A single-cell model for CHO cells.
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Animals↗
Biomedical subjects
M L Shuler
Publications and source records attributed to M L Shuler.
Equilibrium and kinetic analysis of Autographa californica nuclear polyhedrosis virus attachment to different insect cell lines.
The kinetic and equilibrium attachment of Autographa californica nuclear polyhedrosis virus (AcMNPV) to seven insect cell lines was evaluated. Kinetic experiments revealed differences of up to 10-fold in the infection rates among cell lines. Equilibrium binding also varied between cell lines and was saturable. The Tn 5B1-4 and Tn F cell lines had the highest virus binding affinities and infection rates and exhibited diffusion-limited attachment. The rate of infection appears to be limited by the rate of attachment. For the Tn 5B1-4 cells the physical to infective particle ratio for AcMNPV was 5.3. From the Scatchard analyses, the cell lines Tn 5B1-4 and Tn F displayed affinities of 2.35 x 10(10) M-1 and 1.60 x 10(10) M-1, respectively, with 6000 and 13,700 binding sites per cell. The insect cell line Hz 1075, which is not susceptible to AcMNPV infection, displayed a much lower, but saturable, binding of AcMNPV with 900 sites/cell and an affinity of 1.1 x 10(10) M-1. Unlabelled AcMNPV, but not Lymantria dispar MNPV could compete with labelled AcMNPV for binding sites. There were 93 to 96% reductions in virus cell binding following pretreatments of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, an inhibitor of N-linked glycosylation and expression of some membrane proteins on the cell surface, reduced virus binding in a dose-dependent manner suggesting a role for glycoprotein(s) in binding. However there was no evidence for the direct involvement of oligosaccharides in attachment. Metabolic inhibitors of oligosaccharide trimming and competition binding assays using simple sugars caused no measurable reductions in virus binding. These findings suggest that AcMNPV attachment to insect cells is receptor-mediated via a glycoprotein component(s); the direct involvement of oligosaccharide moieties in binding is unlikely.
Animals↗
Experimental apparatus for selection of adherent microorganisms under stringent growth conditions.
A bioreactor apparatus is described for studying bacterial attachment. A cyclic, on-off, flow regime was imposed within the apparatus. Model calculations illustrate the utility of this flow pattern in the selection and maintenance of slow-growing, adherent organisms. The apparatus is believed to have general utility in testing bacterial attachment influenced by many types of experimental or environmental constraints, including variations in fluid dynamics, presence of toxic substances (metals or organics), nature of the substratum surface, concentrations of limiting nutrients, and competition between bacterial strains. As an example application, the apparatus was employed to test 14 bacterial strains for surface attachment in a nutrient-limited growth medium. The medium was developed, using the chemical equilibrium program MINEQL, for planned studies of biofilms in a solution with a chemically defined composition that permits calculation of trace metal speciation. The apparatus was used to select organisms with growth and attachment characteristics that could not be evaluated by conventional batch, or chemostat, culture conditions. When supplied with acetate, pyruvate, or succinate as a carbon and energy source, the gram-negative strains Pseudomonas cepacia 17616 and Zoogloea sp. WGO4 showed superior attachment characteristics to glass surfaces in the chemically defined medium but only moderate fluid-phase growth. The gram-positive Arthrobacter sp. strain 9G4D and gram-negative species P. pickettii and Zoogloea sp. WNJ8, when supplied with pyruvate as a carbon and energy source, were capable of superior growth in the fluid phase but formed only a low to moderate biofilm surface coverage.
Bacteria↗
General analysis of receptor-mediated viral attachment to cell surfaces.
Viruses are multivalent particles that attach to cells through one or more bonds between viral attachment proteins (VAP) and specific cellular receptors. Three modes of virus binding are presented that can explain the diversity in binding data observed among viruses. They are based on multivalency of attachment and spatial versus receptor saturation effects which are easily distinguished based upon simple criteria. Mode 1 involves only monovalent virus/receptor binding. Modes 2 and 3 involve multivalent bonds between the virus and cell; however, in mode 3 space on the cell surface becomes saturated before receptors. A model is developed for viral attachment that accounts for nonspecific binding, receptor/virus interactions, and spatial saturation effects. The model can describe each mode in different limits and can be applied to virus binding data to extract key physical information such as receptor number and affinity. These values are used to postulate the type of VAP/receptor interaction involved and to predict binding at different parameter values. For the mode 2 binding of Adenovirus 2, the model predicts a receptor number of 4-15 x 10(3) on HeLa cells and an affinity of 2-6 x 10(7) M-1 which closely approximate experimental estimates. For the binding of three, broad-host-range, enveloped viruses, Semliki Forest virus, Vesicular Stomatitis virus, and the baculovirus, Autographa californica nuclear polyhedrosis virus, the model predicts receptor numbers of 10(5) or greater and affinities in the range of 10(4) to 10(5) M-1. These values are indicative of a VAP/oligosaccharide interaction which has been documented for a number of other viruses. Experimental evidence is presented that is the first to demonstrate that baculovirus binding is mediated by a cell surface receptor.
Animals↗
Bioreactor development for production of viral pesticides or heterologous proteins in insect cell cultures.
The insect cell-baculovirus expression system has significant potential for producing proteins requiring some degree of posttranslational modification. T. ni cells appear to be as good a host as S. frugiperda cells for heterologous protein production as demonstrated by production of beta-galactosidase. Attachment-dependent cells of T. ni can be effectively cultured in a packed-bed reactor using glass beads. When cell in such a reactor were infected, they produced 35% of the total protein as beta-galactosidase. No cell detachment was observed even 70 h postinfection. A model of viral entry has been proposed and tested.
Animals↗
Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.
The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.
Amino Acid Sequence↗
Growth behavior and prediction of copy number and retention of ColE1-type plasmids in E. coli under slow growth conditions.
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Biotechnology↗
Mathematical model for the control of ColE1 type plasmid replication.
A mathematical model for the molecular events controlling replication of ColE1 type plasmids is described. All the model parameters can be evaluated independently. The model simulates plasmid replication and accurately predicts the copy-number of ColE1 plasmids carrying a variety of regulatory mutations. The model is used to test the plausibility of hypotheses concerning the interactions of regulatory elements involved in the replication apparatus. The model favorably supports the mechanism proposed by Tomizawa and co-workers concerning the nature of RNA-RNA interactions and that the Rom protein increases the binding between the two RNA species. The hypothesis that the interactions of RNA I-II increases the susceptibility of RNA II to the action of endonucleases is not a plausible mechanism.
DNA Replication↗
Predictions of cellular growth patterns by a feedback model.
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Bacteria↗
Cell size as an indicator of changes in intracellular composition of Azotobacter vinelandii.
Cell size, measured electronically, was correlated to changes in cellular composition, number, and morphology of Azotobacter vinelandii OP during batch growth. The effect of a changing abiotic environment on these features of the cell is discussed. For this organism exponential growth was unbalanced growth and cell-size change was a sensitive indicator of this growth pattern. Cell-size measurements have the potential to give a rapid assessment of intracellular compositional changes.
Azotobacter↗
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↗