Interaction of cycloheximide with 25S ribosomal RNA from yeast.
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Cycloheximide↗
Biomedical subjects
M Cannon
Publications and source records attributed to M Cannon.
Enhancement by tamoxifen of the membrane antioxidant action of the yeast membrane sterol ergosterol: relevance to the antiyeast and anticancer action of tamoxifen.
The anticancer drug tamoxifen inhibits lipid peroxidation in ox-brain phospholipid liposomes, and is a good antiyeast agent, with clinical potential. We now report that the ergosterol-containing lipid fraction derived from yeast microsomal membranes (and the ergosterol separated from it) inhibited lipid peroxidation when introduced into ox-brain phospholipid liposomes. Inhibition of lipid peroxidation by the lipid fraction was greatly enhanced when yeast cell growth was inhibited with tamoxifen prior to lipid extraction. The ability of tamoxifen to enhance the membrane antioxidant ability of ergosterol is expressed in terms of a tamoxifen enhancement coefficient. Enhancement by tamoxifen of the membrane antioxidant action of ergosterol is discussed in relation to the antifungal and anticancer actions of tamoxifen.
Animals↗
Tamoxifen inhibits lipid peroxidation in cardiac microsomes. Comparison with liver microsomes and potential relevance to the cardiovascular benefits associated with cancer prevention and treatment by tamoxifen.
Tamoxifen and 4-hydroxytamoxifen were both good inhibitors of iron-dependent lipid peroxidation in rat cardiac microsomes. Tamoxifen was also a good inhibitor of lipid peroxidation in liposomes prepared from the phospholipid obtained from rat liver microsomes. In a modified rat liver microsomal system containing a sufficiently low amount of peroxidizable phospholipid to make it comparable with the rat cardiac microsomal system, tamoxifen and 4-hydroxytamoxifen were of similar effectiveness as in the cardiac system. Tamoxifen is known to lower serum cholesterol levels, and the findings reported here indicate that the drug might also protect heart cell membranes against peroxidative damage. Potential cardioprotective and antiatherosclerotic benefits of tamoxifen are discussed in relation to the drug's use in cancer prevention and treatment.
Animals↗
Natural cycloheximide resistance in yeast. The role of ribosomal protein L41.
The yeast Kluyveromyces lactis is resistant to high concentrations (1 mg/ml) of the antibiotic cycloheximide. Using in vitro translation studies it was confirmed that this extreme resistance is a property of ribosomes. The resistance determinant from K. lactis was cloned into Saccharomyces cerevisiae. Nucleotide sequence analysis of the determinant demonstrated that resistance was conferred by the K. lactis ribosomal protein L41. K. lactis was shown to contain only one copy of the gene that encodes this protein and the gene was located to chromosome III. In contrast, S. cerevisiae was found to contain multiple copies of the gene for the corresponding ribosomal protein L41 which mapped to two of the three chromosomes V, XIV and VIII. Since the cycloheximide-resistance gene of K. lactis causes essentially complete protection against inhibition by the drug, it is likely to be particularly useful as a selective marker in eukaryotic gene transfer studies.
Amino Acid Sequence↗
Droloxifene (3-hydroxytamoxifen) has membrane antioxidant ability: potential relevance to its mechanism of therapeutic action in breast cancer.
Droloxifene (3-hydroxytamoxifen), is a triphenylethylene derivative recently developed for the treatment of breast cancer. Droloxifene was found to exhibit a membrane antioxidant ability in that it inhibited Fe(III)-ascorbate dependent lipid peroxidation in rat liver microsomes and ox-brain phospholipid liposomes. It also inhibited microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH. Droloxifene was a better inhibitor of lipid peroxidation than tamoxifen, but was less effective than 17 beta-oestradiol in the two microsomal systems and in the preformed liposomal system. When introduced into ox-brain phospholipid liposomes, droloxifene inhibited Fe(III)-ascorbate induced lipid peroxidation to approximately the same extent as similarly introduced cholesterol and tamoxifen, although to a lesser extent than 17 beta-oestradiol. This inhibition of lipid peroxidation by droloxifene may result from a membrane stabilization that could be associated in cancer cells with decreased plasma membrane fluidity. This mechanism may be related to the clinically important antiproliferative action of droloxifene on cancer cells.
