Tumor therapy with Amanita phalloides (death cap): stabilization of B-cell chronic lymphatic leukemia.

BACKGROUND: Molecular events that cause tumor formation upregulate a number of HOX genes, called switch genes, coding for RNA polymerase II transcription factors. Thus, in tumor cells, RNA polymerase II is more active than in other somatic cells. Amanita phalloides contains amanitin, inhibiting RNA polymerase II. Partial inhibition with amanitin influences tumor cell--but not normal cell--activity. OBJECTIVES: To widen the treatment spectrum, homeopathic dilutions of Amanita phalloides, containing amanitin, were given to a patient with leukemia. Monitoring the leukemic cell count, different doses of amanitin were given. RESULTS: The former duplication time of leukemic cells was 21 months. Within a period of 21 months, the cell count is stabilized to around 10(5)/µL. No leukemia-associated symptoms, liver damage, or continuous erythrocyte deprivation occur. CONCLUSIONS: This new principle of tumor therapy shows high potential to provide a gentle medical treatment.

Genes associated with opening and senescence of Mirabilis jalapa flowers.

A modest ethylene climacteric accompanies flower senescence in Mirabilis jalapa L., and exogenous ethylene accelerates the process. However, inhibitors of ethylene action and synthesis have little effect on the life-span of these ephemeral flowers. Treatment with alpha-amanitin, an inhibitor of DNA-dependent RNA synthesis, substantially delays the onset of senescence. This effect falls linearly between 7 h and 8 h after the start of flower opening. Subtractive hybridization was used to isolate transcripts that were up- and down-regulated during this critical period. Eighty-two up-regulated and 65 down-regulated transcripts were isolated. The genes identified encode homologues of a range of transcription factors, and of proteins involved in protein turnover and degradation. Real-time quantitative RT-PCR was used to examine expression patterns of these genes during flower opening and senescence. Genes that were identified as being down-regulated during senescence showed a common pattern of very high expression during floral opening. These genes included a homologue of CCA1, a 'clock' gene identified in Arabidopsis thaliana and an aspartyl protease. Up-regulated genes commonly showed a pattern of increase during the critical period (4-9 h after opening), and some showed very strong up-regulation. For example, the abundance of transcripts encoding a RING zinc finger protein increased >40 000 fold during the critical period.

GABA induces activity dependent delayed-onset uncoupling of GABA/benzodiazepine site interactions in neocortical neurons.

Changes in the function of type A gamma-aminobutyric acid receptors (GABA(A)Rs) are associated with neuronal development and tolerance to the sedative-hypnotic effects of GABA(A)R positive modulators. Persistent activation of GABA(A)Rs by millimolar concentrations of GABA occurs under physiological conditions as GABAergic fast-spiking neurons in neocortex and cerebellum exhibit basal firing rates of 5 to 50 Hz and intermittent rates up to 250 Hz, leaving a substantial fraction of synaptic receptors occupied persistently by GABA. Persistent exposure of neurons to GABA has been shown to cause a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine (BZD) site interactions with a half-life of approximately 24 h. Here, we report that a single brief exposure of neocortical neurons in primary culture to GABA for 5-10 min (t(1/2) = 3.2 +/- 0.2 min) initiates a process that results in uncoupling hours later (t(1/2) = 12.1 +/- 2.2 h). Initiation of delayed-onset uncoupling is blocked by co-incubation with picrotoxin or alpha-amanitin but is insensitive to nifedipine, indicating that uncoupling is contingent upon receptor activation and transcription but is not dependent on voltage-gated Ca2+ influx. Delayed-onset uncoupling occurs without a change in receptor number or a change in the proportion of alpha1 subunit pharmacology, as zolpidem binding affinity is unaltered. Such activity dependent latent modulation of GABA(A)R function that manifests as delayed-onset uncoupling may be relevant to physiological, pathophysiological, and pharmacological conditions where synaptic receptors are transiently exposed to GABA agonists for several minutes.

The toxicology of Amanita virosa: the destroying angel.

