Linear Dichroism Characteristics of Ethidium and Proflavine Supercoiled DNA Complexes

DTIC Science & Technology

1990-01-01

of Ethidium-and Proflavine -Supercoi led DNA Complexes CHARLES E. SWENBERG, 1 SUSAN E. CARBERRV, 2 * and NICHOLAS E. GEACINTOV’ SRadliationl Biochenitr...drug were stained with EB, photographed under uv light, molecules, EB and proflavine (PF) (Figure 1), and the bands were quantitated by densitometry

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1.

PubMed

DeJong, Eric S; Chang, Chia-en; Gilson, Michael K; Marino, John P

2003-07-08

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.

Change of the binding mode of the DNA/proflavine system induced by ethanol.

PubMed

García, Begoña; Leal, José M; Ruiz, Rebeca; Biver, Tarita; Secco, Fernando; Venturini, M

2010-07-01

The equilibria and kinetics of the binding of proflavine to poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) were investigated in ethanol/water mixtures using spectrophotometric, circular dichroism, viscometric, and T-jump methods. All methods concur in showing that two modes of interaction are operative: intercalation and surface binding. The latter mode is favored by increasing ethanol and/or the proflavine content. Both static and kinetic experiments show that, concerning the poly(dG-dC).poly(dG-dC)/proflavine system, intercalation largely prevails up to 20% EtOH. For higher EtOH levels surface binding becomes dominant. Concerning the poly(dA-dT).poly(dA-dT)/proflavine system, melting experiments show that addition of proflavine stabilizes the double stranded structure, but the effect is reduced in the presence of EtOH. The DeltaH degrees and DeltaS degrees values of the melting process, measured at different concentrations of added proflavine, are linearly correlated, revealing the presence of the enthalpy-entropy compensation phenomenon (EEC). The nonmonotonicity of the "entropic term" of the EEC reveals the transition between the two binding modes. T-jump experiments show two relaxation effects, but at the highest levels of EtOH (>25%) the kinetic curves become monophasic, confirming the prevalence of the surface complex. A branched mechanism is proposed where diffusion controlled formation of a precursor complex occurs in the early stage of the binding process. This evolves toward the surface and/or the intercalated complex according to two rate-determining parallel steps. CD spectra suggest that, in the surface complex, proflavine is bound to DNA in the form of an aggregate.

Intercalation complex of proflavine with DNA: Structure and dynamics by solid-state NMR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Pei; Juang, Chilong; Harbison, G.S.

1990-07-06

The structure of the complex formed between the intercalating agent proflavine and fibrous native DNA was studied by one- and two-dimensional high-resolution solid-state nuclear magnetic resonance (NMR). Carbon-13-labeled proflavine was used to show that the drug is stacked with the aromatic ring plane perpendicular to the fiber axis and that it is essentially immobile. Natural abundance carbon-13 NMR of the DNA itself shows that proflavine binding does not change the puckering of the deoxyribose ring. However, phosphorus-31 NMR spectra show profound changes in the orientation of the phosphodiester grouping on proflavine binding, with some of the phosphodiesters tilting almost parallelmore » to the helix axis, and a second set almost perpendicular. The first group to the phosphodiesters probably spans the intercalation sites, whereas the tilting of the second set likely compensates for the unwinding of the DNA by the intercalator.« less

RESISTANCE AND CROSS-RESISTANCE OF ESCHERICHIA COLI S MUTANTS TO THE RADIOMIMETIC AGENT PROFLAVINE

PubMed Central

Woody-Karrer, Pearl; Greenberg, Joseph

1964-01-01

Woody-Karrer, Pearl (Palo Alto Medical Research Foundation, Palo Alto, Calif.), and Joseph Greenberg. Resistance and cross-resistance of Escherichia coli S mutants to the radiomimetic agent proflavine. J. Bacteriol. 87:536–542. 1964.—All 50 of the first-step mutants of Escherichia coli S selected for resistance to proflavine were resistant to ultraviolet light and each of five different radiomimetic chemicals. The mutants were classified into eight types on the basis of their relative resistance to six different radiomimetic drugs and on the basis of the shape of their ultraviolet survival curves. Three of these types are identical to types previously isolated with other radiomimetic drugs; five of the types are new. A high proportion of the clones surviving proflavine treatment were phenotypically but not genetically resistant, and no strains were isolated which were resistant to proflavine but were not resistant to radiation. PMID:14129667

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA.

