Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.     

Effects of proflavin and photoactivated proflavin on the template function of single-stranded DNA

DNA context-specific effects of the association of proflavin, single-stranded DNA and DNA polymerase on DNA polymerization reactions were examined. Frameshift mutations induced by the presence of proflavin during in vitro DNA replication of a single-stranded DNA template by the Klenow fragment of Escherichia coli DNA polymerase I were sequenced. More than 80% of the frameshifts were one base-pair deletions opposite purine bases that were immediately 3' to pyrimidines. Purines (Pu) that were not adjacent to pyrimidines (Py) were not deletion sites. The remaining deletions were opposite template pyrimidines that were also immediately 3' to another pyrimidine. All pyrimidine site deletions occurred in the context 5' PyPyPu 3'. In additional experiments, the site-specific inhibition of processive DNA polymerization by proflavin was examined. A novel inhibition of polymerization was found opposite all pyrimidines in the template when proflavin-template complexes were exposed to ten seconds of white light. This inhibition of polymerization is reversible. Longer photoactivation led to an altered pattern of DNA sequence-specific inhibition that was not reversible. The role of DNA sequence-specific interactions of proflavin with DNA in proflavin mutagenesis is discussed.     

A structural model for sequence-specific proflavin-DNA interactions during in vitro frameshift mutagenesis.

Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5' pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it. Because the most frequent mutations are opposite Pu in the template sequence 5' Py Pu 3', a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin. The model seeks to rationalize the roles of the 5' pyrimidine and proflavin in facilitating the bypass. Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5' pyrimidine; 4) hydrogen bonding with the phosphate backbone. These models point to the possibility that a number of proflavin-DNA interactions may be involved. In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.     

A Structural Model For Sequence-Specific Proflavin-DNA Interactions During In Vitro Frameshift Mutagenesis

Abstract

Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5′ pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it. Because the most frequent mutations are opposite Pu in the template sequence 5′ Py Pu 3′, a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin. The model seeks to rationalize the roles of the 5′ pyrimidine and proflavin in facilitating the bypass. Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5′ pyrimidine; 4) hydrogen bonding with the phosphate backbone. These models point to the possibility that a number of proflavin-DNA interactions may be involved. In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.     

Effect of tetrahydrofuran on the binding of the competitive inhibitor proflavin and the storage stability of bovine pancreatic α‐chymotrypsin

The binding of the competitive inhibitor proflavin by bovine pancreatic α‐chymotrypsin in water‐tetrahydrofuran mixtures was studied in the entire range of thermodynamic water activities at 25°C. The data on the binding of proflavin were compared with the results on the storage stability of α‐chymotrypsin in water‐organic mixtures. An analysis of the concentration dependency of these characteristics demonstrated that, at low water activity values, the interprotein contacts in the enzyme formed during its drying largely govern its functional properties, while at high water activity, they are determined by the interaction of the enzyme with the organic solvent. The interplay of these two factors is responsible for the complex shape observed for the isotherm of binding of proflavin, with a maximum degree of binding being attained at medium water activity values.     

Nonintercalative binding of proflavin to Z-DNA: structure of a complex between d(5BrC-G-5BrC-G) and proflavin

The crystal structure of a disordered 1:1 complex between the tetradeoxyoligomer d(5BrC-G-5BrC-G) and proflavin has been determined and refined to an R factor of 26.9% for 474 reflections initially in space group P6(5) and to an R factor of 22.2% for 475 reflections in space group P2(1), both at 2-A resolution with Fobsd greater than or equal to 4.0. The unit cell constants are a = b = 17.9 A, c = 44.5 A, and gamma = 120 degrees. The final models are essentially the same in the two space groups with greater disorder in space group P6(5). In space group P2(1), the asymmetric unit is a tetranucleotide duplex, two sandwiched proflavin molecules, and four "outside-bound" proflavins. The tetranucleotide duplex is in the Z conformation and is located at the origin of the unit cell with a pair of proflavins sandwiched between the tetranucleotides. Thus, the tetranucleotides and proflavin dimers stack alternatively forming a quasi-continuous helix with the helix axis coincident with the c axis. The structure analysis revealed the presence of outside-bound proflavins as well. It is interesting that one type of outside-bound proflavins occupies a similar environment as the cobalt hexaammines in their complex with the decadeoxyoligomer d(CGTACGTACG) [Brennan, R. G., Westhof, E., & Sundaralingam, M. (1986) J. Biomol. Struct. Dyn. 3, 649]. Crystals of the latter are isomorphous to the present complex. The outside-bound proflavins penetrate the deep minor groove, thereby closing it off, and provide a visualization of a quasi-internal mode of binding of proflavin to a nucleic acid.     

