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D W Jayme

Publications and source records attributed to D W Jayme.

11 recordsLinked to original sources
PubMed1999

An animal origin perspective of common constituents of serum-free medium formulations.

Serum-free formulations may be << re-engineered >> to eliminate traditional protein constituents and replace their biological function with non-protein substitutes. Non-protein additives may also be obtained from animal sources. Nutrient formulations totally free of exogenous protein and containing no materials of animal origin may be designed for high density cell culture and biological production. Cell-culture medium production requires (i) strict vendor qualification and raw material specifications; (ii) scrupulous maintenance of media kitchen facility and equipment; (iii) monitoring of process water; (iv) air-handling systems and technical personnel; (v) clearly-defined manufacturing protocols to ensure correct formulation and dispensing and (vi) validated sanitization processes to guard against cross-contamination within a multi-use facility.

Animals↗
PubMed1988

Fetal bovine serum alternatives.

With the current shortage in fetal bovine serum, researchers are turning to a variety of options for cell and tissue culture.

Animals↗
PubMed1985

Culture media for propagation of mammalian cells, viruses, and other biologicals.

In this brief review, we have illustrated the historical development of the growth media commonly employed for the propagation of cultured mammalian cells. While substantial progress has been achieved, the field may best be described as conservative and pragmatic. To date, the function of many components of the growth medium essential for cellular proliferation and biological production has not been precisely defined at the molecular level. Thus, for most large-scale biological production requirements, as well as for routine cell culture and bench-scale pilot development, the traditional enriched culture medium supplemented with fetal bovine serum represents the most convenient culture system. Many cell types may be more economically grown without reduction in biological yield by substituting alternative mammalian sera. Where reduction of total protein or greater definition of growth medium components outweighs the use of more universally applicable culture media, substitution of serum-free, customized formulations of highly enriched growth medium plus defined growth factors may be of significant utility. Optimization of mammalian cell culture media for large-scale biological production should include the following: An initial time investment to optimize the cell culture medium by enriching intermediary metabolite composition (rather than expecting serum or additional growth factors to perform nutritional functions) may result in higher productivity and reduced cost. When screening potential growth media for biological production applications, proliferative rate should not be the sole criterion for performance. Although rapid, logarithmic growth is advantageous to establish large-scale cultures, the maximal cell density and duration of the viable, productive period must also be weighed. Many cell types generate the highest titers of biological product either at stationary phase or under mildly stressful ("controlled death") conditions suboptimal for cellular replication. Thus, the ultimate determinant of growth medium efficacy is neither the degree of definition of medium composition nor the cellular proliferative rate, but the ability to support synthesis of substantial titers of the desired product at reasonably high purity.

Animals↗
PubMed1984

Furosemide-sensitive potassium efflux in cultured mouse fibroblasts.

Transfer of LM(TK-) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50-70% inhibited by furosemide or bumetanide. The diuretic-sensitive component of K+ efflux requires both Na+ and Cl-, and is presumably mediated by a K+, Na+, Cl- cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic-sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic-sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low-K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl-.

Amiloride↗
PubMed1982

Regulation of amino acid transport system L in Chinese hamster ovary cells.

The regulation of the activity of transport System L for neutral amino acids has been investigated in Chinese hamster ovary cells. Incubation of the temperature-sensitive leucyl-tRNA synthetase mutant CHO-tsH1 at marginally permissive temperatures results in leucine-limited growth and increased transport of branched chain and aromatic amino acids. This temperature-dependent transport enhancement is restricted to transport System L and results in increased Vmax values of uptake of System L substrates with unchanged Km values of uptake. Several lines of evidence suggest that trans-stimulation by intracellular amino acids cannot account for the increased System L activity in CHO-tsH1. Other temperature-sensitive aminoacyl-tRNA synthetase mutants of the Chinese hamster ovary line show increased transport of the amino acid corresponding to the synthetase defect after incubation at elevated temperatures. Increased transport by System L occurs in CHO-S, the parental line of CHO-tsH1, after growth in limiting levels of leucine. The System L enhancement can be prevented by cycloheximide but not, at early times, by actinomycin D. We conclude that the activity of transport System L in Chinese hamster ovary cells is regulated by a mechanism which appears to act at the level of translation.

