Search PubMed⌕ Search

Biomedical subjects

A R Morgan

Publications and source records attributed to A R Morgan.

At least 55 records · Page 3Linked to original sources
PubMed1991

Fluorometric assays for DNA topoisomerases and topoisomerase-targeted drugs: quantitation of catalytic activity and DNA cleavage.

We have introduced the novel application of a simple ethidium fluorescence assay, using covalently closed circular DNA, for the study of topoisomerase-targeted drugs. With the specificity of camptothecin for eukaryotic topoisomerases I and of VM26 for eukaryotic topoisomerases II, the two classes of enzymes can be assayed independently in crude extracts and during purification. These assays are fast, sensitive, and quantitative, have a large sample capacity, and eliminate the need for radioactive materials, filters, and agarose gels. We have demonstrated the use of this fluorescence assay to measure the inhibition of the relaxation and supercoiling activities of purified mammalian topoisomerases I and II and bacterial gyrase by nonintercalating drugs. Similarly, the production of drug-induced topoisomerase-mediated cleavable complexes was readily quantitated with both nonintercalating and intercalating drugs. When inhibition and cleavage with VM-26 were measured concurrently as a function of topoisomerase II concentration, a clear inverse relationship between topoisomerase II inhibition and cleavable complex production was observed. When the physiologically relevant salt K+L-glutamate- was used, quantitative relaxation by topoisomerase II was observed up to twice the salt concentration obtained with KCl. The enantiomer K+D-glutamate- gave exactly the same results, indicating that the enhancing role of glutamate- is non-stereospecific.

Amsacrine↗
PubMed1990

Interconversion of replication and recombination structures: implications for terminal repeats and concatemers.

Replication and recombination structures can be interconverted by branch-migration. Using this simple concept a novel mechanism is proposed for generating concatemers through an initial single-strand DNA invasion into a duplex. Only DNAs with terminal repeats can form concatemers, and Herpes Simplex Virus DNA replication is considered in detail. The model is more parsimonious than other models such as Watson's for concatemer formation.

Base Sequence↗
PubMed1990

Hemodynamic effect of the metallopurpurin SnET2 and light on transplantable FANFT induced bladder tumor.

Changes in blood flow to transplantable N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide induced bladder tumors growing subcutaneously in the flanks of Fischer CDF (F344/CrlBR) rats were measured after photodynamic therapy with the photosensitizer tin (II) etiopurpurin dichloride using the radioactive microsphere technique. As with the other photosensitizers, hematoporphyrin derivative and chloroaluminum tetrasulfophtalocyanine, tin (II) etiopurpurin dichloride and light caused a rapid decrease in tumor blood flow in this tumor model. The decrease in blood flow occurred whether the vehicle for photosensitizer delivery was an emulsion or a liposome. Systemic heparinization of animals prior to light treatment did not alter changes in tumor blood flow.

Animals↗
PubMed1990

Diels-Alder adducts of vinyl porphyrins: synthesis and in vivo photodynamic effect against a rat bladder tumor.

Benzoporphyrin derivatives have been proposed as potential photosensitizers for photodynamic therapy. We have prepared a number of benzoporphyrin derivatives and tested their effect, in combination with red light, on the N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) induced rat bladder tumor (AY-27) transplanted into Fischer CDF(F344)/CrlBr rats. Tetracyanoethylene (TCNE) adducts showed very little activity, which may be attributable to their poor tumor uptake or to chemical instability of the adduct under physiological conditions. However, dimethyl acetylenedicarboxylate (DMAD) adducts tested showed greater cytotoxic effect than hematoporphyrin derivative and similar activities to metallopurpurins when tested under the same protocol.

Acetylene↗
PubMed1990

Tin etiopurpurin dichloride-sensitized lipid photooxidation of erythrocyte membranes.

Tin(IV) etiopurpurin dichloride (SnET2 x 2Cl) is a photosensitizer which has been shown to be an effective photodynamic agent for the treatment of transplantable animal tumors in vivo. The purpose of this study was to understand the effect of SnET2 x 2Cl on membrane lipid peroxidation. When erythrocyte membranes were exposed to visible light in the presence of SnET2 x 2Cl, lipid peroxidation was observed. An accumulation of lipid hydroperoxides and an increase in lipid fluorescence were also observed. Thin layer chromatography of lipid extracts from photooxidized membrane revealed photoperoxide products derived from phospholipid. Investigations into the mechanism(s) of lipid peroxidation by SnET2 x 2Cl and light-sensitized membranes were also performed. Results indicate that singlet oxygen (1O2) plays a major role in lipid peroxidation.

