Structure-activity dependence in some novel ring-substituted 3,3-dimethyl-1-phenyltriazenes. Genetic effects in Drosophila melanogaster and in Saccharomyces cerevisiae by a direct and a host-mediated assay

The structure-activity dependence of ten ring-substituted 3,3-dimethyl-1-phenyltriazenes (DMPT), 3,3-dimethyl-1-(3-pyridyl)-triazene (3-PyDMT) and of 3,3-dimethyl-1-(3-pyridyl-N-oxide)-triazene (3-PyODMT) was investigated by the induction of recessive lethal mutations in Drosophila melanogaster and of mitotic gene conversions in Saccharomyces cerevisiae using both direct and host-mediated assays. Significant differences in genetic effectiveness were detected not only between structurally related compounds but also between the responses of each test system to the same mutagen. Triazenes which are easily cleaved at physological conditions showed the highest genetic activity in the direct yeast test whereas stable triazenes, especially those with ortho and para positions blocked by a halogen, were most active in Drosophila. We have concluded that (1) the released arenediazonium cation is most probably responsible for the convertogenic activity in yeast; (2) metabolites, arising from hydroxylation of the methyl group, are essential for the mutagenic activity in Drosophila. A possible molecular basis which could account for the diversity in genetic effectiveness is discussed in terms of reaction mechanisms which can be predicted from the structural features of the tested triazenes.     

Induction of mitotic gene conversion by 3,3-dimethyl-I-phenyltriazene, I-(3-hydroxyphenyl-)-3,3-dimethyltriazene and by I-(4-hydroxyphenyl)-3,3-dimethyltriazene in Saccharomyces cerevisiae

The induction of mitotic gene conversion by 3,3-dimethyl-1-phenyltriazene (DMPT), 1-(3-hydroxyphenyl)-3,3-dimethyltriazene (3-HO-PDMT) and by 1-(4-hydroxyphenyl)-3,3-dimethyltriazene (4-HO-PDMT) in the diploid strain D4 of Saccharomyces cerevisiae was investigated. The frequencies of the non-reciprocal intragenic recombinations at two unlinked loci ade2 (adenine) and trp5 (tryptophan) were determined. Although all three triazenes showed marked convertogenic activities, significant differences in their genetic effectiveness have been observed. Thus both phenolic triazenes were found to be much stronger convertogens than the unhydroxylated parent compound, DMPT. An attempt is made to account for the established differences in convertogenicity by chemical reactivity that could be expected from the structural features of the tested alkaryltriazenes.     

Cytogenetic effects in the mouse of 17 chemical mutagens and carcinogens evaluated by the micronucleus test.

2 dialkylnitrosamines, 4 oxazaphosphorines, 6 aryldialkyltriazenes, urethane, N-hydroxyurethane, 4-nitroquinoline-1-oxide, procarbazine (natulan) and the inorganic carcinogen potassium chromate were studied for cytogenetic activity in the micronucleus test on mouse bone marrow. Except diethylnitrosamine, all chemicals were active. The results are compared with those known from studies in other mammalian and sub-mammalian test systems. The results of the micro nucleus test correlate well with results from other mutagenicity tests and with the carcinogenicity of the chemicals. The lack of an effect on N-nitrosodiethylamine (DENA) is discussed with regard to the short life-time of the ultimate mutagen.     

Genetic characterization of ad-3 mutants induced by chemical carcinogens, I-phenyl-3-monomethyltriazene and I-phenyl-3,3-dimethyltriazene, in Neurospora crassa

180 ad-3 mutants of Neurospora crassa induced by 1-phenyl-3-monomethyl-triazene (PMMT) and 56 ad-3 mutants induced by 1-phenyl-3,3-dimethyltriazene (PDMT) were characterized by dikaryon, trikaryon and complementation tests. Results show that the spectrum of genetic alterations induced by PMMT is different from that of PDMT. This suggests that enzymatic dealkylation of PDMT to PMMT does not occur within Neuropsora crassa conidia, and that the mechanism of mutation induction of PDMT in N. crassa is different from that of PMMT. Hydrolytic breakdown products or its intact molecule or some other converted forms might be responsible for the mutagenic activity of PDMT.Mutation induction of PMMT in N. crassa appears to be via alkylation of DNA by carbonium ions produced by this compound, the same mechanism proposed for its carcinogenic activity. The frequencies of leakiness, allelic complementation and nonpolarized complementation patterns among PMMT-induced ad-3 mutants are similar to those of ad-3 mutants induced by other potent chemical carcinogens, such as MNNG and the aflatoxins.     

