Donald F. Haggerty

Studies on the effects of essential fatty acids on growth rate, fatty acid composition, oxidative phosphorylation and respiratory control of HeLa cells in culture

Abstract 1. Mammalian cells in culture provide a simple model to study the function of polyunsaturated fatty acids. 2. HeLa S 3 cells cultured in a lipid-deficient medium showed a pattern of essential fatty acid deficiency represented by an increase in 18:1 and a decrease in 20:4 and 18:2, a lack of growth and impaired mitochondrial function. 3. Addition of albumin-bound linoleic or arachidonic acid partially or totally prevented these changes.

The protein concentration of crude cell and tissue extracts as estimated by the method of dye binding: Comparison with the lowry method

Abstract The protein concentration of crude extracts from cultured cells and of subcellular fractions from rat tissues has been estimated both by the method of Bradford, involving the binding of Coomassie brilliant blue G-250, and by the procedure of Lowry et al. with the same preparation of bovine serum albumin as a common protein standard. Whereas the method of dye binding yielded significantly lower values than did the Lowry procedure, a consistent quantitative relationship existed between the values obtained by the two techniques. Evidence is provided that this discrepancy does not result from the presence in such complex biological mixtures of a class of small peptides, undetected by the method of dye binding, that react with the Lowry reagents.

Differential expression of multiple forms of arginase in cultured cells

SummaryArginase (EC 3.5.3.1), the final enzyme in the urea cycle, catalyzes the cleavage of arginine to orthinine and urea. At least two forms of this enzyme, Al and All, have been described and are probably encoded by discrete genetic loci. The expression of these separate genes has been studied in mammalian cells grown in culture. The permanent rat-hepatoma line H4-II-E-C3 contained exclusively the Al enzyme; the form in mammals comprising about 98% of the arginase activity in liver and erythrocytes but catalyzing only about one half of that reaction in kidney, gastrointestinal tract, and brain. By contrast, human-embryonic-kidney and -brain cells, after transformation with the human papovavirus BK, contained only the All species of arginase, which form contributes the remaining half of that catalysis in those mammalian tissues in vivo. We report here the results of an extensive study on the properties of these two forms of arginase in the three cell lines, including Km values for arginine, behavior on polyacrylamide gels under non-denaturing conditions, and cross-reactivity with lapine antibodies against the arginases from either rat or human liver.[/p]

The requirement for biotin in mouse fibroblast L-cells cultured on serumless medium

Abstract Mouse L-cells, Clone 929, were adapted to growth on a chemically defined medium, Waymouths Medium MAP 954 1 , in the absence of serum. When biotin was excluded in the preparation of this medium, the resulting medium proved to be incapable of sustaining continuous cell proliferation: A biotin requirement was therefore demonstrable in these cells within a three week period.

A tyrosine-free medium for the selective growth of cells expressing phenylalanine hydroxylase activity☆

Abstract Tyrosine-free modified Hams F-12 medium supplemented with charcoal-extracted serum permits the exclusive propagation of cultured cells containing detectable levels of phenylalanine hydroxylase. The selective properties of this medium have been verified with two phenylalanine hydroxylase-positive (H4-II-E-C3 and MH1C1) and two phenylalanine hydroxylase-negative (HTC and BRL) cell lines. Although cocultivation experiments revealed that BRL cells could replicate slowly in the selective medium in the presence of sufficient numbers of H4-II-E-C3 cells, the former cells did not survive subcultivation in this medium at low cell inoculum (

The effect of population density on phenylalanine hydroxylase activity in rat-hepatoma cells in culture.

Abstract The expression of phenylalanine hydroxylase activity in rat-hepatoma cells in culture (line H4-II-E-C3) is a function of culture density: under normal growth conditions in the presence or in the absence of exogenously added hydrocortisone, the levels of this enzyme are low in subconfluent cell populations, but increase steeply as cultures attain confluency. This phenomenon (i) is not an artifact of the subcultivation process and (ii) is not produced by some alteration in the growth medium effected by high-density cultures. The levels of phenylalanine hydroxylase in high-density cultures of H4-II-E-C3 cells in the presence of serum plus added hydrocortisone are at least 80% of those seen in adult-rat liver. It is concluded that this density-associated phenomenon is the result of an intrinsic property of H4-II-E-C3 cells and possibly constitutes a form of epigenetic control governing the sensitivity of these cells to stimulation by serum or by serum plus hydrocortisone.

Regulation of expression of genes for enzymes of the mammalian urea cycle in permanent cell-culture lines of hepatic and non-hepatic origin

SummaryWe present here the results of investigations conducted by ourselves and others on the regulation of the expression of genes encoding the enzymes of the mammalian urea cycle as manifest in cultured cells of both hepatic and extrahepatic origin. Upon consideration of the recently discovered discrete non-hepatic arginase genetic locus in man and our consequent hypothesis that the form of arginase thus transcribed in such extrahepathic cells functions principally in providing ornithine for protein anabolism and polyamine biosynthesis, rather than in detoxifying ammonia through urea formation, we have chosen instead to study permanent cell lines that are derived from liver and continue to perform a variety of hepatic functions in culture as experimental models for probing the molecular mechanisms underlying the control of ureagenesis within the mature liver cell. Of two such arginase-positive rat-hepatoma lines, we have characterized extensively in one (H4-11-E-C3) the mode of action of glucocorticoids in augmenting the cellular levels of this enzyme as well as of argininosuccinate synthetase. To this end, we have recently demonstrated that these stimulations are both mediated by binding of the hormones to classical cytoplasmic steroid receptors in a specific and saturable fashion and have thus concluded that the H4-11-E-C3 line will provide a suitable cell culture system for subsequent more detailed experiments from which the information garnered will continue to be relevant to the ureagenic pathway as modulated in the differentiated hepatocyte in vivo.

Regulation by glucocorticoids of rat-liver phenylalanine hydroxylase In Vivo

Phenylalanine hydroxylase, a liver-associated enzyme, is induced markedly by glucocorticoids in two permanent rat-hepatoma cell lines. In order to gain evidence that this phenomenon also occurs in vivo, we examined the effect of adrenalectomy and/or hormone supplementation on the levels of phenylalanine hydroxylase in the livers of adult rats: glucocorticoid administration increases, and adrenal ablation reduces, the activity of the hepatic enzyme, and the diminution occurring in the latter instance is entirely prevented by concurrent hormone replacement. These results thus corroborate earlier findings from a single experiment and are consistent with the hypothesis that adrenal corticosteroid hormones participate in modulating phenylalanine-hydroxylase levels within the diploid hepatocyte.