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At least 19 recordsLinked to original sources

PubMed1975

Isolation and characterization of an alpha-amanitin-resistant rat myoblast mutant cell line possessing alpha-amanitin-resistant RNA polymerase II.

Cultures of the rat skeletal muscle myoblast cell line, L6, were treated with the mutagen ethylmethanesulfonate and grown in the presence of alpha-amanitin, an inhibitor of RNA polymerase II in vitro. One clonal cell line, Ama102, resistant tc the cytotoxic action of 2 mu-g/ml of alpha-amanitin was isolated and extensively characterized. Ama102 cells were about 30-fold more resistant to alpha-amanitin than their Ama+ parent cells based on a comparison of the concentration of alpha-amanitin required to reduce their plating efficiencies to similar extents. The RNA polymerase activities from Ama+ and Ama102 cells were solubilized and separated by DEAE-Sephadex chromatography. Whereas all of the Ama+ RNA polymerase II activity was inhibited by 0.1 mu-g/ml of alpha-amanitin, about 30% of the activity in the Ama102 RNA polymerase II peak was resistant to this concentration of alpha-amanitin and was inhibited only by much higher concentrations (25 mu-g/ml) of alpha-amanitin. This alpha-amanitin-resistant activity in Ama102 cells was identified as a bona fide RNA polymerase II by its chromatographic behavior on DEAE-Sephadex, salt optimum, preference for denatured DNA as template, insensitivity to inhibition by potassium phosphate, thermal inactivation kinetics, and inactivation by anti-RNA polymerase II antiserum. Both RNA polymerase IIa and IIb from Ama102 cells exhibited the partial alpha-amanitin resistance, as did this activity when purified further on phosphocellusose. Unlike the parental Ama+ cells, Ama102 cells neither fused at confluence nor showed an increase in the specific activity of creatine kinase. The altered sensitivity of the Ama102 RNA polymerase II to alpha-amanitin appears to account for the drug-resistant phenotype of these cells.

Amanitins↗

PubMed1979

Determination of alpha-, beta-, and gamma-amanitin by high performance thin-layer chromatography in Amanita phalloides (Vaill. ex Fr.) secr. from various origin.

A fast, sensitive high performance thin-layer chromatographic method for the determination of alpha-, beta-, and gamma-amanitin in crude, methanolic extracts of Amanita phalloides is described. The limit of detection is 50 ng of each amanitin. With this method amanitin was determined in 24 pooled samples of Amanita phalloides, collected between 1970 and 1977 in Germany and Switzerland. The total amanitin content varied between 2010 and 7300 mg/kg dry weight and the average value was 4430 mg/kg of which 43% was alpha-amanitin, 49% beta-amanitin and 8% gamma-amanitin. The origin of the fungi hardly influenced their amanitin content: in samples collected during the same year at different sites it fluctuated within a factor of 1.7. The amanitin content of samples from the same site, but collected in different years, maximally varied within a factor of 3.7. The partial decomposition of amanitins during prolonged storage of the lyophilized samples undoubtedly contributed to this variation. Phalloidin, which was determined by conventional thin-layer-chromatography, could not be detected in a sample from 1970, whereas its concentration in material collected during 1977 amounted to 2400 mg/kg dry weight. The toxicity of the samples (LD50 of lyophilized defatted methanolic extracts intravenously for mice) varied within a factor of 2.5.

Agaricales↗

PubMed1979

Alpha-amanitin-resistant D. melanogaster with an altered RNA polymerase II.

Following EMS mutagenesis we recovered a mutant of D. melanogaster that grows at concentrations of alpha-amanitin lethal to wild-type. To our knowledge this mutant represents the first example of an amanitin-resistant eucaryotic organism. The amanitin resistance of the mutant (AmaC4) is due to an alteration in its DNA-dependent RNA polymerase II, which is approximately 250 times less sensitive to inhibition by amanitin than the wild-type polymerase II whether tested in nuclei, in partially-fractionated extracts or as a highly purified enzyme. While the wild-type enzyme activity is inhibited 50% by 2.1 x 10(-8) M alpha-amanitin, inhibition of 50% of the AmaC4 RNA polymerase II activity requires a toxin concentration of 5.6 x 10(-6) M. The mutation responsible for the amanitin resistance of AmaC4 is on the X chromosome near the vermillion locus.