Antibiotics and compounds affecting translation by eukaryotic ribosomes. Specific enhancement of aminoacyl-tRNA binding by methylxanthines

Summary The mode and site of action of inhibitors of translation (initiation, elongation and termination of protein synthesis) in eukaryotic systems is reviewed. The isolation and characterization of a factor is described that binds Ac-Phe-tRNA to form a complex made up of binding factor, Ac-Phe-tRNA, and ribosome. The binding of Ac-Phe-tRNA probably occurs at the ribosomal site involved in the binding of the initiator substrate Met-tRNA_F. The effect of inhibitors of the initiation phase of protein synthesis on the nonenzymic Ac-Phe-tRNA binding to ribosomes is investigated. The two sites translocation model for translation in eukaryotic cells is presented and the effects of inhibitors on the various steps of protein synthesis are determined empirically. The site of action of inhibitors of peptide bond formation at the ribosomal peptidyl transferase center is elucidated. The action of inhibitors of translocation is studied in model cell-free systems from human cells. In addition, a number of methylxanthines are shown to enhance the elongation phase in polypeptide synthesis by stimulating the enzymic binding of aminoacyl-tRNA. The effect of caffeine, theophylline and its derivatives are shown to be fairly specific and dependent on the ribosome concentration. Aminophylline is shown to have a similar effect but also enhances aminoacyl-tRNA synthetase activity at low Mg^{+ +} concentrations, probably by displacing the optimal concentration of Mg^{+ +} in the reaction. This second effect of aminophylline appears to be due to the ethylenediamine moiety of aminophylline since it is also observed in the presence of different polyamines but not in the presence of caffeine or theophylline.An invited article.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Selection of Small Peptides, Inhibitors of Translation

Identification of small molecular weight compounds targeting specific sites in the ribosome can accelerate development of new antibiotics and provide new tools for ribosomal research. We demonstrate here that antibiotic-size short peptides capable of inhibiting protein synthesis can be selected by using specific elements of ribosomal RNA as a target. The ‘h18’ pseudoknot encompassing residues 500-545 of the small ribosomal subunit RNA was used as a target in screening a heptapeptide phage-display library. Two of the selected peptides could efficiently interfere with both bacterial and eukaryotic translation. One of these inhibitory peptides exhibited a high-affinity binding to the isolated small ribosomal subunit (K_d of 1.1 μM). Identification of inhibitory peptides that likely target a specific rRNA structure may pave new ways for validating new antibiotic sites in the ribosome. The selected peptides can be used as a tool in search of novel site-specific inhibitors of translation.     

Age-related changes in different steps of protein synthesis of liver and kidney of rats

Protein synthesis in cell-free systems of rat liver and kidney decreases markedly with age. Examination of activity changes of the different steps revealed for both types of organs that reduced binding of aminoacyl-tRNA to ribosomes and reduced peptidyl transfer might be of major importance for the decrease in overall protein synthesis whereas ageing has only little effect on translocation as well as on initiation and termination.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

25-Hydroxylation of 1-α-hydroxyvitamin D-3 in rat and human liver

1α-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1α-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent K_m value of 2·10^?5 M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100°C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1α-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1α-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.     

Reduced turnover of the elongation factor EF-1 · ribosome complex after treatment with the protein synthesis inhibitor II from barley seeds

The effect of the protein synthesis inhibitor II from barley seeds (Hordeum sp.) on protein synthesis was studied in rabbit reticulocyte lysates. Inhibitor treatment of the lysates resulted in a rapid decrease in amino acid incorporation and an accumulation of heavy polysomes, indicating an effect of the inhibitor on polypeptide chain elongation. The protein synthesis inhibition was due to a catalytic inactivation of the large ribosomal subunit with no effect on the small subparticle. The inhibitor-treated ribosomes were fully active in participating in the EF-1-dependent binding of [¹⁴C]phenylalanyl-tRNA to poly(U)-programmed ribosomes in the presence of GTP and the binding of radioactively labelled EF-2 in the presence of GuoPP[CH₂]P. Furthermore, the ribosomes were still able to catalyse peptide-bond formation. However, the EF-1- and ribosome-dependent hydrolysis of GTP was reduced by more than 40% in the presence of inhibitor-treated ribosomes, while the EF-2- and ribosome-dependent GTPase remained unaffected. This suggests that the active domains involved in the two different GTPases are non-identical. Treatment of reticulocyte lysates with the barley inhibitor resulted in a marked shift of the steady-state distribution of the ribosomal phases during the elongation cycle as determined by the ribosomal content of elongation factors. Thus, the content of EF-1 increased from 0.38 mol/mol ribosome to 0.71 mol/mol ribosome, whereas the EF-2 content dropped from 0.20 mol/mol ribosome at steady state to 0.09 mol/mol ribosome after inhibitor treatment. The data suggest that the inhibitor reduces the turnover of ribosome-bound ternary EF-1 · GTP · aminoacyl-tRNA complexes during proof-reading and binding of the cognate aminoacyl-tRNA by inhibiting the EF-1-dependent GTPase.     

