WORDS)
Antiviral Agents↗
Induction of cytochrome P-450IA1 activity in response to sublethal stresses in microcarrier-attached Hep G2 cells.
Cell damage for cells grown on microcarriers in suspension is a critical problem for scale-up of microcarrier reactors. In order to study cell damage as a mechanistic process, a cellular response that is more sensitive than changes in growth and death rates and would be more closely related to cell regulatory mechanisms would be advantageous. We have observed the induction of a specific cytochrome P-450 monooxygenase, P-450IA1 (CYP1A1), to be a sensitive method for assessing the response of microcarrier-attached Hep G2 cells to stress resulting from hydrodynamic shear and oxygen deprivation. The kinetics of induction and amount of CYP1A1 formed in response to subtle shear stress, moderate shear, and hypoxia are described. Increased stress results in increased CYP1A1 formation.
Biotechnology↗
Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture.
Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 10(5) cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of beta-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production.
Alkaline Phosphatase↗
Production of a sialylated N-linked glycoprotein in insect cells.
Under High Aspect Ratio Vessel (HARV) bioreactor culture conditions designed to simulate the microgravity of orbital space flight, insect tissue culture cells infected with a baculovirus expression vector produced a human glycoprotein with tri- and tetra-antennary complex N-linked oligosaccharides containing terminal sialic acid residues. The oligosaccharide structures were similar to those produced in human placental cells. Insect cells cultured in T-flasks only performed incomplete oligosaccharide processing. The mechanism of HARV-mediated changes in the eukaryotic N-linked glycosylation pathway was investigated and could be mimicked under T-flask growth conditions with the addition of N-acetylmannosamine to the culture medium. The significance of these investigations is discussed with respect to the production of recombinant therapeutic glycoproteins, insect physiology, and orbital space flight.
Animals↗
Metabolic inhibitors, elicitors, and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures.
Inhibition of biosynthetic enzymes and translation and translocation processes, elicitation, and precursor feeding were used to probe biosynthetic pathway compartmentation, substrate-product relationships, and yield limitation of the diterpenoid taxanes in cell cultures of Taxus chinensis (PRO1-95). The results suggest the following: (i) the source of isopentenyl pyrophosphate in taxane production is likely plastidic rather than cytoplasmic; (ii) baccatin III may not be a direct precusor of Taxol (Taxol is a registered trademark of Bristol-Myers Squibb for paclitaxel); (iii) baccatin III appears to have cytoplasmic and plastidic biosynthetic components, while Taxol production is essentially plastidic; and (iv) arachidonic acid specifically stimulates Taxol production but does not have a significant effect on baccatin III yield. Semiempirical mathematical models were used to describe these results and predict potential yield-limiting steps. Model simulations suggest that, under current operating conditions, Taxol production in Taxus chinensis (PRO1-95) cultures is limited by the ability of the cells to convert phenylalanine to phenylisoserine rather than by the branch-point acyl transferase. This result is supported by the lack of improvement of Taxol yield by feeding phenylalanine or benzoylglycine. The methods described in this article, while specifically expanding our knowledge of taxane production in PRO1-95 cultures, could be generally useful in investigating complex aspects of secondary metabolic pathways in plant cell cultures, especially when details of the pathway and compartmentation are sparse.
Alkaloids↗
Possible role of arachidonic acid in stress-induced cytochrome P450IA1 activity.
We have previously reported that a microcarrier-attached human hepatoma (Hep G2) cell line responds to hydrodynamic shear upon transfer to an agitated, clean, autoclaved spinner flask with a transient increase in cytochrome P450IA1 (CYPIA1) activity. Physiological changes induced by hydrodynamic stress could be problematic in the scaleup of microcarrier cultures. A better understanding of how stress alters cell physiology may assist in reactor scaleup. The induction of CYPIA1 activity was dependent on the agitation level of the cultures, and the level of CYPIA1 induction was comparable to that obtained with exposure to approximately 0.1 nM TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin). It has been well documented that hydrodynamic shear stress can cause alterations in the metabolism of phospholipid membrane-bound arachidonic acid (AA) in adherent cells in a parallel plate system. The present study was carried out to determine if either AA or a metabolite of AA was involved in the induction of CYPIA1 activity in the microcarrier cultures of Hep G2 cells. Addition of exogenous AA followed by initiation of the stress resulted in an increase in the level of CYPIA1 activity. Pretreatment of the cultures with quinacrine, an inhibitor of phospholipase A2, reduced the stress-induced CYPIA1 activity. Furthermore, addition of propranolol, an inhibitor of phosphatidic acid phosphohydrolase, resulted in an increase in the response in addition to sustaining the induced enzyme activity. Pretreatment with the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, caffeic acid, had no effect on the response, suggesting that the cyclooxygenase and lipoxygenase pathways were not involved in generating AA metabolites that alter CYPIA1 activity. The agent, nordihydroguaiaretic acid, blocks the monooxygenase pathway and blocks CYPIA1 activity increases. These observations suggest a possible mechanism where the stress on the cells induces phospholipase D, resulting in the formation of phosphatidic acid which then activates phospholipase A2, resulting in the release of AA. Further, these results are consistent with a mechanism in which the metabolism of AA, most likely through the monooxygenase pathway, results in a metabolite that by a yet unknown mechanism induced CYPIA1.
5,8,11,14-Eicosatetraynoic Acid↗