This paper examines the biology and medical consequences of ingesting the potentially lethal poisonous mushroom, Amanita virosa, the Destroying Angel. The fungus, its structure, distribution and toxic components are described. Symptoms of human poisoning by A. virosa are described, following the order of Homeopathic Repertories. Laboratory values for comparison with normal values of haematology, biochemistry and urine analyses are given.

In vitro high-throughput screening assay for modulators of transcription.

We developed a 96-well microtiter-plate high-throughput screening (HTS) assay for the detection of modulators of transcription. This HTS assay consists of three steps: (1) the in vitro transcription reaction; (2) modification and hybridization of RNA products; and (3) washing and quantification. During the first step, a DNA template containing the promoter of interest upstream of a cassette lacking guanosine residues in one of its strands (G-less cassette) is incubated with nuclear extract and the necessary cofactors/activators and substrates. During the second step, the in vitro synthesized transcripts are digested with RNase T1 and hybridized to two DNA oligonucleotides. One oligonucleotide is biotinylated for trapping of the RNA products to a streptavidin-coated plate, and the other is europium-labeled for detection by time-resolved fluorescence. We show that this assay is highly reproducible and robust, yielding results comparable to those obtained by standard methodologies employing radioactive nucleotide incorporation and gel electrophoresis while offering a very significant advantage in terms of throughput (>2000 assay points per operator per day). We demonstrate the usefulness of the assay for the discovery of small molecule inhibitors of transcription, and applications of this approach for the high-throughput discovery of transcriptional modulators are discussed.

Effect of amino acids and alpha-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES.

Four experiments were conducted to test the effects of Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor, alpha-amanitin, on the development of preimplantation rabbit embryos in modified protein-free KSOM medium. Embryos were distributed randomly into different treatments and cultured in 5% O2:5% CO2:90% N2. In experiment 1, 100% of the embryos became blastocysts in the medium with Eagle's 1X NEAA and 0.5X EAA, but 100% stopped development at the morula stage in KSOM without amino acids. These morulae failed to develop further when transferred to amino acid supplemented medium after 72 hr of culture. Glycine alone in modified KSOM (experiment 2) was ineffective in supporting development of 8-16-cell stage embryos past the morula stage. In experiment 3, the addition of 1X NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resulted, respectively, in 57, 65, 65, 44, and 14% blastocysts on Day 3 (P < 0.05) and 86, 77, 77, 78, and 69% on Day 5 (P > 0.05). Omission of Eagle's amino acids until 48 hr clearly delayed embryo development. In experiment 4, when alpha-amanitin (20 microM) was added to the medium containing Eagle's amino acids after 0, 12, 24, 36, and 48 hr of culture most embryos cleaved only once or twice after adding the alpha-amanitin. Without the inhibitor, 94% of the zygotes developed into blastocysts. These results indicate that modified KSOM or KSOM plus glycine could not support rabbit embryo development past the morula stage, but this block was overcome by adding Eagle's amino acids. An exogenous source of amino acids was not critical for embryo development during the first 24 hr of culture, but was required after that for development to equal controls. Addition of alpha-amanitin at multiple pre-blastocyst stages limited further embryo development to one or two cleavage divisions, with no blastocyst development.

The regulation by iron of the synthesis of adhesins and cytoadherence levels in the protozoan Trichomonas vaginalis.

Levels of adherence of Trichomonas vaginalis to epithelial cells was found to be modulated by iron. Cytoadherence values were greater than or equal to twofold higher for trichomonads grown in a complex cultivation medium supplemented with iron. This increase in adherence levels was specifically mediated by iron; parasites cultured in a low-iron medium in the presence of salts other than iron were unresponsive to changes in adherence levels. Expression of the higher adherence property, by parasites grown first in low-iron medium followed by supplementation with iron, was a function of time, and the extent of cytoadherence was proportional to the concentration of iron added to the medium. Lactoferrin, an important iron source for trichomonads at the site of infection, elevated adherence of the parasite to epithelial cells, demonstrating the likely in vivo modulation of adherence by iron. The alteration of levels of adherence caused by iron was determined to be a reflection of gene expression of previously characterized trichomonad adhesins. Parasites grown under iron-replete conditions had higher quantities of surface-exposed adhesins, and this was a result of increased synthesis of adhesins. Actinomycin D and alpha-amanitin prevented expression of adhesin molecules, which resulted in decreased cytoadherence, showing that adhesin synthesis was dependent on gene transcription. Data indicated that genes encoding the four trichomonad adhesins are coordinately regulated by iron.