PubMed

Piette, J; Calberg-Bacq, C M; Lopez, M; van de Vorst, A

1984-04-05

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.

Determination of proflavine in rat whole blood without sample pretreatment by laser desorption postionization mass spectrometry.

PubMed

Chen, Jiaxin; Hu, Yongjun; Lu, Qiao; Wang, Pengchao; Zhan, Huaqi

2017-04-01

A novel pretreatment-free method involving laser desorption postionization (LDPI) coupled with time-of-flight mass spectrometry (MS) was developed for the monitoring of proflavine level in rat whole blood. It comprises a protocol for dosing via intravenous administration and collection of whole blood, followed by direct LDPI-MS analysis without any sample pretreatment. An intense ion signal at m/z 209 was observed from whole blood without any interference signals, except some background signals below m/z 100. The calibration curve was established with use of 9-phenylacridine as the internal standard for proflavine determination from the plotting of the peak ratios of proflavine to the internal standard, with a correlation coefficient (R 2 ) greater than 0.99. The limit of detection was estimated to be 0.48 pmol/mm 2 and the quantification range was 0.5-16.5 μg/mL for proflavine. In addition, only a minimal matrix effect was observed, as expected from considerations of the desorption and ionization mechanism. Interday and intraday accuracy and precision were calculated to be within 13% and 82-114%, respectively. Estimated concentrations of proflavine residue in whole blood were also successfully obtained at selected time points after dosing. The proposed method is simple, low cost, and sensitive, and should be seen as a complementary method for monitoring drug levels in blood. Graphical Abstract Monitoring proflavine levels in rat whole blood at different time points using laser desorption postionization mass spectrometry (LDPI-MS).

Proflavine sensitivity of RNA processing in isolated nuclei.

PubMed Central

Yannarell, A; Niemann, M; Schumm, D E; Webb, T E

1977-01-01

The intercalating agent proflavine inhibits the processing and subsequent release of preformed messenger RNA and ribosomal RNA from isolated liver nuclei to surrogate cytoplasm. The direct effect of proflavine on these processes, as monitored in a reconstituted cell-free system, supports the theory that base-paired segments (i.e. hairpin loops) in the precursor RNA's are involved as recognition sites in nuclear RNA processing. PMID:866181

[Experimental study of proflavine acetate phototransformation processes].

PubMed

Zholdakova, Z I; Sinitsyna, O O; Lebedev, A T; Kharchevnikova, N V

2009-01-01

Changes in proflavine acetate phototransformation processes upon exposure to visible-range irradiation were studied by high performance liquid chromatography. Proflavine acetate was offered as a photosensitizer during photodynamic water disinfection. Dye transformation products upon time-varying exposure to irradiation were identified. By using structure-activity relationships and information from toxicity databases, the authors evaluated the hazard of the identified products and identified the most hazardous ones.

Interaction of proflavine with DNA studied by colloid surface enhanced resonance Raman spectroscopy

NASA Astrophysics Data System (ADS)

Koglin, E.; Séquaris, J.-M.

1986-03-01

The interaction of the mutagenic highly fluourescing proflavine (3,6-diaminoacridine: PF) dye with calf thymus DNA has been studied by Surface Enhanced Resonance Raman Scattering (SERRS). Since the Ag-colloids almost completely quenche the strong fluorescence it is possible to obtain excellent vibrational spectra in a wide frequency range providing valuable information about the intercalation. The intercalation does not affect the vibrational frequencies of the proflavine dye. On the other hand, intensity changes are observed in some of the ring- and NH 2-modes of proflavine upon intercalation. This Raman hypochromism is characteristic for ring stacking interactions and in the SERRS spetroscopy for an additional effects of the dye orientation to the surface.

EFFECT OF PROFLAVINE ON THE SYNTHESIS OF ADENOVIRUS, TYPE 5, AND ASSOCIATED SOLUBLE ANTIGENS

PubMed Central

Wilcox, Wesley C.; Ginsberg, Harold S.