Animals↗
PubMed1990

Metallopurpurins and light: effect on transplantable rat bladder tumors and murine skin.

Purpurins are modified chlorins with photodynamic properties. Their strong absorption in the red region of the visible spectrum makes them candidates for use in photodynamic cancer therapy. A series of metal derivatives of the free base purpurins have been synthesized and shown to cause tumor necrosis in transplantable tumors when exposed to visible light. In the following set of experiments, the effects of two metallo-derivatives (tin and zinc) of two purpurins, octaethylpurpurin (NT2) and etiopurpurin (ET2), and light on the N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide transplantable tumors in Fischer CDF(F344)/CrlBr rats were studied. The photodynamic activity was assessed by a short term assay using tumor dry weight 12 days after purpurin-PDT as a criterion of response. From these experiments it appears that SnET2 greater than SnNT2 greater than ZnET2 greater than ZnNT2 in photodynamic activity. SnET2 was further characterized by attempting to determine the time interval after systemic injection at which maximum therapeutic effect occurred. These studies shown that 24 h after metallopurpurin injection was the optimum time for treatment of tumors with visible light. In a final set of experiments, the effect of solar light on the skin of hairless mice injected with SnET2 was found to be much less injurious than with hematoporphyrin derivative.

Animals↗
PubMed1989

Uracil-DNA glycosylase in insects. Drosophila and the locust.

It has been reported that Drosophila lacks a uracil-DNA glycosylase but that a direct incising activity on uracil-containing DNA appeared developmentally only in third instar larvae. In contrast we have found by two independent assays, that uracil-DNA glycosylase exists in both Drosophila eggs as well as in third instar larvae. The first assay shows the liberation of [3H] uracil from a d(AT)n polymer randomly substituted with [3H]uracil by its synthesis in the presence of [3H] dUTP. The second fluorometric assay for uracil-DNA glycosylase depends on the unique topological properties of circular DNAs and has the advantage of detecting apyrimidinic/apurinic (AP) endonuclease activity as well. To test one other insect, locust eggs were also assayed for uracil-DNA glycosylase. The amount of uracil-DNA glycosylase correlated well with the amount of DNA in actively replicating cells.

Animals↗
PubMed1989

New sensitizers for photodynamic therapy: controlled synthesis of purpurins and their effect on normal tissue.

Purpurins are a class of porphyrin derivative that have been shown to have good in vivo cytotoxicity to N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) induced rat bladder tumors (AY-27) implanted into Fisher 344 rats. The synthesis of purpurins from etioporphyrin I and coproporphyrin I proceeds in high yield and with a high degree of regioselectivity. Product formation can be rationalized in terms of relief of steric strain about the periphery of the purpurin macrocycle. The effect of therapeutic light doses using the rat footpad model suggests that, at therapeutic sensitizer doses, normal tissue damage is within acceptable limits, particularly for metalated purpurins.

Animals↗
PubMed1988

Conformational isomerization of the Holliday junction associated with a cruciform during branch migration in supercoiled plasmid DNA.

The variable positions of a branch-migrating cruciform junction in supercoiled plasmid DNA were mapped following cleavage of the DNA with bacteriophage T7 endonuclease I. T7 endonuclease I specifically cleaved, and thereby resolved, the Holliday junction existing at the base of the cruciform in the circular bacterial plasmid pSA1B.56A. Cruciform extrusion of cloned sequences in pSA1B.56A (containing a 322 base-pair inverted repeat insert composed of poxvirus telomeric sequences) topologically relaxed the plasmid substrate in vitro. Thus, numerous crossover positions were identified within the region of cloned sequences, reflecting the range of superhelical densities in the native plasmid preparation. Endonuclease I-sensitive crossover positions, mapped to both strands of the viral insert following the T7 endonuclease I digestion of either plasmid preparations or individual topoisomers, were regularly separated by approximately ten nucleotides. The appearance of sensitive crossovers every ten nucleotides corresponds to a change in linking difference (delta Lk) of +/- 2 in the circular core domain of the plasmid during branch point migration. In contrast, individual topoisomers of a plasmid preparation differ in linking number in increments of +/- 1. Thus, the observed linearization of each individual topoisomer following enzyme treatment, as a result of resolution of the crossovers associated with each topoisomer, showed that branch point migration to sensitive crossover positions must have occurred facilely. T7 endonuclease I randomly resolved across either axis of the cruciform, though some discrimination (related to the sequence specificity of the enzyme) was observed. The ten-nucleotide spacing between sensitive crossover positions is accounted for by an isomerization of the cruciform junction on branch point migration. An hypothesis is that this isomerization was imposed upon the cruciform junction by the change in helix twist (delta Tw) in the two branches that compose the topologically closed, circular domain of the plasmid. T7 endonuclease I may discriminate between the various isomeric forms and cleave a sensitive conformation that appears with every turn of branch migration which leads to the extrusion, or absorption, of two turns of helix from the circular core.

