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L W Yielding

Publications and source records attributed to L W Yielding.

33 records · Page 2Linked to original sources
PubMed1981

Antitrypanosomal action enhanced by photoaffinity labeling with ethidium analogs.

The trypanocidal activity of photoreactive azido analogs of ethidium was tested to determine the suitability of using such compounds as in vivo probes to study the mechanism of the antitrypanosomal activity of ethidium. Eight ethidium analogs, including three nonphotoreactive compounds, were tested for their ability to kill T. brucei both with and without photolytic activation. Two analogs tested, the monoamino-monoazido isomers, showed greater that 100-fold enhancement of trypanocidal activity following photolytic activation in situ. Without photolytic activation, only the nonphotoreactive monoamino precursor analogs showed activity greater than the parent ethidium compound. The availability of suitable ethidium analogs which can be covalently attached by in situ photoactivation provides a new approach for studying the mechanism by which ethidium exerts its trypanocidal activity.

Animals↗
PubMed1980

Comparative studies of the binding of ethidium bromide and its photoreactive analogues to nucleic acids by fluorescence and rapid kinetics.

The binding studies of the interaction of ethidium bromide with DNA have been hampered by its reversibility, which prevents direct isolation and thus characterization of the complex. The recent development of photoaffinity labeling has provided a means to circumvent this problem. However, to be useful as a probe for the parent compound, a photosensitive analogue must be shown to interact in vivo and in vitro just as the parent analogue. These studies demonstrate by steady-state and nonosecond fluorescence and stopped-flow kinetics that one of the azido analogues of ethidium, 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride, binds nucleic acids quite similarly to ethidium bromide. The interaction of the other azide, 3,8-diazido-5-ethyl-6-phenyl-phenanthridinium chloride, with DNA is qualitatively different from that of the monoazide and ethidium bromide. These results suggest that the monoazide would serve as an ideal probe for determining the actual target sites of ethidium bromide in vivo and in vitro.

Animals↗
PubMed1980

Mutagenesis by photoaffinity labeling using selected azidofluorenes.

Several 2-azidofluorenes have been synthesized for use as photoaffinity labels inside bacteria. In the dark they were not mutagenic for any Salmonella typhimurium tested. When photolyzed inside the bacteria, all were mutagenic for strain TA1538 to varying degrees, and were considerably less mutagenic in the corresponding repair positive TA1978. None were mutagenic for strain TA1535 or TA1537, although most compounds were toxic for those strains when photolyzed.

Affinity Labels↗
PubMed1980

Structure-function characterization for ethidium photoaffinity labels as mutagens in Salmonella.

The development of photoaffinity probes to characterize the binding process and subsequent biological activity of a drug has recently been emphasized by the synthesis of two ethidium azide analogs. The initial finding showed that one of the azido analogs, the 8-azido-3-amino derivative, was at least 40-fold more mutagenic and toxic in Salmonella tester strain TA1538 than the other analog, the 3.8-diazido derivative. These observations suggested the need to examine the structural requirements of ethidium photoaffinity labels for frameshift mutagenic activity in Salmonella. Thus, the isomer of the monoazide, the 3-azido-8-amino derivative, and two deaminated monoazide derivatives were synthesized and all of the ethidium analogs were screened in two Salmonella frameshift tester strains, TA1537 and TA1538, and in their excision-repair positive isogenic strains. The results presented in this paper demonstrate that two substituents are needed to produce significant mutagenicity and toxicity by the compound. One substituent, usually the amino group, is required for mutagenic activity, perhaps by orienting the phenanthridinium ring into its mutagenic configuration. The other substituent, the azido group, is required for covalent attachment, a requisite for mutagenic activity. Thus, photoaffinity labeling has provided a means of comparing structure with mutagenic activity for ethidium compounds.

Affinity Labels↗
PubMed1980

Acridine structure correlated with mutagenic activity in Salmonella.

