Inhibition of RNA synthesis in salivary glands of Drosophilamelanogaster by 5′-methylthioadenosine

5′-Methylthioadenosine (MTA) inhibits the incorporation of [³H] uridine into RNA in salivary glands of $\text{Drosophila}\text{melanogaster}$. This effect is not due to an inhibition of [³H] uridine uptake into the glands. The inhibition of RNA synthesis by MTA is concentration dependent and maximum inhibition is observed after 45 minutes of incubation in the presence of 1 mM MTA. Experiments utilizing α-amanitin suggest that the synthesis of heterogeneous RNA is completely inhibited.     

The synthesis of freezing-point-depressing protein of the winter flounder Pseudopleuronectusamericanus in Xenpuslaevis oocytes

The serum of the winter flounder $\text{Pseudopleuronectus}$$\text{americanus}$ contains a freezing-point-depressing protein of a molecular weight approximately 10,000 with 60% alanine in its composition. When injected into Xenopus o?cyte, a 6–10 S, poly A-rich RNA preparation isolated from the fish liver polysomes stimulated 3–4 fold the incorporation of [³H] alanine into 10% trichloroacetic acid-soluble, non-dialysable proteins. Analysis of the protein fractions showed a translation product similar in molecular weight and electrophoretic mobility to flounder freezing-point-depressing protein. These observations indicated that the 6–10 S RNA from the flounder contained mRNA for the synthesis of flounder's freezing-point-depressing protein.     

Pleiotropic effect of anticapsin on HeLa S3 cells

Anticapsin, the terminal epoxyaminoacid moiety of tetaine, inhibits irreversibly growth of HeLa S3 cells. The antibiotic decreases to a similar extent incorporation of 3H-labelled precursors into nucleic acids and protein in intact cells: inhibition of protein synthesis prevails on prolonged incubation. Also incorporation of [3H]dTTP and [3H]UTP is inhibited in the presence of anticapsin into permeabilized cells. These effects, however, are not due to the interference with DNA or RNA polymerases since anticapsin only slightly suppresses RNA polymerase activity and has no effect on DNA polymerase in the cell-free systems. The results indicate that the mechanism of antiproliferative action of anticapsin in HeLa S3 cells differs from that of tetaine and imply that inhibition of protein synthesis might be the primary effect of anticapsin.     

Inactivation of SV40 replication by derivatives of benzo[a]pyrene.

When African green monkey kidney cell lines, infected with simian virus 40, were exposed to benzo[a]pyrene-7,8-dihydrodiol or $\text{anti}$-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, inhibition of progeny virus formation was observed. Alkylation of SV40 DNA with $\text{anti}$-BPDE inhibits the infectivity of this viral DNA; however, the inactivation does not follow a single-hit mechanism. Studies on [³H]thymidine incorporation indicate that SV40 DNA synthesis is markedly impaired for the first 12 hours following BPDE treatment; 24 to 36 hours later, however, SV40 DNA synthesis is almost normal. These data suggest that the inhibition of SV40 DNA synthesis by BP derivatives is reversible and that the observed reduction in viral titer requires some other explanation.     

Controlled reduction of cytochrome b in succinate-cytochrome c reductase complex by succinate in the presence of ascorbate and antimycin.

$\text{Bacillus subtilis}$ membranes can transfer either N-acetylmuramyl-pentapeptide phosphate or N-acetylglucosaminyl phosphate from UMP directly onto undecaprenyl phosphate. Tunicamycin blocks only the latter transfer and inhibits peptidoglycan synthesis by toluenized cells of $\text{Bacillus megaterium}$ utilizing added nucleotide sugar precursors or cell wall synthesis by intact cells of $\text{B. subtilis}$. Tunicamycin prevents formation of the cell wall disaccharide lipid intermediate by blocking transfer of N-acetylglucosamine onto undecaprenyl muramyl pentapeptidyl pyrophosphate.     

Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent RNA polymerase.     

A simple method for following the fate of alanine-containing components in murein synthesis inEscherichia coli