Animals↗
The structural mimicry of membrane sterols by tamoxifen: evidence from cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anti-cancer agent.
The anti-cancer drug tamoxifen is a potent inhibitor of lipid peroxidation induced by Fe(III)-ascorbate in ox-brain phospholipid liposomes. Similar anti-oxidant effects, but with varying potencies, are also shown by 4-hydroxy-tamoxifen, cholesterol, ergosterol and 17-beta-oestradiol. We now describe a computer-graphic fitting technique that demonstrates a structural similarity between the five compounds. In addition, we have quantified the differences (relative to cholesterol) between the anti-oxidant activities of the compounds in terms of a novel expression referred to here as the cholesterol coefficient (Cc) Finally, we discuss how the inhibitory effect of tamoxifen on lipid peroxidation may result from a membrane stabilization that is associated with a decrease in membrane fluidity. This action may be related to the anti-proliferative effect exerted by tamoxifen on cancer and fungal cells.
Antifungal Agents↗
Drug-induced hypothyroidism: the thyroid as a target organ in hypersensitivity reactions to anticonvulsants and sulfonamides.
Inherited defects in detoxification of reactive metabolites of drugs predispose patients to "hypersensitivity" reactions. Covalent interaction of metabolites with cell macromolecules leads to cytotoxic and immunologic outcomes, manifested clinically by multisystem syndromes with variable organ involvement. Hypothyroidism developed in 5 of 202 patients (age range, 1 to 81 years) we investigated for hypersensitivity reactions to anticonvulsants or sulfonamides shortly after their reaction. None had previous personal or family histories of autoimmune disease. All had low thyroxine levels, elevated levels of thyroid stimulating hormone, and autoantibodies including antimicrosomal antibodies. Patients were 2 to 18 years of age at presentation, and two were male. All returned to a euthyroid state within a year of presentation, and all remain well. The demographics, clinical presentation, and course of the patients is atypical of idiopathic lymphocytic thyroiditis. We investigated the pathogenesis of thyroid toxicity using the hydroxylamine metabolite of sulfamethoxazole as a model. The hydroxyalmine was toxic to thyroid cells in vitro, which did or did not express thyroid peroxidase activity, whereas the parent sulfonamide was toxic only to cells with active thyroid peroxidase. The purified enzyme converted sulfamethoxazole to the hydroxylamine. Formation of reactive drug metabolites by thyroid peroxidase in a host who is genetically unable to detoxify the metabolites may lead directly to cytotoxicity. Covalent binding to macromolecules, including thyroid peroxidase, also may lead to expression of neoantigens and formation of autoantibodies. Patients who have sustained hypersensitivity reactions to drugs should be investigated for possible involvement of the thyroid.
Adolescent↗
In-vitro assessment of a hypersensitivity syndrome associated with sorbinil.
Sorbinil is a hydantoin aldose reductase inhibitor that has shown promise as therapy for patients with diabetic complications such as neuropathy and retinopathy. However, as many as 10% of patients receiving sorbinil have had adverse reactions characterized by fever, skin rash, and myalgia. Our previous studies of phenytoin suggested that susceptibility to reactions might result from an inherited detoxification defect. We did the current study to determine if sorbinil is metabolized to reactive intermediates and if cells from patients with a history of a reaction to sorbinil are appropriate for the in-vitro investigation of susceptibility. Microsome-generated metabolites of sorbinil (50 microM) were toxic to normal peripheral blood lymphocytes (7.9% +/- 0.3% dead cells [mean +/- SE]). Toxicity was increased in the presence of an epoxide hydrolase inhibitor (17.5% +/- 0.3% dead cells) and abolished by an inhibitor of cytochrome P-450. In contrast to cells from healthy controls and diabetics who tolerated sorbinil (7.9% +/- 0.7% and 7.8% +/- 0.4% dead cells, respectively), cells from the six patients who had sorbinil reactions showed significantly increased toxicity from metabolites of sorbinil and phenytoin (19.7% +/- 2.3% dead cells, P less than 0.001). Cells from three patients who had reactions to phenytoin were similarly sensitive to sorbinil metabolites (23.4% +/- 0.3% dead cells). We conclude that sorbinil is oxidatively metabolized to a potentially toxic intermediate. Certain patients may be at increased risk for developing hypersensitivity reactions. Development of this important new drug has been hampered by uncommon but potentially severe reactions. An increased understanding of the steps involved in the development of adverse reactions could lead to screening tests or to the development of safer compounds.
