Cyclic AMP dependent regulation of mitosis in human lymphoid cells

Intracellular levels of cyclic AMP (cAMP), cAMP-dependent phosphodiesterase activity, and adenylate cyclase activity are examined in an established line of human lymphoid cells synchronized by either excess thymidine or by colcemid treatment. cAMP levels and adenylate cyclase activities during the two G periods are high when compared with the values in M. cAMP-dependent phosphodiesterase activity, which is low during early G 2, is shown to increase during G 2 and reach a maximum activity during M. Agents such as dibutyryl cAMP, 1-methyl-3-isobutyl xanthine, noradrenaline, and isopropyl noradrenaline, which increase the levels of intracellular cAMP were examined to determine their effects on mitosis and on DNA synthesis. In thymidine-synchronized cells the onset of mitosis is prevented by increasing or maintaining high levels of cAMP during G 2. The specificity of inhibition of DNA synthesis or mitosis by dibutyryl cAMP is a function of the time, during the cell cycle, when the analogue is added. The elevation of cAMP by methyl xanthine results in a more general inhibition of nucleic acid synthesis and mitosis. Although both catecholamine hormones inhibit mitosis, isopropylnoradrenaline also inhibits DNA synthesis while noradrenaline treatment does not result in such inhibition.     

The effect of removal or replacement with proflavine of the Y base in the anticodon loop of yeast tRNAPhe on binding into the acceptor or donor sites of reticulocyte ribosomes

Phe-tRNA from yeast has a highly modified nucleoside, called Y, adjacent to the 3′ side of its anticodon, that can be removed or replaced with proflavine. In a protein-synthesizing system from rabbit reticulocytes, poly (U)-directed binding and polyphenylalanine synthesis are low with these modified Phe-tRNA species relative to the corresponding values with unmodified Phe-tRNA. However, polymerization can be increased with relatively large amounts of elongation factor I. The modified Phe-tRNA species bound to the ribosomes with poly(U) either in the presence or absence of elongation factor I and GTP is immediately reactive in the peptidyl transferase reaction measured by the formation of diphenylalanine or phenylalanyl-puromycin. It appears to have been bound directly into the donor ribosomal site by either the nonenzymatic mechanism involving Mg²⁺ or by the enzymatic mechanism involving EF-I and GTP.     

Mischarging single and double mutants of Escherichia coli sup3 tyrosine transfer RNA

Mischarging mutants of Escherichia coli sup3 tyrosine transfer RNA have been isolated by selecting for suppression of bacterial amber mutations not suppressed by sup3. Five of the mutants have single base changes in the amino acid acceptor stem (A1, A2, U80, U81 and G82). Mutants A1 and A2 are weak thermosensitive suppressors from which thermostable derivatives have been isolated. Some of these derivatives affect the amount of tRNA synthesized but not the sequence (precursor or promoter mutations), and others are double mutants A1U81 and A2U80. The latter mutant does not mischarge. The efficiency of suppression of A1 and A2 can also be increased by recombination events that lead to duplication and triplication of the suppressor gene.The amino acid inserted by some of these mutants at the amber site has been determined. Mutant A1 inserts glutamine, while U81 and A1U81 insert both glutamine and tyrosine.Taken together the results show that the terminal part of the amino acid acceptor stem has an important role in the specificity of aminoacylation by the glutamine and tyrosine synthetase.     

Phosphatidohydrolase activity in a solubilized preparation from rat brain particulate fraction.

Phospholipase D activity (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was demonstrated in vitro in a solubilized preparation from rat brain particulate fraction which also possessed the transphosphatidylation activity. The preparation attacked a phosphatidylcholine microdispersion and cleaved the terminal phosphate diester bond of this phospholipid resulting in the formation of phosphatidic acid. The pH optimum for the phosphatidohydrolase activity was broad with an apparent peak aroung 6.0 whereas the transphosphatidylation showed a sharp pH optimum at 7.2 Ca²⁺ was not essential for the hydrolysis, but merely stimulated slightly with an optimum about 5 mm, however, it could be replaced by Mg²⁺. The hydrolysis of phosphatidylcholine by the enzyme was almost completely inhibited in the presence of either diethyl ether (20% by volume) or p-chloromercuriophenyl sulfonate (6 × 10 ^?5m). The latter inhibition was reduced by the addition of dithiothreitol (6 × 10^?4m). The result suggests an essential role of sulfhydryl group in the formation of the enzyme-substrate complex.The apparent K_m for phsophatidylcholine for the phosphatidohydrolase activity was about 8.3 × 10 ^?4m.     

