Showing papers by "Ira Pastan published in 1974"

Cyclic AMP and the transformation of fibroblasts.

Abstract: Publisher Summary Cyclic AMP is found in a wide variety of organisms and cells. It is a chemical switch that regulates the activity of existing enzymes and also can induce the synthesis of new proteins. This chapter reviews the role of cyclic AMP in fibroblasts, especially the role of cyclic AMP in the transformation of these cells. The transformed fibroblasts grow faster than normal cells and have a greatly increased saturation density. The transformed cells look different, generally they are less spindly and more refractile in appearance; they are less adhesive to the substratum on which they are growing and will grow in soft agar, whereas normal cells will not; they have altered surface molecules and are readily agglutinated by plant lectins; they often secrete abnormal amounts of macromolecules; and have abnormal tRNA and increased aerobic glycolysis. Cyclic AMP has an important role in regulating many properties of normal fibroblasts. It is involved in controlling cell shape, adhesiveness to the substratum, motility, growth, agglutinability by lectins, and the synthesis of certain macromolecules. As a result of the transformation by chemical carcinogens, X-rays, oncogenic viruses, or spontaneous selection, cyclic AMP levels are decreased and this decrease produces some of the abnormal properties of transformed cells. Transformation by different viruses decreases adenylate cyclase activity by diverse mechanisms because a variety of transformation factors exist.

Myosin is a component of the cell surface of cultured cells.

Abstract: Antisera to myosin from mouse L929 cells, mouse uterus, and rabbit skeletal muscle have been prepared in goats. Each antiserum shows specific reaction by immunodiffusion only to the myosin against which it was prepared. Antiserum against L cell myosin agglutinates L929 cells and shows surface localization in unfixed cells by indirect immunofluorescence. This reaction is prevented by immunoabsorption with L cell myosin, but not by absorption with mouse uterine or rabbit skeletal muscle myosins. Elution of the antibody from L cell myosin antiserum that absorbs to fixed L cells yields antibody reactive to L cell myosin in immunodiffusion. At the ultrastructural level, surface localization is also evident with a peroxidase bridge immunocytologic method as a uniform distribution of surface label. Antiserum against L cell myosin also shows localization on the surface of other cells of rodent origin (3T3-4, NRK, MNRK, KNRK, and MEF) and HeLa cells, but not with some other cells of human origin (ALL and WI-38). The reactivity with antibody of the plasma membrane myosin is unaffected by trypsin treatment or fixation with 0.5-2.0% formalin. These results strongly suggest that myosin in cultured nonmuscle cells is a component of the plasma membrane, a finding of possible significance in studying the mechanisms regulating the motility, morphology, and growth of these cells.

Cyclic AMP mediates the concanavalin A agglutinability of mouse fibroblasts.

Abstract: We have devised a quantitative way to measure the agglutination of cells which utilizes the size discrimination feature of an automatic particle counter. With this method we have studied the agglutinability by concanavalin A of 3T3 cells, a mutant of 3T3 cells (3T3cAMPtcs) in which cyclic AMP levels fall when the cells are subjected to temperature change or fresh serum, and L929 cells. We find with 3T3cAMPtcs cells that low levels of cyclic AMP correlate with increased agglutinability and that high levels of cyclic AMP correlate with decreased agglutinability. Prior treatment of these cells with a cyclic AMP phosphodiesterase inhibitor or Bt2cAMP blocks the increase in agglutinability induced by temperature change. When 3T3 cells are treated with fresh serum, their agglutinability also increases although to a much smaller extent than with 3T3cAMPtcs cells. Cells change their agglutinability very rapidly. Treatment of L929 cells for 15 min with 1-methyl-3-isobutyl xanthine at 1 mM decreases their agglutinability to the level of normal 3T3 cells. We conclude that in normal and transformed cells the level of cyclic AMP regulates agglutinability.

The 5′-Terminal Nucleotide Sequence of Galactose Messenger Ribonucleic Acid of Escherichia coli

Abstract: The 5′-terminal sequence of mRNA from the galactose operon of E. coli has been determined. gal RNA is synthesized in vitro by means of a kinetically controlled purified transcription system and is isolated by a two-step RNA·DNA hybridization scheme. The following sequence of the first 77 nucleotides has been deduced by analysis of oligonucleotides produced by digestion with T1, pancreatic, and carboxymethylated pancreatic ribonucleases: ppA-U-A-C-C-A-U-A-A-G-C-C-U-A-A-U-G-G-A-G-C-G-A-A-U-U-A-U-G-A-G-A-G-U-U-C-U-G-G-U-U-A-C-C-G-G-U-G-G-U-A-G-C-G-G-U-U-A-C-A-U-U-G-G-A-A-G-U-C-A-U-A-C-C-U-G-U. Residues 27-77 correspond to the amino terminal 17 amino acids of UDPgalactose 4-epimerase (EC 5.1.3.2), the protein specified by the promoter-proximal structural gene of the operon. A self-complementary sequence occurs near the 5′ terminus; 12 of 15 nucleotides between residues 4 and 18 are symmetrically located about position 11. The sequence of residues 22-33 closely resembles part of the lactose operator sequence reported previously [Gilbert, W. & Maxam, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3581-3584].

[50] The purification and analysis of mechanism of action of a cyclic AMP-receptor protein from Escherichia coli

Abstract: Publisher Summary Both cyclic AMP (cAMP) and a specific cAMP receptor protein are needed by Escherichia coli to make normal amounts of a variety of inducible enzymes as well as other dispensable proteins. cAMP levels are controlled by the carbon source the organism uses for growth. The levels are particularly low when the organism grows on glucose. The inability of glucose-grown cells to make adequate amounts of inducible enzymes has been called the “glucose effect” or “catabolite repression.” It is now established that catabolite repression is caused by low intracellular levels of cAMP. One class of mutants has lower or absent levels of cAMP because of a defective adenylate cyclase. The inability of these cells to synthesize inducible enzymes is corrected by adding exogenous cAMP. The second class of mutants produces a defective cAMP binding protein. This protein has been called the cAMP receptor (CRP), or the catabolite gene activator protein (CAP or CGA).