Adult↗
Interaction of antibiotics with A- and P-site-specific bases in 16S ribosomal RNA.
We have studied the interactions of the antibiotics apramycin, kasugamycin, myomycin, neamine and pactamycin with 16S rRNA by chemical probing of drug-ribosome complexes. Kasugamycin and pactamycin, which are believed to affect translational initiation, protect bases in common with P-site-bound tRNA. While kasugamycin protects A794 and G926, and causes enhanced reactivity of C795, pactamycin protects G693 and C795. All four of these bases were previously shown to be protected by P-site tRNA or by edeine, another P-site inhibitor. Apramycin and neamine, which both induce miscoding and inhibit translocation, protect A1408, G1419 and G1494, as was also found earlier for neomycin, gentamicin, kanamycin and paromomycin. A1408 and G1494 were previously shown to be protected by A-site tRNA. Surprisingly, myomycin fails to give strong protection of any bases in 16S rRNA, in spite of having an apparently identical target site and mode of action to streptomycin, which protects several bases in the 915 region. Instead, myomycin gives only weak protection of A1408. These results suggest that the binding site(s) of streptomycin and myomycin have yet to be identified.
Aminoglycosides↗
The antioxidant action of ketoconazole and related azoles: comparison with tamoxifen and cholesterol.
The azole antifungal drug ketoconazole was found to inhibit Fe(III)-ascorbate dependent lipid peroxidation using either rat liver microsomes or ox-brain phospholipid liposomes as the substrate. It also inhibited microsomal peroxidation induced by the Fe(III)-ADP/NADPH system. The related azoles, miconazole and clotrimazole, were much weaker inhibitors than ketoconazole. Ketoconazole was approximately equipotent with the triphenylethylene anticancer drug tamoxifen in the microsomal system and was almost as effective as 4-hydroxytamoxifen in the liposomal system. Ketoconazole introduced into phospholipid liposomes during their preparation inhibited Fe(III)-ascorbate induced lipid peroxidation to a greater extent than similarly introduced cholesterol, ergosterol or tamoxifen. Miconazole and clotrimazole were again poor inhibitors of lipid peroxidation in this system. These antioxidant effects of ketoconazole may be due to membrane stabilization in the systems used. The implications of our findings for the clinical applications of these drugs are discussed.
Animals↗
Abuse of neuroleptic drugs.
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Administration, Oral↗
Bioactivity of plasma prolactin in ovariectomized, diethylstilbestrol-treated Long-Evans and Holtzman rats after thyrotropin-releasing hormone or bromocriptine administration.
The objective of this study was to determine the effects of thyrotropin-releasing hormone (TRH) and bromocriptine on plasma levels of biologically active prolactin in ovariectomized, diethylstilbestrol (DES)-treated rats. Female Long-Evans and Holtzman rats were ovariectomized and each was given a subcutaneous implant of diethylstilbestrol (DES). One week later, groups of DES-treated rats were fitted with indwelling intra-atrial catheters, and 2 days later blood samples were withdrawn before and at 1, 2, 5, 10, and 20 min after intravenous administration of TRH (250, 500, or 1000 ng/rat). Blood samples were obtained from other groups at 4 weeks of DES treatment by orbital sinus puncture under ether anesthesia before and at 30, 60, and 120 min after bromocriptine administration (2.5 mg/rat sc). Plasma was assayed for prolactin by conventional radioimmunoassay (RIA) and by Nb2 lymphoma bioassay (BA). Holtzman rats released significantly more prolactin following TRH than did Long-Evans rats when the RIA was used to measure prolactin. However, when the BA was used to assay prolactin in the same samples, the Long-Evans rats released more prolactin than did the Holtzman rats. In addition, the ratio of the BA to RIA values was significantly increased in both strains following TRH, but the greatest increase was observed in the Long-Evans rats, in which the ratio was 4.5 at the peak of the TRH-induced rise in plasma prolactin. Gel filtration chromatography of plasma obtained at 5 min after TRH treatment in Long-Evans rats revealed large molecular forms of prolactin with BA to RIA ratios of 4-5. In addition, monomeric prolactin had a BA to RIA ratio of 2. Bromocriptine treatment reduced prolactin levels in both strains, but the effect was more rapid in Holtzman than in Long-Evans rats. In addition, bromocriptine treatment of Holtzman, but not Long-Evans, rats significantly reduced the BA to RIA ratio of plasma prolactin. The results indicate that TRH and bromocriptine affect the release of biologically active prolactin to a greater extent than prolactin detected by antibody in the RIA, and that Long-Evans and Holtzman rats respond to these secretagogues differently with regard to BA to RIA comparisons.