Amino Acids↗
PubMed1981

Neutral amino acid transport systems in Chinese hamster ovary cells.

The neutral amino acid transport systems A, ASC, and L have been characterized in the Chinese hamster ovary cell. System A, defined by its sodium ion dependency and inhibition by 2-methylaminoisobutyric acid, was found to be extremely sensitive to the pH of the external medium and to increase in response to starvation for amino acids. System ASC, identified by its sodium ion dependency and intolerance of N-methylation of substrates, was found to be relatively insensitive to external pH and nutrient limitation. System ASC in Chinese hamster ovary cells has been shown to be the major mode of entry of neutral amino acids. A much broader substrate specificity was observed for System ASC than has been reported for other mammalian cell types, with nearly every amino acid tested showing significant uptake by this system. In addition, the highest observed velocities of uptake were for System ASC. System L, defined by its sodium ion independency and inhibition by 2-aminobicyclo-[2,2,2]-heptane-2-carboxylic acid, was enhanced in activity by lowered pH. The starvation-induced enhancement in System A activity was prevented by the presence of a single substrate of this system, the nonmetabolizable analog 2-methylaminoisobutyric, or by the presence of the protein synthesis inhibitor cycloheximide.

Alanine↗
PubMed1981

Reduction of K+ efflux in cultured mouse fibroblasts, by mutation or by diuretics, permits growth in K+-deficient medium.

The mouse fibroblastic cell line LM(TK-) is unable to grow at external K+ concentrations below a threshold value of 0.4 mM. At subthreshold K+ concentrations, LM(TK-) cells rapidly lose intracellular K+ and eventually lyse. We have analyzed the pathway primarily responsible for K+ efflux under these experimental conditions and reports its specific inhibition by two diuretics, furosemide and bumetanide. Bumetanide, an analog of furosemide, was a more potent inhibitor (by several orders of magnitude) than was furosemide itself. The effects of ouabain and bumetanide were additive, suggesting independence of diuretic-sensitive K+ efflux from Na+/K+ pump-mediated fluxes. Characterization of K+ efflux in LTK-5, a mutant derived from LM(TK-) and selected for its ability to grow at 0.2 mM K+ indicated that the mutant had lost the diuretic-sensitive K+ efflux pathway. Net cation fluxes, steady-state intracellular cation concentrations, and growth at reduced K+ concentrations were comparable for LM(TK-) cells maximally inhibited by diuretics and for the LTK-5 mutant grown either in the presence or absence of diuretics. Thus, reduction in K+ efflux, either by diuretic addition diuretics. Thus, reduction in K+ efflux, either by diuretic addition or by genetic alteration, can permit the cell to maintain normal cation gradients and to grow at otherwise subthreshold external K+ concentrations.

Animals↗
PubMed1977

A role for aminoacyl-tRNA synthetases in the regulation of amino acid transport in mammalian cell lines.

The regulation of neutral amino acid transport was examined utilizing two temperature-sensitive aminoacyl-tRNA synthetase mutants, by comparison of their transport activities at permissive and marginally permissive temperatures. When the temperature-sensitive leucyl-tRNA -synthetase mutant tsH1 was shifted from a normal growth temperature of 34 degrees to a marginally permissive temperature for growth of 38%, a significant enhancement in the initial rate of uptake of leucine and other L system amino acids was observed when compared with that of the parental cells (CHO-S). In contrast, the uptake of A system-specific amino acids (alanine, glycine, alpha-aminoisobutyric acid) showed no significant differences relative to the parent strain. In a similar manner, a temperature-sensitive asparaginyl-tRNA synthetase mutant RJK-4 exhibited a significant enhancement of the transport activity of A system amino acids relative to its parental strain RJK-0 when the growth temperature was shifted from 33 degrees to 39.5 degrees. Preliminary kinetic data suggest that the Vmax for transport is increased when the mutants are grown under conditions of amino acid limitation. These experiments suggest that the aminoacyl-tRNA synthetase or a related product may function in the regulation of neutral amino acid transport in Chinese hamster cells.

Amino Acids↗