Biomedical subjects

E T Papoutsakis

Publications and source records attributed to E T Papoutsakis.

80 records · Page 5Linked to original sources

PubMed1988

Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli.

A 13.6-kilobase (kb) Sau3AI restriction endonuclease fragment of Clostridium acetobutylicum DNA cloned into pBR322 enabled Escherichia coli ato mutants to grow on butyrate as a sole carbon source (But+). Complementation of the ato defect by the recombinant plasmid pJC6 was due to expression of the genes for phosphotransbutyrylase (PTB) and butyrate kinase (BK). Both genes were efficiently expressed in E. coli, as their products were readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. PTB was found to have a polypeptide subunit molecular weight of approximately 31,000, while that of BK was approximately 39,000. Deletion analysis and Tn5 mutagenesis of plasmid pJC7 (a But+ subclone containing a 4.4-kb BamHI fragment from the insert of pJC6) localized the PTB and BK genes within a region spanning approximately 2.9 kb. Preliminary evidence suggests that the two genes may form an operon that is transcribed as a single unit from a promoter of clostridial origin within the 4.4-kb insert of pJC7.

Acetyltransferases↗

PubMed1987

Transport of substrates and metabolites and their effect on cell metabolism (in butyric-acid and methylotrophic fermentations).

The two components, delta pH and delta psi, of the membrane protonmotive force (delta p) effect and are affected by the transport of many substrates and metabolites. Because the integrity (or restoration) of the delta p requires the expenditure of metabolic energy, such transport processes affect the overall cell bioenergetics. However, the transport or high concentrations of certain substrates and metabolites can have more serious effects on cell metabolism because they partially or completely abolish either or both the delta pH and delta psi. If the cells cannot eventually restore the collapsed component(s) of the delta p, complete growth inhibition and cell death become inevitable. In the butanol/acetone fermentation of Clostridium acetobutylicum, the transport and the presence of key metabolites (acetic and butyric acids, and butanol) have serious and some necessary effects on the delta p. Acetic and butyric acids act as uncouplers of the delta pH, thereby reducing the internal pH. Using other acid uncouplers (such as acetoacetate, which is metabolized by the cells, or FCCP, which is not metabolized by the cells), we found that a lower pHo combined with the metabolic-energy drain of the uncoupling effect and high internal acid concentrations are implicated in the mechanism(s) of solventogenesis. Thus, the production or presence (or both) of the two acids (acetic and butyric) is beneficial to the initiation of solvent production. The transport mechanisms of CH3OH, CH2O, and HCOOH in obligate CH3OH utilizers (methylotrophs) were also discussed in detail. We showed that CH3OH is actively transported by the cells at the expense of metabolic energy and that its transport significantly affects the dynamics of continuous bioreactors. The accumulation of CH2O was found to be driven by the membrane delta p. Finally, formate was accumulated by the delta pH according to the general transport mechanism of short-chain fatty acids. The inhibition of growth by formate was explained by its uncoupling effect on the cells. Growth inhibition by CH3OH appeared to be related to the severe reduction of the membrane delta pH and cell pHi by relatively low CH3OH concentrations.

Biological Transport↗

PubMedDate not supplied

Shear sensitivity of hybridoma cells in batch, fed-batch, and continuous cultures.

Previously, we observed that CRL-8018 hybridoma cells were more sensitive to well-defined viscometric shear during the lag and stationary phases than during the exponential phase of batch cultures. Some potential hypotheses for explaining the increase in shear sensitivity are (1) nutrient limitations that result in a decrease in production of specific cellular components responsible for the mechanical strength of the cell, (2) nutrient limitations that lead to synchronization of the culture in a cell cycle phase that is more sensitive to shear, or (3) a link between cell growth and shear sensitivity, such that slowly growing cells are more sensitive to shear. Here, the duration of the exponential phase was increased with use of fed-batch, and the effect on shear sensitivity of the cultures was measured with a viscometric technique. Extension of exponential growth resulted in an increased period during which the cells were insensitive to shear. Additionally, the shear sensitivity of the cells was constant over a wide range of growth rates and metabolic yields in chemostat cultures. These observations suggest that as long as the cells are actively (exponentially) growing, their shear sensitivity does not depend on the growth rate or metabolic state of the cell as expressed by metabolic yields. Thus, hypothesis 3 above can be dismissed.

Cell Division↗

PubMedDate not supplied

The lactate issue revisited: novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures.

It is well established that cell proliferation in batch (unfed) hematopoietic cell cultures is greatly inhibited relative to that in cultures with feeding. What is not known, however, is the nature of this inhibition. On the basis of our observations in hematopoietic cultures that cell proliferation ceases when the lactate concentration ([lactate]) exceeds 20 mM (accompanied by a decrease in culture pH), we investigated the effect of lactate accumulation on cell proliferation, metabolism, and differentiation. We differ in our approach from previous efforts in that we have tried to more accurately recreate the manner in which lactate accumulates in culture by employing a daily feeding protocol in which [lactate] and/or pH in the fresh medium was adjusted to match the conditions prior to feeding. We conclude that the decrease in pH associated with lactate accumulation significantly inhibits both cell proliferation and metabolism. Although inhibition in cultures with high [lactate] and low pH is similar to that in unfed cultures, pH control in unfed cultures does not alleviate the inhibition, indicating that other inhibitory factors are also present. Thus, pH control is necessary, but not sufficient, to eliminate inhibition of cell growth and metabolism in unfed hematopoietic cell cultures. We also conclude that high [lactate] and low pH have little effect on cell differentiation in fed cultures, although there is evidence to suggest that low pH may play a role in monocyte differentiation in unfed cultures.