Elucidation of a minimal immunoreactive site of vertebrate calmodulin

The heptapeptide AsnTyrGluGluPheValGlnNH₂ corresponding to residues 137–143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem.256, 4205–4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127–144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only sevenresidue segment in the 135–145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.     

Synthesis and maturation of cross-reactive glycoprotein in fibroblasts deficient in arylsulfatase a activity

The biosynthesis of arylsulfatase A was studied in cultured fibroblasts by pulse-chase labeling with [2-³H]mannose; the enzyme was isolated by immunoprecipitation and denaturing polyacrylamide gel electrophoresis. In normal fibroblasts, and in fibroblasts from a patient with multiple sulfatase deficiency, the enzyme was synthesized as a glycoprotein of apparent molecular weight of 59,000; half of it was processed over a period of 4 days to M_r= 57,000. The precursor chain of M_r= 59,000 was secreted in the presence of 10 mM NH₄Cl. An immunoprecipitable glycoprotein of normal size was synthesized by fibroblasts from two unrelated patients with metachromatic leukodystrophy, but this material disappeared within twenty hours. In fibroblasts from an individual with pseudodeficiency of arylsulfatase A, the immunoprecipitable precursor glycoprotein was smaller (M_r= 56,000). The synthesis of cross-reactive proteins with altered properties supports the concept of allelic mutations as the genetic basis of metachromatic leukodystrophy and of arylsulfatase A pseudodeficiency.     

Differentiation antigens in human T-cell activation: Evidence that anti-VH antibodies react with a membrane structure on human T Lymphocytes distinct from the antigens detected by monoclonal antibodies of the OKT and Leu series

The effect of a panel of monoclonal antibodies and heteroantibodies on T-cell proliferation in various assay systems has been examined. The antibodies tested were directed against T-cell differentiation antigens, HLA-DR antigens, and structures defined by an anti-human V_H antiserum. As the test cell system highly purified subpopulations of T-cell growth factor (TCGF)-dependent T-cell lines activated either by mitogen or antigen were used. A survey of the data indicates the following: (1) Mitogenic and antigenic triggering of T lymphocytes are mediated through partly different membrane structures. (2) Antigenic stimulation by purified protein derivative (PPD) as well as polyclonal activation induced by OKT3/anti-Leu 4 monoclonal antibodies can be inhibited by heteroantibodies raised against human immunoglobulin V_H fragments thus pointing to a possible connection between the antigens detected by these antisera. (3) There does not seem to be differences between the two major subpopulations of T lymphocytes (i.e., helper/inducer and suppressor/cytotoxic cells) as to how they respond to antigens or mitogens in the investigated assay systems. (4) A clear distinction was found between T blasts specific for PPD and allogeneic cells as compared to cytotoxic T cells (CTL), as the T4 and T8 antigens seem to be functionally important for antigen recognition among CTL but not for the blasts proliferating in response to PPD and allogeneic cells. (5) An inhibitory effect of OKT3/anti-Leu 4, OKIal, and anti-HLA-DR on TCGF-dependent growth was detected, possibly indicating a steric relationship between these antigens and TCGF receptors on mitogen-induced T blasts. (6) Soluble factors obtained after incubating adherent cells with OKIal and anti-HLA-DR antibodies seemed to have an inhibitory effect on overall T-cell proliferation stressing the importance of studying the T-cell activation process at different levels in these kinds of experiments. (7) The results further suggest a complexity in the build up of antigen receptors on the various T-effector cells, perhaps also involving receptors for growth factors, HLA-DR antigens, and receptors for the latter.     

Lymphokine and phorbol (PMA) regulation of complement (C2) synthesis using U937

Human monocytes synthesize large amounts of the second complement component (C2) after incubation with a T-lymphocyte product called monocyte complement stimulator (MCS). The human monocyte-like cell line, U937, also synthesizes C2 and can be stimulated to increase this synthesis by lymphokine-rich culture supernates. Additionally, phorbol myristate acetate (PMA), an agent which induces maturational changes in other macrophage-like cell lines, also stimulates C2 synthesis by U937 cells. Lymphokine and PMA stimulation of C2 secretion by U937 are both reversibly inhibitable by cycloheximide. At optimal concentrations for stimulation of C2 synthesis, PMA inhibits [³H]thymidine incorporation by U937 indicating that increased C2 is not due to increased numbers of U937 cells.     

Stimulation of Ca2+ efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10^?10 M to 10^?7 M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.