Thrombin inhibitor design: X-ray and solution studies provide a novel P1 determinant

The crystal structures of proflavin and 6-fluorotryptamine thrombin have been completed showing binding of both ligands at the active site S1 pocket. The structure of proflavin:thrombin was confirmatory, while the structure of 6-fluorotryptamine indicated a novel binding mode at the thrombin active site. Furthermore, speculation that the sodium atom identified in an extended solvent channel beneath the S1 pocket may play a role in binding of these ligands was investigated by direct proflavin titrations as well as chromogenic activity measurements as a function of sodium concentration at constant ionic strength. These results suggested a linkage between the sodium site and the S1 pocket. This observation could be due to a simple ionic interaction between Asp189 and the sodium ion or a more complicated structural rearrangement of the thrombin S1 pocket. Finally, the unique binding mode of 6-fluorotryptamine provides ideas toward the design of a neutrally charged thrombin inhibitor.     

Characterization of the interaction between chymotrypsin and heparin.

Heparin forms a complex with chymotrypsin which is active towards glutaryl-L-phenylalanine-p-nitroanilide (GPANA) and glutaryl-L-phenylalanine-beta-naphthylamide (GPNA) at pH 7.6. The activity of chymotrypsin towards GPANA at pH 7.6 is enhanced in the presence of heparin. Heparin does not bind at the active site of the enzyme since proflavin is not displaced from the active site of chymotrypsin upon complex formation. The heparin-chymotrypsin complex migrates under basic polyacrylamide disc gel electrophoresis conditions to a position intermediate between heparin and free chymotrypsin. The complex is dissociable under acidic polyacrylamide gel electrophoresis conditions. It is estimated that one to three molecules of heparin can bind to each chymotrypsin molecule on the basis of electrophoretic and enzymic activity data.     

Proflavin and microwave radiation: absence of a mutagenic interaction

The potential ability of radiofrequency electromagnetic radiation (RFR) in the microwave range to induce mutagenesis, chromosomal aberrations, and sister chromatid exchanges in mammalian cells is being explored in our laboratories. In addition, we have also been examining the ability of simultaneous exposure to RFR and chemical mutagens to alter the genotoxic damage induced by chemical mutagens acting alone. We have performed experiments to determine whether there is an interaction between 2.45-GHz, pulsed-wave, RFR and proflavin, a DNA-intercalating drug. The endpoint studied was forward mutation at the thymidine kinase locus in L5178Y mouse leukemic cells. Any effect on the size distribution of the resulting colonies of mutated cells was also examined. The exposures were performed at net forward powers of 500 or 600 W, resulting in a specific absorption rate (SAR) of approximately 40 W/kg. The culture-medium temperature reached a 3 degrees C maximal increase during the 4-h exposure; appropriate 37 degrees C and convection-heating temperature controls (TC) were performed. In no case was there any indication of a statistically significant increase in the induced mutant frequency due to the simultaneous exposure to RFR and proflavin, as compared with the proflavin exposures alone. There was also no indication of any change in the colony-size distribution of the resulting mutant colonies, neither, and there was no evidence in these experiments of any mutagenic action by the RFR exposure alone.     