In vivo effects of transcriptional and translational inhibitors on duodenal vitamin D-dependent calcium-binding protein messenger ribonucleic acid stimulation by 1,25-dihydroxycholecalciferol.

The in vivo stimulation of vitamin D-dependent calcium-binding protein (9 K CaBP) synthesis by 1,25-dihydroxycholecalciferol [1,25(OH)2D3] in the rat duodenum has been analyzed using a specific [32P]complementary DNA probe for rat 9 K CaBP and inhibitors of RNA transcription (actinomycin D, alpha-amanitin) or protein synthesis (cycloheximide). The relative amounts of 9 K CaBP messenger RNA (mRNA) were assayed by dot-blot hybridization and the relative amounts of 9 K CaBP by RIA. Both inhibitors were injected at doses which significantly inhibited by 80-95% [35S]methionine or [3H]uridine incorporation into protein and RNA, respectively. In vitamin D-deficient rats, a single 1,25(OH)2D3 injection (650 pmol/100 g BW) resulted in a rapid production of 9 K CaBP mRNA which was significantly detectable as early as 3 h, and was followed by an increase of 9 K CaBP levels. Injection of actinomycin D (25 micrograms/100 g BW) 1 h before 1,25(OH)2D3 treatment and repeated every 4 h did not prevent the hormone-induced elevation of duodenal CaBP mRNA, even when the actinomycin dose was doubled and given 2 h before hormonal treatment. alpha-Amanitin (2 micrograms/100 g BW) also failed to block the hormonal stimulation. The protein synthesis inhibitor cycloheximide (25 micrograms/100 g BW) did not cause any change in the 1,25(OH)2D3-induced CaBP mRNA but blocked the CaBP increase after hormone injection. Thus, transcription inhibitors did not prevent the in vivo hormone-induced elevation of 9 K CaBP mRNA, which suggests that 1,25(OH)2D3 increases 9 K CaBP synthesis by increasing 9 K CaBP gene expression at one or more posttranscriptional steps. More precise data will be obtained by measuring the rate of 9 K CaBP gene transcription on isolated nuclei from rat duodenum.

Rapid induction of specific mRNAs by auxin in pea epicotyl tissue.

DNA sequences complementary to three indoleacetic acid (IAA)-inducible mRNAs in pea epicotyl tissue were isolated by differential plaque filter hybridization of cDNA libraries constructed in the vector lambda gt10. Clone pIAA6 hybridized to an mRNA encoding the previously identified translational product polypeptide 6 (Mr 22,000), and clone pIAA4/5 hybridized to one or two mRNAs, encoding polypeptides 4 and 5 (Mr 23,000 and 25,000, respectively). The cDNA clones were subsequently used to characterize the hormonally mediated mRNA accumulation. The induction of the mRNAs was rapid, within 15 minutes of exposure to the IAA, and specific to auxins. Anaerobiosis, heat and cold stress did not induce the mRNAs. Other plant hormones, such as gibberellic acid, kinetin, abscisic acid and ethylene were also unable to cause or interfere with the IAA-induced mRNA accumulation. The hormonally regulated mRNAs were induced at least 50 to 100-fold above control levels after two hours of treatment with IAA and the accumulation was (1) independent of protein synthesis, (2) completely abolished by alpha-amanitin, (3) not due to polyadenylylation of pre-existing RNAs, and (4) independent of IAA and fusicoccin-induced H+ secretion. The IAA-induced mRNAs returned to control levels within three hours after removal of IAA, and the hormonally regulated genes were primarily expressed in the third and second internode of the seven-day-old etiolated pea seedling. The data indicate that IAA increases the amount of specific mRNAs rather than alters the translatability of pre-existing mRNAs. Auxin-induced H+ secretion appears not to have a potential role in mediating the induction and perhaps is a consequence of the enhanced biosynthetic activity induced by the hormone. The IAA-mediated mRNA induction is the fastest known for any plant growth regulator and may represent a primary hormonal response to auxin.

