Babette S. Radner

Suppression of X-ray induced transformation by vitamin E in mouse C3H/10T12 cells

Vitamin E (d-alpha-tocopherol) was shown to decrease X-ray induced transformation in mouse C3H/10T1/2 cells. The d-alpha-tocopherol was active in the form of succinate diluted in ethanol, but was inactive at the highest nontoxic concentration of the pure substance dissolved in oil and diluted in acetone. Vitamin E succinate was effective when present throughout the entire assay period or when treatments began after confluence was reached at day 12 post-irradiation. It was ineffective if present only for the early portion of the radiation transformation assay period, indicating that its effect may be reversible. Vitamin E did not suppress the growth and expression of transformed C3H/10T1/2 cells as foci when the transformed cells were surrounded by a monolayer of normal cells.

The biogenesis of anthocyanins. VII. The requirement for both purines and pyrimidines.

Abstract Previous evidence that anthocyanin formation in Spirodela is dependent on the synthesis of a complex nucleotide or nucleic acid has been extended through a survey of the effects of a variety of purine and pyrimidine analogs. Almost all such compounds have been found to inhibit anthocyanin formation. The group includes analogs of all four bases which make up ribonucleic acid (RNA): 8-azaguanine, 8-azaadenine, 6-azauracil, and 2-thiocytosine, as well as Antrycide (2,6′-dimethyl-6,4′-pyrimidylamino-4, 2′-diaminoquinoline 1,1′-dimethosulfate), an inhibitor of purine incorporation into nucleic acid. The most effective inhibitor is still, as previously reported, 8-azaguanine. It decreases the anthocyanin formation by 50% at 3 × 10 −7 M . Subinhibitory concentrations of azaguanine, azaadenine and Antrycide, but not in general of the natural bases, clearly promote pigment formation. The inhibitions are reversed in each case by the corresponding purine or pyrimidine, or its riboside or ribotide. There is a specific requirement for a purine, not satisfiable by pyrimidines, and conversely a requirement for a pyrimidine, not wholly satisfied by purines. There is indication of a requirement for both uracil and cytosine. Experiments using simultaneously two types of inhibitors, namely, analogs of both purines and pyrimidines, confirm that both purine and pyrimidine cooperate in the synthesis of anthocyanin. It has been demonstrated that the reversing compounds function within the plant, rather than by preventing the absorption of the antagonist. It is concluded that an unstable nucleic acid, which must be continually resynthesized, is the catalyst or limiting factor in the synthesis of anthocyanin in Spirodela .

The biogenesis of the anthocyanins. III. The role of sugars in anthocyanin formation.

Abstract Anthocyanin formation in growing cultures of Spirodela is promoted by sucrose but not by glucose; conversely, growth is promoted by glucose but not by sucrose. Fructose is intermediate in both respects. In non-growing cultures, however, all three sugars are equally effective in promoting anthocyanin formation. A number of treatments which increase or decrease the anthocyanin content have parallel effects on the reducing sugar content. A plot of anthocyanin content against reducing sugar content shows a smooth relationship. Variations in the sucrose content are smaller and show no parallelism with pigmentation. It is deduced that anthocyanin may be formed independently from any of the three sugars, but that glucose is preferentially consumed for growth. Phosphate apparently does not participate in the formation of anthocyanin, and if the process does take place directly from sugars it probably does not proceed via the usual glycolytic pathway, since none of a number of glycolytic intermediates gives rise to any anthocyanin. There is some evidence for a participation of meso -inositol in the biogenesis of anthocyanin. The effects of quinic and shikimic acids appear too small for them to be considered as intermediates. Phosphate appears to be no more required with inositol than with the sugars.

The biogenesis of anthocyanins. V. Evidence for the mediation of pyrimidines in anthocyanin synthesis

Abstract Anthocyanin synthesis in Spirodela oligorrhiza is inhibited by thiouracil and also by other compounds which interfere with pyrimidine or purine metabolism. These include (in order of increasing effectiveness) benzimidazole, 2,6-diaminopurine, quinine, azaadenine and azaguanine, the last-named compound being 650 times stronger as an inhibitor than thiouracil. With the exception of that due to benzimidazole, these inhibitions of pigment formation are partially or largely reversed by certain pyrimidines and purines. Another adenine derivative, 6-furfuryl-adenine or “kinetin” also causes clear inhibition. In the case of thiouracil, the inhibition is exerted in the light but not in the dark. The inhibition in the light is completely reversed by copper ions or by uracil or thymine, and partially reversed by adenine and hypoxanthine. However, plants preilluminated in a thiouracil solution produce no pigment in the dark even in the presence of uracil. The evidence, together with that previously presented, supports the idea that there are at least two stages in anthocyanin production: (a) a light reaction in which a copper enzyme participates and which probably involves the synthesis either of a nucleotide, or of one or more pyrimidines or purines, and (b) a dark reaction utilizing the products of this light reaction for formation of anthocyanin.

The biogenesis of anthocyanin. VI. The role of riboflavine.

