Judith K. Christman

Purification and characterization of a cellular fibrinolytic factor associated with oncogenic transformation: The plasminogen activator from SV-40-transformed hamster cells

Abstract Neoplastic cells, whether transformed by oncogenic viruses or chemical agents, release a fibrinolytic factor not released by normal cells. We have purified this factor 14 000-fold from the supernatant culture fluid of SV-40-transformed hamster cells. It is a plasminogen activator with a molecular weight of approx. 50 000 consisting of subunits linked by disulphide bridges. It is irreversibly inhibited by diisopropylfluorophosphate, indicating that it is a serine protease. The subunit contianing the diisopropylfluorophosphate binding site has a molecular weight of approx. 25 000.

Effect of elevated potassium level and amino acid deprivation on polysome distribution and rate of protein synthesis in L cells

Abstract The effect of incubation in several defined amino acid deficient media on the rate of protein synthesis and proportions of monomeric and polysomal ribosomes in L cells was examined. The sedimentation distribution of ribosomes isolated from L cells deprived of any or all of the essential amino acids showed a sharp and rapid shift ( min ) from polysomes to the 80-S region. Concurrently the rate of protein synthesis decreased by 50–70 %, largely due to slowing of the rate of initiation of peptide chains. Replacement of Na+ in the growth medium with K+ had the same effect and, when combined with amino acid starvation, reduced the rate of protein synthesis to less than 5 % of the normal level. It is demonstrated that this severe inhibition of protein synthesis is rapidly reversible when cells are restored to normal growth conditions and that polysomes reform even in the presence of actinomycin D. Evidence is presented to confirm the hypothesis that all of the components of the protein synthesizing system retain their functional integrity and that even under the most stringent conditions tested by us, peptide chain initiation is the rate limiting event when L-cell protein synthesis is inhibited.

Distribution of 5-S RNA in erythroid cells

Abstract 1. 1. The distribution and proportion of 5-S RNA in erythroid cell ribosomes were studied by Sephadex-gel filtration and polyacrylamide-gel electrophoresis in an attempt to find some correlation between 5-S RNA content of ribosomes and cell maturation. 2. 2. Ribosomes were isolated from phenylhydrazine-induced reticulocytes. Both free and membrane-bound ribosomes were examined. These ribosomes all contained 5-S RNA as a constant proportion of the total rRNA. Ribosomes isolated from normal circulating erythroid cells and reticulocytes of varying ages also contained the same proportion of 5-S RNA. 3. 3. It is concluded that one cannot distinguish between ribosomes from cells of varying age and presumably of different functional capabilities on the basis of gross 5-S RNA content.

Characterization of a viral messenger ribonucleoprotein particle accumulated during inhibition of polypeptide chain initiation in reovirus-infected L cells

Abstract A prior study has shown that L cells deprived of essential amino acids in isotonic Tris-buffered KCl show a marked and rapid inhibition of protein synthesis which is due to a decrease in the rate of initiation of new peptide chains. The inhibition, which is manifested by a disaggregation of polysomes with apparent conservation of mRNA, is rapidly reversed when the cells are resuspended in complete growth medium. This phenomenon has been applied in studies on the mechanism of mRNA storage and reutilization in reovirus-infected L cells. When reovirus-infected L cells are incubated in KCl-Tris medium, the viral messengers accumulate as ribonucleoprotein particles which sediment at approximately 50 S in sucrose density gradients and have a density in CsCl of 1.40. These 50-S messenger-containing ribonucleoprotein particles do not appear to be complexed with ribosomal subunits. The particles contain all 10 species of viral mRNA and have no demonstrable viral proteins. The mRNA from these particles is readily and rapidly mobilized into functional polysomes when the infected cells are restored to normal growth medium. Moreover, the mRNA moiety of partially purified 50-S ribonucleoprotein particles can direct the incorporation of amino acids into protein in a cell-free system.

Mechanism of inhibition of DNA methyltransferase by 5-azacytosine substituted DNA

There are two lines of evidence indicating that enzymatic methylation of cytosine residues in DNA may influence the expression of genes in vertebrate cells. First, a general correlation exists between the persistance of unmodified methylation sites in or near coding sequences of specific genes and the expression of these genes. Such sites are methylated in DNA of cells where the genes are inactive and lack methyl groups in cells where the genes are expressed. (For recent reviews see Razin and Riggs, 1980; Ehrlich and Wang, 1981.) Second, at least two agents which inhibit methylation of cytosine residues in DNA, L-ethionine (Christman, et al, 1977) and 5-azacytidine (Jones and Taylor, 1980), are able to induce stable alterations of gene expression in mammalian cells. 5-azacytidine (5-aza-CR) is of particular interest in this respect because it is not a general inhibitor of transmethylation reactions but instead specifically inhibits methylation of cytosine residues in DNA and RNA (Friedman, 1979; Lu and Randerath, 1978).

An apparent identity of 5-S RNA from free and membrane-bound reticulocyte ribosomes

Abstract 1. 1. A comparison of the chemical and physical properties of reticulocyte 5-S RNA isolated from free and membrane-bound ribosomes was made to clarify further the relationship of 5-S RNA to ribosome function. 2. 2. Although reticulocyte 5-S RNA could be distinguished from Escherichia coli B 5-S RNA by its nucleotide composition, optical properties, response to urea treatment and electrophoretic mobility on polyacrylamide gels, no such differences were found between 5-S RNA isolated from free and membrane-bound reticulocyte ribosomes.

Effect of tumor promoters on induction of aryl hydrocarbon hydroxylase in human lymphocytes.

The induction of aryl hydrocarbon hydroxylases in lymphocytes is dependent on their activation. The tumor-promoting phorbol esters which induce blast formation and DNA synthesis in lymphocytes enable polycyclic aromatic hydrocarbons to induce aryl hydrocarbon hydroxylases. Melittin, the major constituent of bee venom, acts synergistically with these phorbol esters in enhancing both lymphocyte activation and hydroxylase synthesis. Since aryl hydrocarbon hydroxylases convert procarcinogens to carcinogens these results suggest that tumor promotion by phorbol esters may be associated with their ability to affect the induction of these enzymes. This hypothesis is supported by the finding that phorbol and phorbol esters which lack tumor-promoting activity fail to enhance induction of aryl hydrocarbon hydroxylases in lymphocytes. However mezerein, although rather ineffective in promoting tumors, activates lymphocytes and permits polycyclic aromatic hydrocarbons to induce their hydroxylases effectively.