Kinetics of proflavin binding to bacteriophages T2L and T4D

We have measured the kinetics of proflavin binding to T-even bacteriophages—the 700 S and 1000 S forms of T2L, T4D, and T4D os41—by difference spectroscopy at 430 nm. Measurements were carried out from 22° to 37°C. Binding is very slow to encapsulated DNA compared to free DNA, requiring hours to reach equilibrium. The kinetic data are compatible with the two-step mechanism where P is proflavin, N is nucleotide, and PN₁ and PN₂ are complexes. Computer integration of the rate equations allows evaluation of the rate constants; previous equilibrium measurements gave thermodynamic parameters. For all phage studied, the bimolecular step is endothermic with high positive entropy; the second, unimolecular step is highly exothermic with small negative entropy change. Both forms of T2L bind proflavin with essentially the same rate, as do T4D and the osmotic shock resistant mutant T4D os41. This suggests that the encapsulated DNA is equally accessible to proflavin in both forms of each phage. However, T4D binds dye appreciably faster than T2L, indicating that capsid permeability or DNA environment (glucosylation or packing) is different in the two species.     

Interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT): thermodynamic analysis from (1)H NMR data]

Temperature relationships of chemical shifts of protons of proflavin mixed with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water solution were investigated on impulse NMR spectrometer (500 MHz). Procedure is suggested for calculating values of mole fractions of various associates in solution as a function against temperature. Free energies of Gibbs, entalpy and entropy were determined in the reactions of complex formation 1:1, 1:2, 2:1 of proflavin with tetranucleotide. The results point to a significant role of hydrophobic interactions during the formation of dye--tetramere duplex complexes.     

Effect of malathion on the initiation and elongation steps of transcription

In isolated cell nuclei of pig thymus malathion, S-1,2-bis(ethoxycarbonyl)-ethyl-O,O-dimethyldithiophosphate inhibited both initiation and elongation of all three classes of nuclear RNA polymerases; proflavin was used as an inhibitor of initiation, and actinomycin, as an inhibitor of elongation.     

The effects of N-cadherin misexpression on morphogenesis in Xenopus embryos

N-cadherin is a calcium-dependent, cell adhesion molecule that has been proposed to play a role in morphogenesis in vertebrate embryos. Throughout early neural development, N-cadherin is expressed during the morphogenetic changes that occur when ectoderm, in response to neural induction, forms a neural plate and tube. To study the role of N-cadherin in these processes, cDNA clones encoding Xenopus laevis N-cadherin were isolated and used to study the expression of N-cadherin in frog embryos. These studies showed that N-cadherin RNA is not expressed at detectable levels in early cleavage embryos or in isolated ectoderm in the absence of neural induction. However, N-cadherin RNA rapidly appeared in ectoderm exposed to a heterologous neural inducer, indicating that N-cadherin expression, as an early response to induction, precedes the morphogenetic events associated with early neural development. The role of N-cadherin in these morphogenetic events was studied by ectopically expressing N-cadherin in the ectoderm of embryos prior to induction. The ectopic expression of this protein in ectoderm led to the formation of cell boundaries and to severe morphological defects. These results are consistent with the hypothesis that the morphogenetic changes associated with early neural development are controlled, in part, by the induced expression of N-cadherin in the neural plate.     

Effect of cationic drugs on suprahelical organization of DNA

Interaction of a minor groove-binding drug Hoechst-33258, and an intercalating drug, proflavin, with the PSI (+) form of DNA, was studied using CD spectroscopy. Both drugs are shown to relax the suprahelical organization of DNA, leading to the formation of a B-like structure, above a certain drug to phosphate ratio. However, unlike proflavin, Hoechst-33258 brings about further structural changes after formation of the B-like structure whereas proflavin does not. A reversal of the CD signal in the 300–450-nm spectral region is also observed with Hoechst-33258, indicating a change in the handedness of the suprahelical organization of DNA. To the best of our knowledge, drug-mediated changes as presented in this paper have not been reported so far. © 1993 John Wiley & Sons, Inc.     