Bone Marrow Cells↗

PubMedDate not supplied

Characterization of hematopoietic cell expansion, oxygen uptake, and glycolysis in a controlled, stirred-tank bioreactor system.

Cultures of umbilical cord blood and mobilized peripheral blood mononuclear cells were carried out in a stirred bioreactor with pH and dissolved oxygen control. Expansion of total cells and colony-forming units granulocyte-macrophage was greatly enhanced by the use of a cell-dilution feeding protocol (as compared to a cell-retention feeding protocol). The specific oxygen consumption rate (qO2) for these cultures ranged from 1.7 x 10(-8) to 1.2 x 10(-7) micromol/(cell.h). The maximum in qO2 for each culture closely corresponded with the maximum percentage of progenitor or colony-forming cells (CFCs) present in the culture. The maximum qO2 values are slightly less than those reported for hybridomas, while the lowest qO2 values are somewhat greater than those reported for mature granulocytes. Examination of the ratio of lactate production to oxygen consumption in these cultures suggests that post-progenitor cells of the granulomonocytic lineage obtain a greater portion of their energy from glycolysis than do CFCs. The different metabolic profiles of CFCs and more mature cells suggest that monitoring the uptake or production of oxygen, lactate, and other metabolites will allow estimation of the content of several cell types in culture.

Bioreactors↗

PubMedDate not supplied

Decreasing extracellular pH increases CD13 receptor surface content and alters the metabolism of HL60 cells cultured in stirred tank bioreactors.

Flow cytometry and Northern blotting were used to examine the effects of extracellular pH on CD13 receptor surface content and mRNA levels of HL60 (human promyelocytic leukemia) cells. Decreasing culture pH (7.4, 7.2, and 7.0) increased the CD13 receptor surface content of HL60 cells cultured at low agitation intensity (80 rpm) in 2-L bioreactors. Unlike our earlier findings on the effects of increasing agitation rates and serum concentrations, changes in CD13 receptor content in response to decreasing culture pH did not correlate with changes in CD13 mRNA levels. Decreasing culture pH also decreased the average HL60 cell size. HL60 cells cultured at pH 7.0 and 7.2 exhibited glucose consumption and lactate production rates that were approximately 30-40% and 20-30% lower, respectively, than values of cells cultured at pH 7.4.

Bioreactors↗

PubMedDate not supplied

Transport in a grooved perfusion flat-bed bioreactor for cell therapy applications.

This study considers the transport of oxygen (a growth-associated solute) and lactate (a metabolic byproduct) in a flat-bed perfusion chamber modified to retain cells through the addition of grooves, perpendicular to the direction of flow, at the chamber bottom. The chamber has been successfully applied to hematopoietic cell culture and may be useful for other basic and applied biomedical applications. The objective of this study is to characterize the culture environment in terms of solute transport under various operational conditions. This will allow one to improve the design and operating strategy of the perfusion system for maximizing cell numbers. The system is numerically simulated using the finite element package FIDAP. The reaction kinetics describing oxygen uptake by cells are simplified to zero order to give an upper bound for the oxygen consumption. A flat-bed chamber without grooves is considered here as a benchmark. We show that the growth environment is not oxygen limited (local oxygen concentration above 10 microM) for a variety of flow rates and culture conditions (qO2 = 0.1 micromol/(10(6) cells h)). With a medium flow rate of 2.5 mL/min through the reactor, the model predicts that the 29-cm2 reactor can support at least 33.4 x 10(6) total cells when the inlet medium is in equilibrium with high (20%) oxygen concentration. The culture becomes oxygen limited however for the same flow rate for low (5%) oxygen concentration and can only support 7.2 x 10(6) total cells. Comparison of grooved vs nongrooved chambers reveals that the presence of grooves only affects solute transport on a local scale. This result is attributed to the small size (200 microgram) of the cavities relative to the chamber dimensions. The comparison also yields an empirical relation that allows for rapid estimation of oxygen and lactate concentrations in the grooves using only the numerical simulation of the simpler nongrooved chamber. Finally, our investigation shows that, while decreasing the spacing between cavities decreases the total number of cells the reactor can support, the efficiency of the reactor is increased by 25% (on an area basis) without growth restriction.

Bioreactors↗

PubMedDate not supplied

Extracellular pH affects the proliferation of cultured human T cells and their expression of the interleukin-2 receptor.

Ex vivo expansion of T cells is an important aspect of many cellular immunotherapy protocols, and the effects of the culture environment on the cells must be understood to produce large numbers of functional cells. Extracellular pH is a fundamental parameter that has many different effects on cultured cells. In this study, peripheral blood mononuclear cells were stimulated with phytohemagglutinin and cultured at pH values of 7.0, 7.2, or 7.4. The effects of pH on the cells were studied during the 2 to 3 weeks of proliferation resulting from phytohemagglutinin stimulation, in order to examine the culture kinetics over realistic time scales for ex vivo expansion. The proliferation capacity of the T cells increased more than three-fold for the pH 7.0 and 7.2 cultures compared with the pH 7.4 cultures. The culture pH also affected the kinetics of the interleukin-2 receptor down-regulation process. The faster receptor down-regulation in both the pH 7.2 and 7.4 cultures resulted in a more than twofold greater fraction of interleukin-2 receptor(+) cells in the pH 7.0 cultures. Although the fraction of apoptotic cells (using the Annexin V flow-cytometric method) remained less than 10%, we observed 27% more apoptosis in the pH 7.4 cultures than in the 7.2 cultures and 49% more apoptosis in the pH 7.4 cultures than in the 7.0 cultures. These effects on interleukin-2 receptor expression and cellular apoptosis may partially explain the observed effects of pH on T-cell proliferation.

Apoptosis↗