In vitro transcription of a cloned vaccinia virus gene by a soluble extract prepared from vaccinia virus-infected HeLa cells.

Faithful transcription of a vaccinia virus gene was accomplished in vitro by using a soluble extract prepared from vaccinia virus-infected HeLa cells. Specific transcription of the cloned vaccinia virus gene was detected by using template DNA restricted within the transcribed region. The vaccinia virus gene was not transcribed by extracts prepared from uninfected HeLa cells even with supplementation by purified vaccinia virus RNA polymerase, nor was a clone of adenovirus 2 DNA bearing the major late promoter transcribed by the extract from vaccinia virus-infected HeLa cells. Thus, infection by vaccinia virus altered cellular transcriptional specificity to favor expression of vaccinia virus genes. RNA synthesis by the infected cell extract was resistant to alpha-amanitin but strongly inhibited by beta, gamma-imido ATP and novobiocin.

[Synthesis of RNA and DNA in isolated nuclei of Colpoda cucullus (Protozoi Ciliati)]. / Sintesi di RNA e DNA nei nuclei isolati di Colpoda cucullus (Protozoi Ciliati).

The ciliated protozoan Colpoda cucullus has been cultivated at 27 degrees C with gentle shaking in a baked lettuce infusion supplemented with Klebsiella suspensions. Under these conditions cells had a mean generation time of about 7 hours and could attain densities up to 20,000/ml and 45,000/ml in the log and stationary phase of growth, respectively. Nuclear preparations obtained from exponentially growing cells by the gum arabic-octanol method showed a satisfactory degree of purity and integrity. They consisted primarily of the large macronuclei attached to which the small micronuclei were sometimes visible. Upon incubation at 27 degrees C in conventional reaction mixtures nuclear preparations actively incorporated 3H-UTP and 3H-dTTP into acid-insoluble material. alpha-amanitin caused a 50% inhibition of RNA synthesis whereas aphidicolin did not affect at all DNA synthesis.

Nuclear ligation of RNA 5'-OH kinase products in tRNA.

Mouse L-cell nuclei incorporate gamma-32P from ATP in vitro predominantly in 5'-monophosphoryl termini and internal phosphodiester bonds with a nonrandom nearest-neighbor distribution. In the presence of 1 microgram of alpha-amanitin per ml the gamma-32P showed a time-dependent appearance in RNA bands which migrated with mature tRNA species but not with pre-tRNA and 5S RNA. The gamma-32P was found in internal phosphodiester bonds as shown by alkaline phosphatase resistance and was identified in 3'-monophosphates after RNase T2, T1, and A digestion. The specificity of this incorporation was indicated by a limited number of labeled oligonucleotides from a T1 digest and identification of 70 to 80% of the 32P label as Cp on complete digestion of the eluted tRNA band. We also observed transiently [gamma-32P]ATP-labeled RNA bands (in 5'-monophosphate positions) that were 32 to 45 nucleotides long. The results presented suggest splicing of several mouse L-cell tRNA species in isolated nuclei which involve the RNA 5'-OH kinase products as intermediates.

Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor.

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.

In vitro RNA synthesis and expression of vitellogenin gene in isolated chicken liver nuclei.

Optimal conditions for prolonged in vitro synthesis of RNA in isolated chicken liver nuclei have been described. It is shown by incorporation of gamma32P-GTP into RNA, analysis of the product on sucrose density gradient, and digestion with alkaline phosphatase and ribonuclease A that there is reinitiation of RNA synthesis. Polynucleotide kinase activity has been ruled out as explanation for the incorporation of gamma32P-GTP. alpha-Amanitin inhibits RNA synthesis by about 50%. Nuclei prepared from estradiol-treated chicks have twice the RNA synthesis activity as the controls. RNA is synthesized in the presence of Hg-UTP and the mercurated product separated by affinity chromatography on sulfhydryl-Sepharose column under stringent conditions. Vitellogenin mRNA sequences are measured by hybridization with DNA complementary to vitellogenin mRNA. Estradiol treatment leads to a 10-fold increase in vitellogenin mRNA sequences.