Animals↗
A comparison of plasma prolactin levels in young female Long-Evans and Holtzman rats as measured by Nb2 lymphoma bioassay and radioimmunoassay.
This study was conducted to determine the plasma levels of prolactin in prepubertal and young, postpubertal, proestrus rats of mammary tumor-susceptible (Sprague-Dawley) and tumor-resistant (Long-Evans) strains using a sensitive bioassay-Nb2 lymphoma cell replication. Prepubertal Long-Evans rats had significantly higher levels of prolactin than did Holtzman Sprague-Dawley rats of the same age. Likewise, Long-Evans rats secreted significantly more prolactin into the blood on the afternoon and evening of proestrus than did Holtzman rats. Finally, ovariectomized Long-Evans rats released more prolactin into the blood at 1 day, but not at 8 or 15 days, of treatment with diethylstilbestrol. Prolactin levels determined by conventional radioimmunoassay and by bioassay were similar except on the afternoon of proestrus, when, in both strains of rats, the bioassay to radioimmunoassay ratio increased significantly above 1.0 during the late evening. In addition, the ratio was significantly less than 1.0 in the early and late afternoon in the Holtzman rats, but not Long-Evans rats. These data indicate that a strain of rats that is resistant to experimentally induced mammary cancer has higher prolactin levels in the blood than does a strain that is susceptible to mammary cancer at a time when mammary gland growth is rapid. Furthermore, there are times during the proestrus prolactin surge when the bioassay yielded higher and lower values of prolactin than radioimmunoassay of the same samples, suggesting functional heterogeneity of prolactin that may impact on mammary gland or other target tissue function.
Animals↗
Mechanism of inhibition of lipid peroxidation by tamoxifen and 4-hydroxytamoxifen introduced into liposomes. Similarity to cholesterol and ergosterol.
The anticancer drug tamoxifen when introduced into phospholipid liposomes during their preparation inhibited Fe(III)-ascorbate induced lipid peroxidation to a greater extent than similarly introduced cholesterol. Ergosterol was equipotent with tamoxifen, but much less effective than 4-hydroxytamoxifen. Possible mechanisms underlying these effects are discussed in relation to structural mimicry of the sterols by these triphenylethylene drugs as membrane stabilizers against lipid peroxidation.
Cholesterol↗
Characterization of Saccharomyces cerevisiae strains displaying high-level or low-level resistance to trichothecene antibiotics.
Biochemical and genetic analyses have been carried out on Saccharomyces cerevisiae strains characterized in vivo as sensitive, low-level-resistant or high-level-resistant to trichothecene antibiotics. Levels of drug resistance in vitro were determined for each strain and for suitable diploids derived from them. Ribosome biogenesis was also studied in selected haploids. It is suggested that resistance in all cases results from a mutation in the gene encoding ribosomal protein L3. If this is indeed the situation, then different mutations in this same gene not only can cause low-level or high-level resistance to trichothecene antibiotics but also can affect the maturation of either 40 S or 60 S ribosomal subunits.
Drug Resistance, Microbial↗
The antioxidant action of tamoxifen and its metabolites. Inhibition of lipid peroxidation.
The anti-oestrogen drug tamoxifen is an inhibitor of lipid peroxidation in rat liver microsomes and in phospholipid liposomes. Its cis isomer and N-desmethyl form are weaker inhibitors, but 4-hydroxytamoxifen is much more powerful. It is possible that the antioxidant property of tamoxifen might contribute to its biological actions.
Animals↗
Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass.
The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.
Animals↗
Organ-specific modulation of gene expression in transgenic plants using antisense RNA.
We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS 'gene' dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially 'down mutations' in essential genes where only a short 5' sequence of the mRNA is required. They also suggest that the 'position effect' on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.
Base Sequence↗