Characterization of an α-amanitin insensitive RNA polymerase from cauliflower

α-Amanitin insensitive RNA polymerase (polymerase I isolated from apical parts of the cauliflower inflorescence was highly stable for several months at − 18°. The DEAE-cellulose fraction was more effective in utilizing denatured DNA than native DNA as a template. Optimum pH for RNA synthesis was ca 7 in the reaction mixture with Tris-HCl or with Tris-maleate buffer. From the properties examined, it seems that DNA-dependent RNA polymerase I of cauliflower differs from other eucaryotic RNA polymerases.     

Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1. Injection of alpha-amanitin to mice causes a decreased incorporation of [6-(14)C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn(2+) and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with alpha-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg(2+)-activated RNA polymerase is only slightly affected by alpha-amanitin either administered to mice or added in vitro.     

Changes in the patterns of synthesis of ribonucleic acid species in immature rat uterus in response to oestradiol-17β

1. After treatment of immature rats with diethylstilboestrol, the wet weight and RNA content of uterine tissue increased rapidly, reaching a peak at 40hr. After an initial lag of a few hours, the acid-soluble ribose and protein contents also rose to maxima at 40hr. No increase in DNA content occurred until at least 24hr. after treatment. 2. The RNA from immature rat uterus isolated at various times up to 6hr. after administration of oestradiol-17beta was labelled by injecting [(3)H]uridine and [(3)H]guanosine intraperitoneally 30min. before the animals were killed. It was fractionated on columns of kieselguhr coated with methylated serum albumin and the radioactivity in fractions corresponding to transfer RNA, 7s RNA, ribosomal RNA, Q(1)-RNA, Q(2)-RNA and DNA-like RNA was determined. 3. The radioactivity of the whole RNA increased steadily for 6hr. after hormone treatment. The earliest changes occurred in the Q(1)-RNA (ribosomal RNA precursor), whereas at longer time-intervals the radioactivity of the ribosomal RNA, 7s RNA and transfer RNA increased by four- to five-fold. The radioactivity of the DNA-like RNA increased by about 50%, but only at the longer time-intervals. 4. It is concluded that one of the earliest changes in response to oestradiol treatment is a major increase in synthesis of ribosomal RNA followed later by a similar increase in synthesis of transfer RNA and by a much smaller increase in synthesis of DNA-like RNA. The change in synthesis of ribosomal RNA in immature rat uterus may represent one of the most important responses to oestradiol treatment.     

The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17beta action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3mug or more of oestradiol-17beta. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2-2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17beta is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA.     

Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of alpha-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg(2+), Mn(2+) and (NH(4))(2)SO(4). A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of alpha-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the alpha-amanitin-sensitive RNA polymerase was also 50-100-fold more sensitive to exotoxin inhibition than was the alpha-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase.     

Inhibition of DNA polymerase α by gossypol

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase α (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase α with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase α does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase β in a dose-dependent manner, but had no effect on DNA polymerase γ. These results suggest that inhibition of DNA polymerase α may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.     

Induction of chromosome puffs in Drosophila hydei salivary glands after inhibition of RNA synthesis by α-amanitin

The effect of injection of various concentrations (4 ng to 0.5 g/larva) of -amanitin on chromosomal RNA synthesis in larval salivary glands of D. hydei was investigated at subsequent time intervals (90 min to 20 hrs) after injection. — As revealed by autoradiography, ³H-uridine incorporation into the polytene chromosomes was greatly reduced as compared with that in control larvae. In -amanitin injected larvae, new chromosome puffs could be induced by a temperature treatment. These puffs never attained a size comparable to that in the controls. The newly induced puffs did not show specific incorporation of ³H-uridine. — Puffs which were active before the toxin was applied undergo a reduction in size and incorporation of ³H-uridine.     

DNA-dependent in vitro synthesis of ribosomal proteins,protein elongation factors,and RNA polymerase subunit α: Inhibition by ppGpp

We have previously shown that the synthesis of ribosomal proteins (r proteins) in E. coli cells is under stringent control (Dennis and Nomura, 1974). Since guanosine tetraphosphate (ppGpp) had been implicated in stringent control, we examied the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage λfus3 and λrif^d18. λfus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit α, in addition to genes for approximately 27 r proteins. λrif^d18 carries genes for EF-Tu, RNA polymerase subunits β and β^I, and a set of rRNAs, in addition to genes for approximately five r proteins. We have shown that low concentrations of ppGpp (0.2–0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp. In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit α, as well as rRNAs.     

Ribosomal biosynthesis of α-amanitin in Galerina marginata

Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerinamarginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerinahybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G.marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary relationship between AMA1 in the genera Amanita and Galerina is unclear.     

Inhibition mechanism of rat α₃β₄ nicotinic acetylcholine receptor by the Alzheimer therapeutic tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer's disease, on currents elicited by activation of rat α(3)β(4) nAChR heterologously expressed in KXα3β4R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 ± 0.2 μM and 4.3 ± 1.3 for the channel opening equilibrium constant, Φ(-1). Experiments were performed to investigate whether tacrine is able to activate the α(3)β(4) nAChR. Tacrine did not activate whole-cell currents in KXα3β4R2 cells but inhibited receptor activity at submicromolar concentration. Dose-response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 μM. The increase of Φ(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.     

