The effect of 3-aminobenzamide on thymidine incorporation in ultra-violet irradiated chick pigment epithelium cells.

The addition of 3-aminobenzamide (3-AB) to cultures of chick embryo pigmented epithelium rescues these cells after high doses of ultraviolet treatment. The addition of 3-AB prevents cells from losing pre-formed protein and DNA and stimulates thymidine incorporation by the cells after ultraviolet irradiation. Since 3-AB is an inhibitor of poly (ADP) ribosylation, these observations support the conclusion that death of these cells after ultra-violet irradiation depends upon poly (ADP) ribosylation and may be an apoptotic response.

The effect of sodium iodate and melanin on the formation of glyoxylate.

Sodium iodate damages retinal pigment epithelium specifically, but the reason for this specificity is not well understood. The work reported here describes an effect of sodium iodate on melanin, a major component of the retinal pigment epithelium. Sodium iodate increases the ability of melanin to convert glycine to glyoxylate. Almost ten times as much glyoxylate is formed when sodium iodate is present compared to the amount formed with melanin alone, although iodate alone does not convert glycine to glyoxylate. A chemical reaction between sodium iodate and melanin is suggested as a partial explanation of the specificity of iodate toxicity towards retinal pigment epithelium.

In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells.

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl2, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.

The formation of glyoxylate from glycine by melanin.

Natural and synthetic melanins catalyze the conversion of glycine to glyoxylate and formic acid in vitro. The conversion depends upon the concentration of melanin.

Accumulation of ornithine by chick retinal pigment epithelium cells in culture.

This study was designed to measure the accumulation of ornithine in retinal pigment epithelium cells grown in culture. Ornithine accumulated in retinal pigment epithelium cells in which the ornithine aminotransferase activity was inhibited with L-canaline. The effect of L-canaline was eliminated by the concomitant presence of methionine.

Comparison of ornithine aminotransferase activities in the pigment epithelium and retina of vertebrates.

1. The specific activities of ornithine aminotransferase (OAT) in the pigment epithelia, retinas, and livers from several classes of vertebrates were assayed. 2. The specific activities of OAT were much higher in the pigment epithelia from mammals and birds than in their respective retinas or livers. 3. Pigment epithelium from porcine eyes had the highest specific activity measured. The specific activity of OAT in the pigment epithelium from the pig was five times higher than the OAT activity in its retina and 13 times higher than the OAT activity in its liver.

The effect of methionine on the accumulation of ornithine by Chinese hamster cells in culture.

Ornithine was found to be toxic to cells that lack ornithine aminotransferase (EC 2.6.1.13). These cells also accumulated substantial amounts of ornithine. The addition of methionine to these cells protected them from the toxic effects of ornithine and also diminished their accumulation of ornithine. Cells that have an active ornithine aminotransferase were resistant to the toxic effects of ornithine, and accumulated less of this compound.

Ornithine aminotransferase in chick embryo tissues.

Ornithine aminotransferase (E. C. 2.6.1.13) deficiency has been found to be associated with congenital atrophy of the choroid and retina of man. A deficiency in this enzyme could be associated in a causal fashion with atrophy, or it could be a coincidental association. To challenge the hypothesis that ornithine aminotransferase activity is required directly or indirectly for maintaining the eye in a functional state, it was of interest to determine the level of activity of this enzyme in normal eye tissue. We report here that the level of ornithine aminotransferase activity in pigment epithelium of chicken is 11 times that found in liver. The level of enzyme activity in retina is 80% of that found in liver. The level of enzyme present in embryonic pigment epithelium is essentially unchanged between the eighth and eighteenth day of incubation. These observations suggest strongly that ornithine aminotransferase activity is involved in maintaining the structure or function of the eye.

Effect of proline on synthesis of collagen by cells in culture.

Chick embryo cells, human epithelial cells, and mouse cells in culture were tested for response to elevated levels of proline in the growth medium. In none of the cell lines tested was the amount of collagen formed stimulated by addition of excess proline.

Cirrhosis and the synthesis of proline and glycine in rat liver.

The relation between availability of free amino acids and development of cirrhosis in rat liver has been experimentally evaluated. Levels of free glycine and proline were found to increase when animals are treated with phenobarbital and carbon tetrachloride. This increase is concomitant with increase in collagen and loss of activity of enzymes responsible for degradation of amino acids. It is concluded that elevated levels of proline observed in the serum of cirrhotic patients may be a consequence, rather than a cause, of collagen accumulation in the liver.

Alternative pathways for proline synthesis in mammalian cells.

Proline is synthesized in animal cells by way of two alternative pathways. Both of these pathways are functional in many cell lines. The relative contribution of a pathway to total proline synthesis may be altered by a mutational event. Differences in the developmental origin of a cell line are retained as differences in the pathways of proline synthesis.

Inhibitory effect of penicillin on proline synthesis in Escherichia coli.

The production of glutamic gamma-semialdehyde, an intermediate in the synthesis of proline, was inhibited in Escherichia coli by physiological concentrations of penicillin. Sucrose (0.6 m) and sodium chloride (0.1 m) prevented penicillin inhibition of glutamic gamma-semialdehyde synthesis. Cells which were in the stationary phase, or which had been permitted to metabolize without growth, were insensitive to the effects of penicillin on glutamic gamma-semialdehyde synthesis.