Amanitins↗

PubMedDate not supplied

Amanitin content and toxicity of Amantia verna Bull.

The alpha-, beta-, and gamma-amanitin content of 11 samples of Amanita verna Bull., collected during 1975--1978 in Germany and Switzerland, has been determined by high performance thin-layer chromatography (HPTLC) of crude methanolic extracts. The toxicity (i. v. LD50 for mice of defatted, lyophilized, methanolic extracts) of 3 samples has been compared with that of A. phalloides from the same site of collection. The amanitin content of A. verna ranged from 2250 to 4570 mg/kg dry weight; the fungi contained almost as much beta- as alpha-amanitin, whereas the gamma-amanitin content amounted to about 12% of the total amanitin. A verna contained less amanitin (65% on the average) than A. phalloides from the same collection site, but it was not significantly less toxic, since the phallotoxins contributed to the toxicity of either species in our tests.

Agaricales↗

PubMed1979

Synthesis of low molecular weight RNA components A, C and D by polymerase II in alpha-amanitin-resistant hamster cells.

In an attempt to establish which RNA polymerase catalyzes the synthesis of the low molecular weight RNA components A, C and D, Ama 1 cells (mutant Chinese hamster cells) were used in experiments with addition of alpha-amanitin. Ama 1 cells contain an altered RNA polymerase II which is 800 times more resistant towards inhibition by alpha-amanitin than the wild type enzyme. Alpha-amanitin (up to 200 microgram/ml) added to these cells does not affect the synthesis of the low molecular weight RNAs A, C and D. These data together with our previous data showing that alpha-amanitin (0.5 - 5.0 microgram/ml) preferentially inhibits the synthesis of A, C and D in normal cells indicate that RNA polymerase II catalyzes the synthesis of the low molecular weight RNA components A, C and D.

Amanitins↗

PubMed1977

Altered sensitivity of Rous sarcoma virus transformed cells to inhibition of RNA synthesis by alpha-amanitin.

Chick embryo cells transformed by Rous sarcoma virus (RSV) continue to synthesize 40--50 percent of control amounts of RNA following 12--24 hour exposure to 2 microng/ml of the toxin whereas normal chick embryo cells similarly treated synthesize less than 5 percent of control amounts of RNA. Analysis of cells treated with alpha-amanitin, or the resistant forms I and III polymerase, do not increase in infected cells over the levels found in uninfected control cells during the first 24 hours following infection indicating that increase in polymerase levels in infected cells does not account for the observed resistance. No significant difference was detected in the sensitivity to alpha-amanitin in the form II polymerase isolated from normal and transformed cells; The greater sensitivity of normal cells to alpha-amanitin can be reduced by growing the cells at low cell density but the resistance of RSV transformed cells is not significantly altered by changes in cell density. The results suggest that the resistance of RSV transformed cells may be related to altered control of density dependent contact-inhibition of the transformed cells.

Amanitins↗

PubMed1977

Cytochrome C as antidote in mice poisoned with the mushroom toxin alpha-amanitin.

The curative potencies of cytochrome c applied 8 hours after lethal doses of alpha-amanitin in mice are confirmed. In a comprehensive series of experiments, various dosage schedules were explored. Optimal effects with cure rates approaching 100% were obtained either with repeated doses of cytochrome c or by combining it with penicillin. Male mice are more sensitive to alpha-amanitin than female mice but it was established that the antidote is effective in both sexes. Substantial cure rates are seen even when the treatment after supralethal doses of alpha-amanitin is withheld for 12 hours after the poisoning.

Amanita↗

PubMed1979

alpha-Amanitin and 5-fluorouridine inhibition of serum-stimulated DNA synthesis in quiescent AKR-2B mouse embryo cells.