Inhibition by Thiopeptin of Bacterial Protein Synthesis

Thiopeptin, a sulfur-containing antibiotic, was found to inhibit protein synthesis in a bacterial ribosomal system. The pretreatment of ribosomal subunits with the antibiotic revealed that thiopeptin may act on the 50 S ribosomal subunit. The elongation of peptide chain on the ribosome is more profoundly blocked by the antibiotic than the initiation of protein synthesis. It was demonstrated that thiopeptin inhibits elongation factor (EF)-Tu-dependent GTP hydrolysis and binding of aminoacyl-tRNA to the ribosome. The peptidyl transferase-catalyzed puromycin reaction is not significantly affected by the antibiotic. Thiopeptin inhibits EF-G-associated GTPase reaction, and translocation of peptidyl-tRNA and mRNA from the acceptor site to the donor site. Protein synthesis in ribosomal systems, obtained from rat liver and rabbit reticulocytes are insensitive to the antibiotic.     

Effect of polaymines on yeast cell-free protein synthesizing system. I. Influence of spermine and spermidine on aminoacyl-tRNA transfer reaction.

Spermine and spermidine added to a Saccharomyces cerevisiae cell-free protein synthesizing system increased phenylalanine polymerization reaction several-fold at suboptimal concentration of Mg2+ and approximately two-fold at optimal amounts of Mg2+. The addition of polyamines greatly stimulated the enzymatic and nonenzymatic binding of phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA to ribosomes. The binding of the acetylated derivative was higher than phenylalanyl-tRNA, however, as it was shown the former was bound exclusively to the A site of the ribosome. Contrary to the binding process, the puromycin reaction was not stimulated by spermine added at a concentration which enhanced the polyphenylalanine synthesis. These results indicate that polyamines have not only a sparing effect on the Mg2+ requirement for yeast protein synthesis in vitro and suggest that one of the possible sites of polyamines action might be the binding of aminoacyl-tRNA to ribosomes.     

CHARACTERISTICS AND PRODUCTS OF A CELL-FREE POLYPEPTIDE SYNTHESIZING SYSTEM FROM NEONATAL AND ADULT MOUSE BRAIN

—The regulation of protein synthesis by ribosomes isolated from mouse brain tissue was studied using a cell-free polyphenylalanine synthesizing system. Polypeptide synthesis was followed by assaying translocation and analysing the reaction products by BD-cellulose chromatography. The brain ribosomal activity could be divided by these methods into two distinct steps : binding of aminoacyl-tRNA to the ribosome and active translocation leading to subsequent polyphenylalanine synthesis. In comparison to initial binding of aminoacyl-tRNA, translocation in the cell-free system increased the incorporation of labelled phenylalanine by 10-fold. An analysis of the reaction products clearly showed active ribosomal synthesis of oligophenylalanine from [³H]phe-tRNA. Ribosomes isolated from neonatal brain tissue were 2–4 times as active as those obtained from adult brain tissue in polypeptide synthesis. In addition, polypeptides synthesized on the more active ribosomes from neonates tended to be of greater chain length than those from adult. Therefore, the maturation-dependent decrease in ribosomal protein synthetic activity during neural development was shown to be directly associated with the ribosome particles.     

The effect of antiestrogens on egg yolk protein synthesis and estrogen-binding to chromatin in the rooster liver

Nafoxidine and CI-628, two well known antiestrogenic compounds, reduce the stimulating effect of estradiol on the estrogen-binding capacity of the liver chromatin from roosters. In vitro both antiestrogens compete with [³H] estradiol for the binding sites on the liver chromatin. They inhibit the estrogen-induced synthesis of egg yolk proteins (vitellogenin) and fail to induce this estrogen-specific protein synthesis by themselves. They show the ability, however, to increase the estrogen-binding sites on the liver chromatin to some extent.     

Effect of some polycyclic aromatic hydrocarbons on protein synthesis in vitro

1. The effect of the strongly carcinogenic polycyclic aromatic hydrocarbons benzo[a]pyrene, 3-methylcholanthrene and dibenz[a,h]anthracene and of the non-carcinogenic anthracene, pyrene and phenanthrene on protein synthesis was studied in vitro with subcellular systems from rat liver. 2. Both types of hydrocarbons affect amino acid activation and inhibit transfer of labelled amino acids from transfer RNA to ribosomes. 3. Only the carcinogenic compounds stimulate the incorporation of labelled algal-protein hydrolysate and of some individual amino acids into transfer RNA. The most active dose was 10mmug. under the conditions used. This effect is abolished by preincubation of pH5 enzymes with the carcinogens before the addition of radioactive amino acids. 4. The carcinogens stimulate the incorporation of some amino acids into ribosomal protein whereas the non-carcinogenic compounds have no such effects. 5. Polynucleotide-dependent stimulation of protein synthesis is greatly enhanced in the presence of the carcinogenic hydrocarbons when either free amino acids or transfer RNA charged with labelled amino acids are used. The non-carcinogenic compounds induce a partial inhibition of this process. 6. It is concluded, in agreement with other authors, that carcinogens may increase the number of active incorporation sites on both transfer and ribosomal RNA. Possible mechanisms of such an effect are discussed.     