Base Sequence↗
PubMed1988

New photosensitizers for photodynamic therapy: combined effect of metallopurpurin derivatives and light on transplantable bladder tumors.

A series of metallopurpurins was tested for their photodynamic activity against transplantable N-[4-(5-nitro-2-furyl)-2-thiazoyl]formamide-induced urothelial tumors growing in male Fischer CDF (F344/CrlBR) rats. Histological examination of tumors in animals treated with the metallopurpurins and red light (greater than 590 nm, 360 J/cm2) revealed tumor necrosis 24 h after completion of therapy. Control tumors showed no histological change. In 30-day tumor regrowth studies, 70% of animals treated with the metallopurpurin derivative SnET2 were free of tumors while 50% of the animals treated with the free-base purpurin ET2 were free of tumor. Metallopurpurins have intense absorptions in the red region of the visible spectrum, a region with good tissue penetration. The metallopurpurins are easily prepared from the corresponding purpurins with a high degree of purity. This study demonstrates the potential of these photosensitizers for photodynamic cancer therapy.

Animals↗
PubMed1988

Laser flash photolysis of purpurins: novel potential photosensitizers of interest in photodynamic therapy.

Purpurins, a new class of photosensitizers of potential use in the photodynamic therapy of cancer, have been studied by laser spectroscopy to determine the properties of their excited states. For the two molecules studied, generation of triplet states and singlet oxygen (1O2) is very efficient. Singlet oxygen yields are presented for red and blue excitation though no significant wavelength effect is apparent. The promise these sensitizers have shown merits further investigation of other derivatives.

Lasers↗
PubMed1988

Quantitative assays for uracil-DNA glycosylase of high sensitivity.

We have developed a sensitive fluorometric assay using bisulfite deaminated (C----U), covalently-closed circular PM2 DNA as the substrate. We describe a reliable way to prepare this substrate without nicking the PM2 DNA. Methods, which depend on toluenization of the cells, are described for reproducibly and quantitatively assaying uracil-DNA glycosylase. The sensitivity is such that only 200 EL4 mouse thymoma cells or 30,000 Escherichia coli cells are needed for each point in a kinetic assay.

Animals↗
PubMed1988

Characteristics of the interaction of anthrapyrazole anticancer agents with deoxyribonucleic acids: structural requirements for DNA binding, intercalation, and photosensitization.

The binding constants for interaction of several novel anthra[1,9-cd]pyrazol-6(2H)-ones (anthrapyrazoles) with DNA have been determined by an ethidium displacement method. The apparent binding constants range from less than 2 X 10(6) to 2.7 X 10(8) M-1. The binding is influenced not only by the nature of the side chains but also by the number and position of hydroxyl groups on the chromophore. Unwinding angles, determined by a topoisomerase I assay, ranged from 0 degrees to 29.2 degrees. The deshydroxy compound 1 gave the highest unwinding angle, and both substitution of hydroxyl groups in the chromophore and alterations in the side chains decrease the unwinding angle, consistent with a decreased or partial intercalation. Representative anthrapyrazoles cause an increase in sonicated DNA viscosity as expected for intercalators. Spectrophotometric examination of the binding of compound 1 to DNAs of different base composition show that the apparent binding to GC is approximately 3 times that of AT, a result which was paralleled by thermal denaturation studies. Certain of the anthrapyrazoles exhibit marked visible light photosensitization and induce DNA single-strand breakage upon illumination in the presence of NADH. The essential structural requirement for photosensitizing properties with these agents was the absence of hydroxyl groups in the chromophore. By employing 32P-labeled DNA of known sequence, it was possible to examine the anthrapyrazole 1-photosensitized cleavage of DNA at the individual base level employing denaturing polyacrylamide sequencing gels. Smooth sequence neutral photosensitized cleavage of DNA is observed analogous to hydroxyl radical "footprinting."

Anthraquinones↗
PubMed1987

The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I.

Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence.

Base Sequence↗
PubMed1987

Cruciform extrusion in plasmids bearing the replicative intermediate configuration of a poxvirus telomere.

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (delta Gf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high delta Gf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured delta Gf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.

Base Sequence↗