The structural basis for direct mutagenicity of acridines was studied by testing 50 different analogs in the Ames Salmonella tester strains without the addition of mammalian activating enzymes. These experiments showed that the single most effective substituent for frameshift mutagenesis in strain TA1537 is an amino group at the "9" position, while an amino group at either the "3" or "1" position is less effective. Other substitutions at the "9" position demonstrate decreased frameshifting activity compared to 9-aminoacridine. Furthermore, all substituents in combination with the amino group of 9-aminoacridine also decrease frameshifting activity, except for the addition of another amino group at the "1" position or a methyl at the ring nitrogen. Nitro substituents at the "1" and "3" positions enhance 9-aminoacridine toxocity. All nitro substituents decrease typical acridine-frameshift mutagenesis for strain TA1537, but they induce mutagenic activity either in the other type of frameshift strain, TA1538, or in the base-pair substitution strain TA1535. These studies have provided important structure-function relationships for acridine mutagenicity and toxicity in Salmonella. Consequently, this biological system has provided a sensitive means for determining the structural requirements for mutagenic mechanisms.

Acridines↗
PubMed1980

Petite induction in Saccharomyces cerevisiae by ethidium analogs: distinction between resting and growing cells.

The importance of specific substituents, especially amino azide groups, for ethidium induction of petites was evaluated in resting and dividing cells of Saccharomyces cerevisiae through the study of a series of ethidium analogs. The structural requirements in resting and growing cells were found to be different, suggesting that at least two mechanisms are responsible for induction. The significance of particular substituents in the induction processes were recognized by: (1) a dependence upon the ethyl substituent at the ring nitrogen in both actively growing and in resting cells; and (2) the implication that amino substituents are important for the effect in dividing cells and especially in resting cells. Photolytic enhancement of petite induction (via a nitrene which forms a covalent linkage to a biological site) was observed for 3 of the azide analogs, which emphasizes the likelihood that metabolic activation of ethidium to a covalent complex is responsible for its effectiveness. Furthermore, these studies indicate that these monoazide analogs should be ideal probes for examining the mitochondrial mutagenic processes.

Ethidium↗
PubMed1979

Ethidium bromide enhancement of frameshift mutagenesis caused by photoactivatable ethidium analogs.

Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.

Azides↗
PubMed1977

Synthesis, separation and characterization of the mono- and diazide analogs of ethidium bromide.

Ethidium bromide is used to characterize nucleic acid secondary and tertiary structural properties and the biological consequences of drug interactions. The mono- and diazido analogs of ethidium have proven valuable as photoaffinity probes in chemical and biological studies on nucleic acids, since they render the ethidium-nucleic acid interaction covalent. Although both of these compounds have been synthesized previously, the published synthesis procedure for the monoazide is inadeqlate since a major portion of the product has been identified as the diazide analog. This lack of purity severely limits the usefulness for nucleic acid research. The procedure presented here for the synthesis, separation, purification and crystallization of these analogs should provide the quantities and quality of these important reagents needed to perform a variety of chemical and biological experiments.

Azides↗
PubMed1976

Production of frameshift mutations in Salmonella by a light sensitive azide analog of ethidium.

Frameshift mutations have been produced in specific repair-negative Salmonella tester strains by photoaffinity labeling technique using ethidium azide. Reversions requiring a +1 addition or a -2 deletion were specially sensitive. Mutagenesis was reduced by the simultaneous addition of non-mutagenic ethidium bromide, and was prevented by photolysis of the azide prior to culture addition. Identical tester strains active in DNA excision repaire were not mutagenized by the azide. These results are consistent with the interpretation that photolysis of the bound ethidium analog converts the drug from its noncovalent mode of binding (presumably intercalation) to a covalent complex with consequent production of frameshift mutations. Such photoaffinity labeling by drugs which bind to DNA not only confirms the importance of covalent drug attachment for frameshift mutagenesis, but also provides powerful techniques for studying the molecular deatils of a variety of genetic mechanisms.

Azides↗