In vivo short-term assays for tumor initiation and promotion in the glandular stomach of Fischer rats

Here we summarize the data on 55 compounds tested in in vivo short-term assays for tumor-initiating and tumor-promoting activity in the glandular stomach of male Fischer (F344) rats. Most of the data has been previously published. Tumor-initiating activity was assayed by measuring the induction of unscheduled DNA synthesis (UDS) and DNA single strand scission; tumor-promoting activity was assayed by measuring the induction of ornithine decarboxylase (ODC) activity, increased replicative DNA synthesis (RDS), and of c-fos and c-myc oncogene expression. The compounds were orally administered. Twenty-nine compounds were tested for UDS. Eight were positive, including 5 glandular stomach carcinogens; 16 were negative, including 5 liver carcinogens; and 5 were equivocal. Twenty compounds were tested for DNA single strand scission. Twelve were positive, including 6 glandular stomach carcinogens; 7 negative, including 2 liver carcinogens; and 1 was equivocal. Thirty-two compounds were tested for RDS. Twenty-six were positive, including 8 glandular stomach carcinogens and 6 glandular stomach tumor-promoters; 4 were negative, including 3 liver carcinogens and a stomach irritant; and 2 were equivocal. Forty-five compounds were tested for ODC. Thirty-seven were positive, including 8 glandular stomach carcinogens and 6 glandular stomach tumor promoters; 7 were negative, including 3 liver carcinogens; and one was equivocal. All glandular stomach carcinogens and tumor-promoters examined were positive in both RDS and ODC. Two compounds were tested for c-fos and c-myc expression; one was a glandular stomach carcinogen and one was a glandular stomach tumor promoter, and both were positive. In addition, 2 compounds inhibited the increase in RDS induced by the tumor promoter NaCl, suggesting anti-tumor-promoter activity. Thus these assays are useful for assessing potential tumor-initiating and tumor-promoting activity in the rat glandular stomach.     

Different reactivity of carboxylic groups of cytochrome c oxidase polypeptides from pig liver and heart

Cytochrome c oxidase isolated from pig liver and heart was incubated with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide and [¹⁴C]glycine ethyl ester in the presence and absence of cytochrome c. Labelling of individual subunits was determined after separation of the enzyme complexes into 13 polypeptides by SDS-gel electrophoresis. Polypeptide II and additional but different polypeptides were labelled in the liver and in the heart enzyme. Labelling of polypeptide II and of some other polypeptides could be partially or completely suppressed by cytochrome c. From the data two conclusions can be drawn: In addition to polypeptide II, other polypeptides take part in the binding of cytochrome c to cytochrome c oxidase; the binding domain for cytochrome c is different in pig liver and heart cytochrome c oxidase.Cytochrome c oxidase isozymeCytochrome c binding domain1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideTissue specificity     

Synthesis and biological evaluation of thiazole derivatives as GPR119 agonists

A series of 4-(phenoxymethyl)thiazole derivatives was synthesized and evaluated for their GPR119 agonistic effect. Several 4-(phenoxymethyl)thiazoles with pyrrolidine-2,5-dione moieties showed potent GPR119 agonistic activities. Among them, compound 27 and 32d showed good in vitro activity with an EC₅₀ value of 49?nM and 18?nM, respectively with improved human and rat liver microsomal stability compare with MBX-2982. Compound 27 & 32d did not exhibit significant CYP inhibition, hERG binding, and cytotoxicity. Moreover, these compounds lowered the glucose excursion in mice in an oral glucose-tolerance test.     

Interaction of antitumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study.

The cytotoxic and mutagenic properties of antitumor drugs such as adriamycin, acridines, diacridine, actinomycin D and Pt compounds are related to their interaction with nucleic acids and inhibition of protein synthesis. We have examined their interaction with human erythrocyte ghost membranes and murine mastocytoma cells using spin labeling techniques. These drugs induce changes in electron spin resonance of the spin labeled ghost membranes and in the mastocytoma cells. These alterations suggest that these drugs induce changes in protein conformation of the membranes. The membrane binding properties of these drugs may be important in their mechanism of action.