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RNA synthesis in isolated bovine thyroid nuclei and nucleoli. alpha-Amanitin effect, a hint to the existence of a specific regulatory system.

DNA dependent RNA polymerase activities in isolated bovine thyroid nuclei and nucleoli have been studied. They retain their RNA synthetic activity for an extended period of time. This RNA synthetic activity is sensitive to actinomycin D and requires the presence of all four ribonucleoside triphosphates. The optimal conditions have been determined. Polyacrylamide gel electrophoresis reveals that the RNA synthesized has a size distribution ranging from 34S to 4S. The production of 18S-8S RNA is very sensitive to low concentrations of alpha-amanitin. However, in isolated bovine thyroid nuclei (not in nucleoli) this drug displays an effect on all RNA classes produced. The alpha-amanitin induced drastic decrease of [3H]-UMP incorporation in RNA of all sizes synthesized by isolated bovine thyroid nuclei is discussed.

Amanitins

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

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

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

Synthesis of influenza virus polypeptides in cells resistant to alpha-amanitin: evidence for the involvement of cellular RNA polymerase II in virus replication.

Influenza virus polypeptides were not synthesized in wild-type CHO-S-infected cells in the presence of alpha-amanitin, but were synthesized in CHO-Amal cells, a mutant cell line whose DNA-dependent RNA polymerase II is specifically resistant to this drug, indicating that this cellular enzyme is involved in influenza virus replication. The results of experiments designed to detect viral polypeptides synthesized from primary transcripts suggest that the synthesis of a cellular RNA species by RNA polymerase II is required for primary transcription of the influenza virus genome.

Amanitins

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

Inhibition of influenza C virus replication by actinomycin D, alpha-amanitin, and UV irradiation.

Actinomycin D and alpha-amanitin caused similar reductions in the yields of influenza A/WSN and influenza C/JBH/1/66 viruses in a chicken kidney cell culture system. Irradiation of host cells with UV light before virus infection also produced a similar reduction in yields of the two viruses. The results indicate a close similarity between the replication processes of influenza C and other orthomyxoviruses.

Amanitins

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

[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

[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

Normal versus alpha-amanitin induced cellular dynamics of the midgut epithelium in female Aedes aegypti L. (Insecta, Diptera) in response to blood feeding.

In midgut epithelial cells (stomach) of untreated female A. aegypti an increase in the surface area of the rough endoplasmic reticulum (rer) and in the ratio of membrane-bound to free ribosomes is morphometrically measured during digestion of the first blood meal. This can be correlated with the synthesis and release of digestive proteases. The dynamics of the ribosomes in A. aegypti are similar to those in A. stephensi. 3 ng alpha-amanitin per mosquito prevent normal blood digestion, the proliferation of the rer and the increase in the ratio of bound to free ribosomes. On the other hand, some synthesis of new ribosomes takes place.

Aedes