Abstract 1. 1. The inhibition of the formation of anthocyanin brought about by methionine, ethionine, thiouracil, azaguanine, etc. can be completely reversed by riboflavine. The amount of riboflavine needed for reversal is approximately constant and independent of the concentration of inhibitor. 2. 2. Riboflavine alone has a small promoting effect on anthocyanin formation, but only after the linear phase of pigment increase has been passed. 3. 3. When the plants are preilluminated, riboflavine increases anthocyanin production in a subsequent dark period; this action is enhanced by sucrose and diminished by the absence of CO2. Yields in the dark almost as high as those in the light can thus be obtained. 4. 4. It is deduced that riboflavine acts, not as a photoreceptor, but as a dark catalyst to produce anthocyanin from sucrose or other precursors. It is calculated that each molecule of riboflavine leads to the formation of 30 to 60 molecules of anthocyanin. 5. 5. The above deduction is confirmed by direct riboflavine determinations, which show that the riboflavine content of the plants varies parallel to their anthocyanin content. When azaguanine inhibits the formation of anthocyanin (in the light), riboflavine formation is also prevented, and the addition of riboflavine solution restores both the riboflavine and the anthocyanin content. Similarly, azaguanine and ethionine, which do not inhibit anthocyanin formation in the dark, do not affect riboflavine formation in the dark either. 6. 6. The light reaction in the formation of anthocyanin is probably, at least in the main, the synthesis of riboflavine.

The biogenesis of anthocyanins. IV. The inhibitory effect of methionine and other sulfur-containing compounds on anthocyanin formation☆

Abstract A survey of sulfur-containing compounds which inhibit anthocyanin formation shows that inhibition cannot be ascribed to the sulfhydryl group, to the SS linkage, to the sulfone grouping, or even probably to the sulfur atom itself. Among the most potent inhibitors are ethionine, methionine, sulfadiazine, and thiouracil. Many known antithyroid compounds inhibit pigment formation, but there is no correlation between antithyroid and “anti-anthocyanin” activity. The inhibition caused by methionine is not due merely to its sulfur atom nor to its amino acid grouping, though both these elements were shown to contribute to the inhibitory activity. Methionine does not inhibit through being oxidized to the sulfoxide or sulfoximine, for these compounds are less active than methionine itself. Ethionine does not reverse methionine inhibition but is itself the most active inhibitor studied. Ethionine does not inhibit the small amount of anthocyanin formation which occurs in the dark. It does, however, inhibit the subsequent formation of anthocyanin in the dark if it is preilluminated with the plants. After eliminating several possible alternatives, it is deduced that ethionine must inhibit a light reaction whose product is converted to anthocyanin in the dark.

The biogenesis of anthocyanins. IX. The effect of ribonuclease on anthocyanin formation in Spirodela oligorrhiza

Abstract Ribonuclease inhibits anthocyanin formation in Spirodela oligorrhiza and, even more powerfully, in corn-leaf discs. Its action is, however, dependent on decreasing the level of heavy-metal ions in the Spirodela tissue. The findings substantiate the conclusion previously drawn from studies with antimetabolites that a nucleic acid is the catalyst or limiting factor in the synthesis of anthocyanin in Spirodela . The finding that deoxyribonuclease does not inhibit anthocyanin production in Spirodela lends support to the hypothesis that the nucleic acid is an RNA, not a DNA.

Effects of agents known to antagonize the enhancement of in vitro transformation by 12-tetradecanoyl-phorbol-13-acetate (TPA) on the TPA suppression of metabolic cooperation

Utilizing the phenomenon of metabolic cooperation in Chinese hamster V79 cells, we have studied the effects of agents which suppress the 12-tetradecanoyl-phorbol-13-acetate (TPA) enhancement of transformation in vitro, on the TPA suppression of cell-cell communication. None of the agents tested, namely all-trans-retinoic acid, the trimethyl methoxyphenyl analogue of N-ethyl-retinamide, soybean trypsin inhibitor, antipain nor superoxide dismutase, decreased the enhanced recovery effect of TPA on metabolic cooperation. One of the compounds, retinoic acid, significantly increased the % recovery above that observed for TPA alone.

Effect of split doses of radiation on mutation frequency in rodent cell lines

Mutation frequencies in mouse cells and Chinese hamster cells were measured following single or split doses of UV light or X-rays. Split doses separated by a few to over 24 h induced no more ouabain- or 6-thioguanine-resistant mutants than did comparable single doses. These data lend no support to the possible existence of an inducible error-prone repair system in rodent cells.

Suppression of X-ray induced transformation by Valium and Aspirin in mouse C3H10T12 cells

Two commonly used drugs, Valium (diazepam) and Aspirin (acetylsalicyclic acid), were shown to suppress X-ray induced transformation in mouse C3H/10T1/2 cells. Valium was studied in an ethanol solution. Aspirin, which is soluble in both water and ethanol, was active only in the ethanol solution. Both drugs were effective only when present throughout the entire assay period.