Induction of cell division in BALB/c-3T3 cells by phorbol myristate acetate or bovine serum: Effects of inhibitors of cyclic AMP phosphodiesterase and Na+−K+-ATPase

Summary Cell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10⁻³ m) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7×10⁻⁶ m), Persantin (5×10⁻⁵ m) or RO-20-1724 (10⁻⁴ m), prevents cell replication induced by PMA or serum. In contrast, ouabain (10⁻⁴ m) and N,N′-dicyclohexylcarbodiimide (10⁻⁵ m), inhibitors of Na⁺−K⁺-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the cell cycle are sensitive to dbcAMP addition. One is early in the G₁ phase at the time of reinitiation of the cell cycle from a stationary (G₀) phase, a second is associated with the G₁-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological changes, responses of plasma membrane ezymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3 cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner. This work was supported by a USPHS Research Grant CA12503, and a Center Grant ES-00260 awarded to the Institute of Environmental Medicine. Mrs. Susan Kulina provided the consistent and excellent technical aid necessary to perform this work. Note added in proof: During the preparation and review of this paper, Boynton reported that PMA appears to sensitize BALB/c-3T3 cells to calcium ion which may play a critical role in the regulation of the DNA synthesis (36).     

Zinc deficiency decreases the activity of calmodulin regulated cyclic nucleotide phosphodiesterases in vivo in selected rat tissues

The effect of zinc deficiency on calmodulin function was investigated by assessing the in vivo activity of two calmodulin regulated enzymes, adenosine 3′,5′-monophosphate (c-AMP) and guanosine 3′,5′-monophosphate (c-GMP) phosphodiesterase (PDE) in several rat tissues. Enzymatic activities in brain, heart, and testis of rats fed a zinc deficient diet were compared with activities in these tissues from pair fed, zinc supplemented rats. In testis, a tissue in which zinc concentration decreased with zinc deficient diet, enzyme activities were significantly decreased over those in rats who were pair fed zinc supplemented diets. In brain and heart, tissues in which zinc concentrations did not change with either diet, enzymatic activities between the groups were not different. These results indicate that zinc deficiency influences the activity of calmodulin-regulated phosphodiesterases in vivo supporting the hypothesis that zinc plays a role in calmodulin function in vivo in zinc sensitive tissues.     

Structural basis of activation of the tumor suppressor protein neurofibromin

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Feeding response of the stable fly, Stomoxys calcitrant L., to blood fractions and adenine nucleotides

Abstract Feeding responses of the stable fly ( Stomoxys calcitrans L., Diptera: Muscidae) to blood fractions and saline-based diets were studied in an artificial feeding device. Flies fed equally on whole blood, plasma and erythrocyte fractions while resuspended platelets or the buffy layer did not stimulate feeding. Evidence from filtration studies indicates that plasma contains both less-than-5000D and greater-than-100,000D phagostimulatory components. ATP, ADP and AMP in saline stimulate feeding in this insect in a dose-dependent manner. cAMP also stimulates feeding in the range of concentrations tested. It is argued that the less-than-5000D phagostimulant may be one or more of these adenine nucleotides.     

Ligation of restriction endonuclease-generated DNA fragments using immobilized T4 DNA ligase

Phosphorylation of terminal deoxynucleotidyl transferase within leukemic cells has been demonstrated, using ³²P labelling of intact cells in culture, followed by immunoprecipitation of the cellular extracts using an anti-terminal transferase antiserum. The phosphate linkage was found to involve serine and threonine residues. Purified calf thymus terminal transferase served as a substrate for cyclic AMP independent protein kinase obtained from leukemic cells. Phosphorylation $\text{in vitro}$ of terminal transferase was accompanied by increased activity and decreased inhibition by excess ribo-ATP. These results indicate that terminal transferase is a physiologic cyclic AMP independent protein kinase substrate, and that this reaction may be important in its control.     

Differential effects of GTP on the coupling of β-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes application of new methods for the analysis of receptor-effector coupling

A detailed comparison of the interaction of β-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.     

Inhibition of 3-phosphoglycerate dehydrogenase by compounds other than l-serine

3-Phosphoglycerate dehydrogenase from etiolated pea epicotyls was not affected during in vitro assay by a range of hexose phosphates, amino group precursors and nucleotides at 1 mM but was significantly inhibited by 1 mM ATP and GTP. ADP and GDP gave slight inhibition at this concentration. NADH caused almost total inhibition at 0.45 mM.     

A structural determinant of mycophenolic acid resistance in eukaryotic inosine 5′‐monophosphate dehydrogenases

Mycophenolic acid (MPA) is a potent natural product inhibitor of fungal and other eukaryotic inosine 5′‐monophosphate dehydrogenases (IMPDHs) originally isolated from spoiled corn silage. MPA is produced by the filamentous fungi Penicillium brevicompactum, which contains two IMPDHs, PbIMPDHA and PbIMPDHB, both of which are MPA‐resistant. The MPA binding sites of these enzymes are identical to MPA‐sensitive IMPDHs, so the structural determinants of resistance are unknown. Here we show that a single residue, Ser267, accounts for the MPA resistance of PbIMPDHA. Substitution of Ser267 with Ala, the residue most commonly found in this position in eukaryotic IMPDHs, makes PbIMPDHA sensitive to MPA. Conversely, Aspergillus nidulans IMPDH becomes MPA‐resistant when the analogous Ala residue is substituted with Ser. These substitutions have little effect on the catalytic cycles of either enzyme, suggesting the fitness costs are negligible despite the strong conservation of Ala at this position. Intriguingly, while only 1% of fungal IMPDHs contain Ser or Thr at position 267, these residues are found in the IMPDHs from several Aspergillus species that grow at the low temperatures also favored by Penicillium. Perhaps Ser/Thr267 is an evolutionary signature of MPA exposure.