The fate of the alanine-containing components in murein synthesis was followed by incorporation of¹⁴C-l-alanine inE. coli under conditions allowing cell-wall synthesis while preventing protein synthesis. The components were separated by chromatography and detected by autoradiography.Spots containing murein, cell-wall precursors, alanine andd-alanyl-d-alanine were identified. A further component was probably identical to pyruvic acid. Two unidentified spots were found in the region where lipid-intermediates in cell-wall synthesis are usually found. However, the absence of turnover of these two components was at variance with the proposed properties of the lipidintermediates. d-Alanyl-d-alanine and the component which is probably identical to pyruvic acid were excreted into the medium, whereas murein and cell-wall precursors were found in the cellular fraction.The influence of the concentration of alanine, and of the number of cells per ml, on the acid-precipitable activity were studied. The latter increased during, at least, the first two hours and represented mainly lysozyme-degradable material.Significant turnover of murein could be detected neither in the presence nor in the absence of protein synthesis.A time course of the activity of the radioactive components is provided. The influence of a number of antibiotics inhibiting cell-wall synthesis on the acid-precipitable activity and on the activity of the main intermediates in murein synthesis was studied.We thank Mrs. Arna van Schijndel-van Dam and Mr. A. A. G. Verweij for excellent assistance. We thank Dr. P. E. Reynolds (University of Cambridge) for teaching one of us (E J. J. L.) several techniques in the field of bacterial cell walls, and Dr. H. J. W. Wijsman for stimulating discussions.     

Amphomycin inhibits the incorporation of mannose and GlcNAc into lipid-linked saccharides by aorta extracts

Amphomycin inhibits the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNac from UDP-[³H]GlcNAc into lipid-linked saccharides by either a particulate or a solubilized enzyme fraction from pig aorta. The solubilized enzyme was much more sensitive to the antibiotic than was the particulate fraction with 50% inhibition being observed at 8–15 μg of amphomycin. Although the antibiotic inhibited mannose transfer from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol, lipid-linked oligosaccharides and glycoprotein, the synthesis of mannosyl-phosphoryl-dolichol was much more sensitive to amphomycin. Amphomycin also inhibited the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryldecaprenol in particulate extracts of $\text{Mycobacterium smegmatis}$.     

2H-1,3-Oxazine-2,6(3H)-dione,a new pyrimidine antimetabolite

2H-1,3-Oxazine-2,6(3H)-dione inhibits the growth of $\text{Escherichia}\text{coli}$ B, the inhibition being complete at a concentration of 10^?4M. It may be relieved with uridine, cytidine and partly with uracil. Orotic acid, cytosine, purine bases and purine ribonucleosides show no effect. At a molar ratio of uridine to the inhibitor of 1:2 the inhibition is completely suppressed. 2H-1,3-Oxazine-2,6(3H)-dione is thus a novel inhibitor of the biosynthesis of pyrimidine precursors of nucleic acids.     

Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular mucin of the mouse

The influence of isoproterenol and pilocarpine on the in vitro incorporation of [³H]leucine and $\text{N-}\text{acetyl}\text{[}{}^{14}\text{C}\text{]}\text{mannosamine}$ into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [³H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [¹⁴C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [¹⁴C]ManNAc relative to [³H]leucine into submandibular mucin of the mouse. During a period of 10 h the [¹⁴C]ManNAc incorporation into the mucin was enhanced 2–3-fold by isoproterenol and 3–4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.     

Growth inhibition of clonal osteosarcoma cell-lines by low concentrations of glucocorticoid hormones

Clonal osteosarcoma cell line, ROS $\text{2}\text{3}$, showed marked inhibition of [³H]thymidine incorporation in response to low concentrations (10^?10 M) of triamcinolone acetonide and dexamethasone. Hydrocortisone and corticosterone induced inhibition at somewhat higher concentrations. The osteosarcoma cell line ROS $\text{17}\text{2}$ responded similarly to triamcinolone acetonide and dexamethasone but at higher concentrations of the hormones. In ROS $\text{2}\text{3}$ the inhibitory effects of triamcinolone acetonide were accompanied by only slight elevation in the amount of intracellular exchangeable Ca²⁺. In contrast, in primary cultures of normal rat-calvarian bone cells, [³H]thymidine incorporation was inhibited to a much lesser extent only at higher concentrations of triamcinolone acetonide (10^?7 M). The difference in the susceptibility of normal and malignant bone cells to the inhibitory effects of glucocorticoids may have potential therapeutic importance.     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Measurement of cyclic 3',5'-adenosine monophosphate synthesis in growing Escherichia coli.

The net synthesis of cAMP by an adenine auxotroph of $\text{Escherichia coli}$ was measured by assaying the incorporation of tritium from [³H]-adenine into cyclic [³H] AMP during exponential growth. Synthesis of cAMP ceased abruptly when glucose was added to cells growing in glycerol and then recovered to an intermediate rate of synthesis after 0.5–1.0 generation. Cyclic AMP appeared to be synthesized from a precursor pool that turned over more rapidly than total cellular ATP. The rates of cAMP synthesis measured by this technique are compatible with the cellular levels of cAMP previously measured in this strain(3).     