Hydrogen-adduct radicals in thymidine and thymidine 5'-monophosphate photosensitized by proflavin. Protection by sulfur compounds (author's transl)]

At pH values 5 and 9, the influence of cysteine, cystine, 3-mercaptopropionic acid and S-methylcysteine on the induction of H-adduct radicals in thymidine and thymidine 5'-monophosphate photosensitized by proflavin is investigated. The results obtained indicate that efficient protection is given when the electrostatic interactions between the charged groups of the molecules present allow the close proximity of a thiol or a disulfide bridge and the proflavin - DNAs complex constituent. The scheme proposed for the mechanism involves the capture by the protector of an electron or a proton, indispensable intermediates in the formation of the H-adduct radicals.     

Binding of proflavin to T2L bacteriophage and its DNA.

We have measured the binding equilibria of proflavin to T2L bacteriophage, in both “slow” and “fast” sedimenting forms, and to free T2L DNA. Measurements were carried out by difference spectroscopy at 430 nm at temperatures from 13 to 43°C and at pH 5.6 and 7.6. We found no significant difference in the binding parameters of the two phage forms. Also, the fraction of nucleotides available as binding sites for proflavin was the same for both free and intraphage DNA. However, the binding constant is about an order of magnitude lower for encapsulated than for free T2L DNA, due to the decreased exothermicity of the binding reaction within the phage head.     

Evidence for heterogeneity in DNA-associated solvent mobility from acridine phosphorescence spectra

The mobility of solvent associated with native DNA in comparison with that of the bulk solvent is monitored from the temperature-dependent red shift in the phosphorescence spectra of acridines bound to DNA and free in glycol–buffer mixtures. Over the temperature range for which the red shift occurs the phosphorescence decay changes with emission wavelength, indicating the time-dependent nature of the process. Moreover, at these temperatures, emission anisotropy measurements establish that motions of the dye itself are not involved. Correspondence between perturbations to the solvent that influence the temperature at which the red shift occurs for free acridine with those for the DNA-bound dye confirm that “bound solvent” is responsible for the spectral changes. For the DNA-bound acridines the extent of the red shift is smaller and the midpoint T_1/2 of the transition is warmer. The reduction in the red shift reveals that the bound dye is less exposed to solvent and varies as 9-aminoacridine < acridine orange ～ proflavin, i.e., 9-amino-acridine is less exposed to solvent. On the other hand, the warmer T_1/2 indicates that DNA-associated solvent is considerably less mobile than bulk solvent. T_1/2 varies for proflavin bound to DNA, poly[d(AT)], poly[d(GC)], and poly(dG): poly(dC), and for proflavin, acridine orange, and 9-aminoacrine bound to DNA. These observations suggest that there is a heterogeneity in the mobility of DNA-associated solvent.     

Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the α-chymotrypsin–proflavin interaction

Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K _obs, ΔH _obs⁰). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between α-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between α-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

L'action differentielle des hormones juveniles sur la metamorphose chez Locusta migratoria

The morphogenetic effect on metamorphosis of the three juvenile hormones is studied under experimental conditions that permit accurate conclusions in Locusta migratoria. Injections are made at the beginning on the last larval instar, which is the best time for action at the optimum level on the control of metamorphosis. Racemic hormones in stereochemical form comparable to that of natural compounds were used. Doses were chosen between 5 and 50 μg in order to give clear morphogenetic effects and give effects at a physiological level.A chronological study showed that juvenile hormones have the most important morphogenetic effect when injected in the first 40 hr of the last larval instar. Oiled solutions, stored at 4°C, lost only a small part of their morphogenetic activity after 9 months.Among the three hormones, JH-III presented the weaker morphogenetic effect, very significantly different from that of JH-I and JH-II. It has been possible to dissociate the effects of JH-I and JH-II on metamorphosis, JH-I giving a more potent action.