Spermine synthesis in the uterus of the ovariectomized rat in response to oestradiol-17β

We reported that spermidine and spermine pools in the uterus both doubled within 24h after oestradiol administration to castrated rats (Russell & Taylor, 1971). Now we have studied the enzymic synthesis of spermine (by spermidine-dependent S-adenosyl-l-methionine decarboxylase) and find that the activity of the enzyme(s) involved is elevated soon after hormone administration. Enzyme activity is increased within 4h and is five times that of controls within 24h. Cycloheximide or actinomycin D administered at the time of oestradiol injection completely blocked the increase in enzyme activity. The enzyme involved in spermine synthesis, S-adenosyl-l-methionine decarboxylase, with S-adenosyl-l-methionine and spermidine as required substrates, was partially purified on Sephadex and DEAE-cellulose columns. The decarboxylation of S-adenosyl-l-methionine could not be separated from the transfer of a propylamine moiety from the decarboxylated S-adenosyl-l-methionine to spermidine to form spermine. We were unable also to separate this system from the enzyme that formed spermidine when S-adenosyl-l-methionine and putrescine are used as substrates. Spermidine-stimulated S-adenosyl-l-methionine decarboxylase has an apparent half-life of 60min, identical with the half-life reported for putrescine-stimulated S-adenosyl-l-methionine decarboxylase. These results strongly suggest that the same enzyme(s) operate in the synthesis of both spermidine and spermine.     

Regulation of RNA polymerase II activity in α-amanitin-resistant CHO hybrid cells

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to α-amanitin with mutant cells containing RNA polymerase II activity resistant to α-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing α-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to α-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the α-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975).A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in α-amanitin exhibited a 2–3 fold increase in the activity of the α-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the α-amanitin-inactivated polymerase II of wild-type sensitivity by ³H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of ³H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.     

The long term effect of α-amanitin on RNA synthesis in adult female Aedes aegypti

The effect of the eukaryotic RNA polymerase inhibitor α-amanitin on RNA synthesis in adult female Aedes aegypti was studied. Administration of the toxin 24 hr, prior to a 2 hr pulse of radioactive precursors inhibited incorporation of the label into total RNA by approximately 40%. Nuclear RNA and microsomal RNA showed 25% and 15% less radioactivity, respectively, than the saline injected controls. Total soluble 4S RNA was unaffected by α-amanitin but heterodisperse RNA incorporated 50% less label than controls. Using both nitrocellulose membrane filters and poly(U)-sepharose chromatography it was found that the synthesis of poly(A)-containing microsomal RNA was severely inhibited by the toxin. It was concluded that the major long term effect of α-amanitin in these adults is to inhibit messenger RNA synthesis and to have little if any effect on ribosomal or 4S RNA.     

Regulation of Akt expression and phosphorylation by 17β-estradiol in the rat uterus during estrous cycle

Background

Ablation of the low-affinity receptor subunit for leukemia inhibitory factor (LIFR) causes multi-systemic defects in the late gestation fetus. Because corticosterone is known to have a broad range of effects and LIF function has been associated with the hypothalamo-pituitary-adrenal axis, this study was designed to determine the role for LIFR in the fetus when exposed to the elevated maternal glucocorticoid levels of late gestation. Uncovering a requirement for LIFR in appropriate glucocorticoid response will further understanding of control of glucocorticoid function.

Methods

Maternal adrenalectomy or RU486 administration were used to determine the impact of the maternal glucocorticoid surge on fetal development in the absence of LIFR. The mice were analyzed by a variety of histological techniques including immunolabeling and staining techniques (hematoxylin and eosin, Alizarin red S and alcian blue). Plasma corticosterone was assayed using radioimmunoassay.

Results

Maternal adrenalectomy does not improve the prognosis for LIFR null pups and exacerbates the effects of LIFR loss. RU486 noticeably improves many of the tissues affected by LIFR loss: bone density, skeletal muscle integrity and glial cell formation. LIFR null pups exposed during late gestation to RU486 in utero survive natural delivery, unlike LIFR null pups from untreated litters. But RU486 treated LIFR null pups succumb within the first day after birth, presumably due to neural deficit resulting in an inability to suckle.

Conclusion

LIFR plays an integral role in modulating the fetal response to elevated maternal glucocorticoids during late gestation. This role is likely to be mediated through the glucocorticoid receptor and has implications for adult homeostasis as a direct tie between immune, neural and hormone function.     

Inhibition of Early Steps in the Gibberellin-Activated Synthesis of α-Amylase

Subsequent production of amylase is severely inhibited if barban [4-chloro-2-butynyl N-(3-chlorophenyl) carbamate] is added to embryo-free half seeds of barley within 4 to 5 hours after gibberellic acid treatment of these seeds. Thirty to 50 mg/L concentrations of barban are effective. Barban inhibition is non-competitive with respect to gibberellic acid. Addition of barban 7 hours or more after gibberellic acid treatment is almost without effect.The delay between gibberellic acid treatment and amylase formation tends to become shorter with more prolonged imbibition periods. Regardless of imbibition period, susceptibility to barban is lost within 7 hours after gibberellic acid treatment.Other herbicidally active phenylurethanes are also inhibitors, but none are as effective as barban. Phenethyl alcohol and 2 arylcarbamates can act as inhibitors.     

Inhibition by N′-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in α-ketoglutarate amination

The effect of N′-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the α-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K_i, estimated as 199?μmol?l^?1 at 25°C and pH 8.0.     

Inhibition Kinetics and the Aggregation of α-Glucosidase by Different Denaturants

Kinetic changes of alpha-glucosidase from Saccharomyces cerevisiae in guanidinium chloride (GdmCl) and SDS solutions were investigated. The results showed both denaturants can lead conformational changes and loss of enzymatic activities. However, the concentrations of denaturants causing loss of activities were much lower than that of conformational changes, which suggested that the conformation of active site of α-glucosidase was more fragile than the whole molecular conformation in response to the two denaturants. According to the different kinetic process of the enzyme in the GdmCl and SDS solutions, the further investigation on the process of denaturation were made, it showed GdmCl and SDS had different types of inhibition and different types of interaction with the enzyme. Furthermore, the mechanisms of the two denaturants were discussed.