Glucagon or cyclic AMP-stimulated synthesis of 5-phosphoribosyl 1-pyrophosphate in isolated hepatocytes and inhibition by antimicrotubular drugs.

Glucagon increased the level of 5-phosphoribosyl 1-pyrophosphate ($\text{PP}$Rib$\text{P}$) in isolated rat hepatocytes; a relatively high concentration of cyclic AMP could replace glucagon. In the presence of glucagon, the rate of incorporation of respective radioactive precursors into purine, pyrimidine, and oxidized pyridine nucleotides was accelerated, indicating that glucagon stimulates the synthesis of $\text{PP}$Rib$\text{P}$. Addition of 10^?6 M colchicine, vinblastin, or podophyllotoxin abolished the glucagon or cyclic AMP-induced increase in the $\text{PP}$Rib$\text{P}$ level. Colchicine did not affect accumulation of cyclic AMP induced by glucagon. These results suggest the involvement of tubulin or microtubules in the signal transfer from cyclic AMP to stimulated synthesis of $\text{PP}$Rib$\text{P}$.     

An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

Lysophosphatidylserine dependent incorporation of acyl CoA into phospholipids in rat brain microsomes

[¹⁴C]OleoylCoA was incorporated into phosphatidylinositol 4$\text{1}\text{2}$ times more efficiently than into phosphatidylserine in rat brain and liver microsomes when incubated with various levels of 1-acyl-sn-glycero-3-phosphoserine. In contrast, 1-acyl-sn-glycero-3-phosphocholine dependent incorporation of oleoylCoA was only into phosphatidylcholine. When [l-³H]serine labeled 1-acyl-sn-glycero-3-phosphoserine was used as the labeled substrate, no phosphatidylserine synthesis could be detected in rat brain microsomes. OleoylCoA incorporation in phospholipids in the presence of lysophosphatidylserine was primarily at the 2-position while stearoylCoA was incorporated at the 1-position. These results are interpreted to suggest that there is no acylCoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase in rat brain microsomes and the lysophosphatidylserine dependent position-specific incorporation of acylCoA into various phospholipids may be due to an exchange reaction. A simple highly reproducible one dimensional thin-layer chromatographic system is described for the separation of all the major phospholipids of brain and liver.     

Ruthenium red as a carrier of electrons between external NADH and cytochrome c in rat liver mitochondria.

We have determined that Co²⁺, Ni²⁺ or Zn²⁺ may substitute for Mg²⁺ during DNA synthesis with $\text{E.}\text{coli}$ DNA polymerase I, sea urchin nuclear DNA polymerase and the DNA polymerase from avian myeloblastosis virus (AMV). In addition, the frequency of non-complementary nucleotide incorporation using AMV DNA polymerase was increased using Co²⁺ or Mn²⁺ as the metal activator. These results suggest that the fidelity of DNA synthesis may be influenced by the metal activator used during catalysis.     

Isolation of relaxed-control mutants of Escherichia coli K-12 which are sensitive to glucose starvation

Mutants of $\text{Escherichia coli}$ K-12 which are sensitive to glucose starvation were isolated by an enrichment procedure using thymine starvation to select for nongrowing cells. Eleven independent isolates were obtained by this method. The mutants are also sensitive to glycerol starvation and to a lesser extent to nitrogen or amino acid starvation. The mutants are more sensitive than the parental strain to inhibitors of protein synthesis but not inhibitors of RNA or DNA synthesis. [³H]-leucine incorporation experiments indicate that protein synthesis is blocked in the mutants during recovery from glucose starvation or chloramphenicol inhibition. Incorporation of [³H]uridine in amino acid-starved cells demonstrates that the mutants are partially relaxed for control of RNA synthesis. Physiological and genetic experiments indicate that these mutants are different from previously isolated relaxed-control mutants.     

Increase in dATP pool in aphidicolin-resistant mutants of mouse FM3A cells

Mutants that were resistant to aphidicolin were isolated from mutagenized mouse FM3A cells at a frequency of about 10^?6. Resistance to aphidicolin in these mutants was not due to an effect on [³H]thymidine incorporation into DNA, DNA synthesis in permeabilized cells, or DNA polymerase α.All the mutants showed a greatly increased dATP pool and decreased ability to incorporate [³H]deoxycytidine into DNA. They also showed cross-resistance to both 1-β-D-arabinofuranosyladenine and 1-β-D-arabinofuranosylcytosine.These results indicate that an enzyme involved in production of dATP or its regulation is altered in these mutants. It is suggested that dATP competes with aphidocolin at its killing site or that dATP reverses the effect of aphidicolin by some unknown mechanism $\text{in}\text{vivo}$.