AKR-2B mouse embryo cells undergoing the serum-stimulated transition from a quiescent to a proliferating state exhibit an increase in the rate of hnRNA synthesis which appears to be mediated through an increase in the actual number of RNA polymerase II molecules. alpha-Amanitin, administered early in the prereplication interval following stimulation, effectively inhibits hnRNA synthesis, polysomal mRNA accumulation, polyribosome formation, and subsequent DNA synthesis, and cell division. Unexpectedly, alpha-amanitin treatment also produces almost complete inhibition of the synthesis of 45S rRNA precursor and the increase in accumulation of cytoplasmic rRNA following serum stimulation. In order to determine whether the inhibition of new ribosomal synthesis might in itself be sufficient to prevent serum-stimulated DNA synthesis, the effects of 5-fluorouridine (5-FU), a specific inhibitor of 45S rRNA processing, were investigated. If added within eight hours following serum stimulation, 5-FU was found to completely inhibit subsequent DNA synthesis. These results suggest that quiescent AKR-2B cells do not contain a sufficient excess of ribosomes to support the synthesis of proteins which are required for DNA synthesis in response to serum growth factors. Furthermore, an early polymerase II mediated synthesis of mRNA(s) coding for some factor(s) necessary for ribosomal gene transcription may be an essential step in the serum-stimulated synthesis of new ribosomes.

Amanitins↗

PubMed1979

alpha-Amanitin: inactivation by bovine lactoperoxidase.

The principle amatoxin, alpha-amanitin, is found to be extremely sensitive toward lactoperoxidase catalyzed degradation, rather than iodination, of the indole nucleus. Extensive attenuation of inhibitor potency against eukaryotic DNA-dependent RNA polymerase II accompanies the treatment of alpha-amanitin with lactoperoxidase, iodide and hydrogen peroxide.

Amanitins↗

PubMed1978

The organization, composition and matrix of hepatocyte nuclei exposed to alpha-amanitin.

Alterations in the structure and molecular composition of avian hepatocyte nuclei were compared following administration in vivo of lethal and sub-lethal doses of alpha-amanitin. This toxin interferes with extranucleolar transcription by direct inhibition of RNA polymerase II activity. the resultant effects include: extensive condensation of chromatin, displacement of nucleoplasmic contents and fragmentation of nucleoli. Changes in nuclear morphology were quantitated by stereometry and related to variations in RNA and residual, non-histone proteins (NHP). Gross alterations in nuclear structure and depletion of RNA and NHP levels were of similar magnitude with both doses of amanitin. The effects were fully reversible, however, with a minimal dose but terminal with a lethal dose. DNA and histone protein levels remained unchanged at all stages. These results imply that the process of transciption may itself keep and/or maintain chromatin in a dispersed state, and that in the absence of transcription chromatin naturally condenses. Modification of nuclear proteins may be necessary only to maintain chromatin compacted permanently or for extended periods of time. A model of nuclear organization is proposed to incorporate these considerations and to identify the probable location of the nuclear matrix in situ.

Amanitins↗

PubMed1976

Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA.

Studies were made on the effects of alpha-amanitin, cycloheximide, and thioacetamide on synthesis and content of low molecular weight nuclear RNA. Cycloheximide, an inhibitor of protein synthesis and the synthesis of 45S pre-rRNA and 5S RNA, also inhibited synthesis of nuclear U1 and U3 RNAs. alpha-Amanitin, an inhibited the synthesis of U1 and U2 low molecular weight nuclear RNA. Thioacetamide, which induces nucleolar hypertrophy and increased nucleolar RNA polymerase activity, markedly increased synthesis of 5.8S RNA and U3 RNA. These results show that syntheses of individual low molecular weight nuclear (LMWN) RNAs are controlled by different regulatory mechanisms. In particular, there appears to be a specific relationship between U3 RNA and functional states of the nucleolus.

Acetamides↗

PubMed1979

The action of silybin on the mouse liver in alpha-amanitine poisoning.

Histochemical and histoenzymological studies were carried out on liver slices from mice which had received alpha-amanitine two days previously, some of which had been treated with silybin, and from control mice. The toxin produced certain changes in the activity of the enzymes involved in different metabolic processes, and in the amounts of lipids and nucleic acids. Treatment with silybin, given 60 min before administering alpha-amanitine or 10 min later alike, prevents the appearance of these changes and gives results comparable to those in the control animals.

Amanitins↗

PubMed1975

Hamster alpha-amanitine-resistant RNA polymerase II able to transcribe polyoma virus genome in somatic cell hybrids.

A hamster cell line resistant to alpha-amanitine has been isolated (alpha-am-r, BHK-T6-G-1). Cell extracts of this mutant have an alpha-amanitine-resistant RNA polymerase II (nucleosidetriphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6) activity as shown by DEAE-cellulose column chromatography. This mutation is dominant in interspecific hybrids with 3T3 mouse cells. In such hybrids polyoma virus can grow with equal efficiency in the presence or absence of the drug, thus indicating that the RNA polymerase of the unsusceptible parental cell can participate in the correct transcription of the viral genome.