On RNA-polymerases of leukemia L 1210 origin and an enzymatic method to screen antitumor antibiotics.

Four DNA-dependent RNA-polymerases were separated from the cell homogenate of moust leukemia L1210 cell by DEAE-cellulose column chromatography and tentatively designated as Peaks I, II, III and IV in the elution order. Peak II was inactivated by the addition of alpha-amanitin and effects of antibiotics and enzymes on the RNA-polymerase activity using Peaks, I, II and a mixture of Peaks I and II were examined. The RNA-polymerases were used to screen for enzyme inhibitors produced by microbes. This enzymatic method was successfully proved to select antitumor antibiotics.

Hypothalamic superfusion with muscarinic drugs: their effects on pressor responses to hypothalamic stimulation.

The posterior hypothalamus of cats anaesthetized with pentobarbital sodium was superfused with artificial cerebrospinal fluid through a push-pull cannula and electrically stimulated with the noninsulated tip of the cannula. The effects of muscarinic drugs on the pressor response to stimulation of the hypothalamus were investigated. Superfusion with muscarine, oxotremorine or N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602) decreased the pressor responses to hypothalamic stimulation. Superfusion with methylatropine did not influence the pressor responses to hypothalamic stimulation; however, superfusion with methylatropine 60 min prior to and during superfusion with the muscarinic drugs abolished the inhibitory effects of muscarine and oxotremorine and temporarily reversed that of AHR 602 on the pressor responses. Superfusion of the posterior hypothalamus with arecoline enhanced the rise of blood pressure elicited by hypothalamic stimulation. When the hypothalamus was superfused with hexamethonium 60 min prior to and during superfusion with arecoline, arecoline reduced the pressor responses to electrical stimulation of the hypothalamus. Superfusion with methylatropine prior to and together with an ineffective concentration of arecoline increased the rise of blood pressure elicited by hypothalamic stimulation. From the drugs studied here only oxotremorine caused a fall of the "resting" arterial blood pressure; it was abolished by the intravenous injection of methylatropine. From these results it was concluded that superfusion of the posterior hypothalamus with muscarinic drugs impairs the pressor responses to hypothalamic stimulation. Drugs possessing both nicotinic and muscarinic properties either enhance or diminish the pressor responses according to their relative potencies on the two types of receptor.

Toxic effects of phallolysin from amanita phalloides.

Phallolysin, a protein from Amanita phalloides with cytolytic effects in vitro, was highly toxic when given intravenously to rats, mice, rabbits and guinea pigs: i.v. LD50 in rats was 85 Haemolytic Units (HU)/kg, corresponding to 0.05 mg protein/kg b.w. Death ensued from intravascular haemolysis. In rats large doses (600 HU/kg b.w.) caused cardiac death within a few minutes due to liberation of potassium from lysed cells. The serum contained lethal concentrations of potassium. There was also histological evidence of severe renal damage as a result of the haemolysis. In addition, phallolysin directly damaged the isolated guinea pig heart and the isolated rat liver, probably by its action on membranes. Given by mouth, phallolysin was not poisonous to rats.

Quantitation of amanitins in Amanita verna with calf thymus RNA polymerase B.

A procedure utilizing the specific inhibition of calf thymus DNA-directed RNA polymerase B has been applied to the quantitation of amanitins. This procedure has permitted the accurate quantitation of alpha-amanitin in amounts as low as 0.05 nanogram, a sensitivity 2000-fold greater than chemical detection methods used following tlc. Analysis of extracts of specimens of Amanita verna identified by morphological criteria has demonstrated that while toxin concentration is variable, some specimens are practically devoid of amanitins and may represent a variety of A. verna or a distinct species.