Animals↗

PubMed1978

Ultrastructural changes in beta-cells of pancreatic islets in alpha-amanitin-poisoned mice.

In mice poisoned by alpha-amanitin nuclear changes typical of this toxin were observed in beta-cells of pancreatic islets. The lesions became progressively more severe and at 48 h after toxin injection some cells were necrotic. The damage to these cells could have implications in the changes in glycogen metabolism which occur after alpha-aminitin poisoning.

Amanitins↗

PubMed1979

Nuclear accumulation of influenza viral RNA transcripts and the effects of cycloheximide, actinomycin D, and alpha-amanitin.

The use of virus-specific (32)P-labeled complementary DNA and (125)I-labeled virion RNA as hybridization probes has allowed us to quantitate the number of molecules of complementary RNA (cRNA) and progeny virion RNA in MDCK cells infected with influenza virus. We compared the distribution of cRNA between the nucleus and the cytoplasm in cycloheximide-treated cells to that found in untreated cells, beginning 1 h after infection. A greater percentage of the total cRNA was detected in the nucleus of the drug-treated cells at all times investigated. For the first 2 h after infection about 50% of the cRNA synthesized in the cycloheximide-treated cells was found in the nucleus. These nuclear cRNA molecules were characterized and shown to be polyadenylated transcripts of each of the genome virion RNA segments. Viral cRNA synthesis was not completely inhibited by the addition of actinomycin D at the beginning of infection, with or without the concomitant addition of cycloheximide. A large fraction (about 90%) of these cRNA sequences were detected in the nucleus. Characterization of these nuclear cRNA molecules showed that they contained polyadenylic acid and represented transcripts of both those segments coding for proteins synthesized predominantly early after infection ("early" proteins) and those virion RNA segments coding for "late" proteins. Also, in vitro translation of these cRNA molecules showed that they were functional virus mRNA's. In contrast to actinomycin D, alpha-amanitin completely inhibited cRNA synthesis when added at the beginning of infection, and addition of this drug after 1.5 h had no effect on further cRNA synthesis.

Amanitins↗

PubMed1975

Early stimulation of human chorionic gonadotropin secretion by dibutyryl cyclic AMP and theophylline in human malignant trophoblast cells in vitro: inhibition by actinomycin D, alpha-amanitin, and cordycepin.

Human malignant trophoblast cells (BeWo line) in culture were employed to investigate the early stimulation of human chorionic gonadotropin (hCG) secretion by 1 mM dibutyryl cyclic AMP and 1 mM theophylline (dbT). The earliest increase in secreted immunoreactive hCG occurred at 3 1/2 h following addition of dbT, and was preceded by an increase in intracellular hCG. These results suggested that dbT stimulated hCG synthesis, rather than release. Addition of either 0.062 mug/ml actinomycin D or 100 mug/ml cordycepin along with dbT, or 3 mug/ml alpha-amanitin 9 h prior to addition of dbT, prevented the increase in hCG secretion at 3 1/2-8 h. It was concluded that synthesis of RNA (possibly messenger RNA) is required for the early stimulation of hCG secretion by dbT.

Amanitins↗

PubMed1977

[Effects of alpha-amanitin on the development of Xenopus (Xenopus laevis Daud.) heart in vitro].

The differentiation of Xenopus heart is studied in vitro, in the presence of alpha-amanitin. The results obtained depend on the concentration of the inhibitor, the length of treatment and the stage of primordium. The mRNA pool assures the differentiation of explants, removed from young stages, for 12 hours. This time is half for the primordia removed from stages greater than 3,5 mm.

Amanitins↗

PubMed1979

[Effect of administration of alpha-amanitine on Mn2+ - dependent poly-A-polymerase in rat liver nuclei].

Mn2+-dependent poly(A) polymerase activity tested in isolated rat liver nuclei is unchanged both in the absence and presence of exogenous poly(A) 30 min. after administration of a dose of alpha-amanitin that inhibits DNA-dependent RNA polymerase B. Longer times of treatment cause poly(A) polymerase to drop to 50% in the absence of poly(A) and to increase almost twice in its presence.

Amanitins↗