1962-01-01

Wilcox, Wesley C. (University of Pennsylvania, Philadelphia) and Harold S. Ginsberg. Effect of proflavine on the synthesis of adenovirus, type 5, and associated soluble antigens. J. Bacteriol. 84:526–533. 1962.—The synthesis of type 5 adenovirus in HeLa cells was suppressed to a considerable extent by low concentrations of proflavine, an acridine dye. In comparison, the processes leading to the production of soluble complement-fixing antigens and toxin were less sensitive to the action of this chemical. Addition of proflavine to infected cells at different times during the virus growth cycle revealed that the processes leading to the synthesis of soluble antigens began prior to the first appearance of newly synthesized virus. This observation is compatible with the hypothesis that the soluble antigens may represent virus subunits or precursor materials. In addition, these data indicate that it is possible to interrupt the latter stages of the virus synthetic process by addition of proflavine late in the eclipse period. PMID:14000661

Absence of Mutagenic Activity of Hycanthone in Serratia marcescens,

DTIC Science & Technology

1986-05-29

repair system but is enhanced by the plasmid pKMl01, which mediates the inducible error-prone repair system. Hycanthone, like proflavin , .1...enhanced by the plasmid pKM10, which mediates the inducible error-prone repair system. Hycanthone, like proflavin , intercalates between the stacked bases...Roth (1974) lave suggested that proflavin , which has a planar triple ring structure similar to hycanthone, interacts with DNA, which upon replication

Structure and dynamics of proflavine association around DNA.

PubMed

Sasikala, Wilbee D; Mukherjee, Arnab

2016-04-21

Proflavine is a small molecule that intercalates into DNA and, thereby, acts as an anticancer agent. Intercalation of proflavine is shown to be a two-step process in which the first step is believed to be the formation of a pre-intercalative outside bound state. Experimental studies so far have been unable to capture the nature of the outside bound state. However, the sub-millisecond timescale observed in fluorescence kinetic experiments is often attributed to the binding of proflavine outside of DNA. Here, we have performed molecular dynamics simulations with multiple proflavine molecules to study the structure and dynamics of the formation of the outside bound state of DNA at different ion concentrations. We observed that the timescale of the outside bound state formation is, at least, five orders of magnitude faster (in nanoseconds) than the experimentally reported timescale (sub-milliseconds) attributed to binding outside DNA. Moreover, we also observed the stacked arrangement of proflavine all around DNA, which is different from the experimentally predicted stacking arrangement perpendicular to the helical axis of DNA in the close vicinity of the phosphate groups. This study, therefore, provides insight into the molecular structure and dynamics of the pre-intercalative outside bound state and will help in understanding the overall intercalation mechanism.

Visualization of drug-nucleic acid interactions at atomic resolution v. structure of two aminoacridine/dinucleoside monophosphate crystalline complexes, proflavine: 5-iodocytidylyl(3'-5') guanosine and acridine orange: 5-iodocytidylyl(3'-5') guanosine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, B.S.; Seshadri, T.P.; Sakore, T.D.

1979-01-01

Acridine orange and proflavine form complexes with the dinucleoside monophosphate, 5-iodocytidylyl(3'-5') guanosine (iodoCpG). The acridine orange-iodoCpG crystals are monoclinic, space group P2/sub 1/, with unit cell dimensions a = 14.36 A, b = 19.64 A, c = 20.67 A, ..beta.. = 102.5. The proflavine-iodoCpG crystals are monoclinic, space group C2, with unit cell dimensions a = 32.14 A, b = 22.23 A, c = 18.42 A, ..beta.. = 123.3. Both structures have been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least squares. Acridine orange forms an intercalative structure with iodoCpG but the acridinemore » nucleus lies asymmetrically in the intercalation site. This asymmetric intercalation is accompanied by a sliding of base-pairs upon the acridine nucleus. Base-pairs above and below the drug are separated by about 6.8 A and are twisted about 10/sup 0/. Proflavine demonstrates symmetric intercalation with iodoCpG. Hydrogen bonds connect amino- groups on proflavine with phosphate oxygen atoms on the dinucleotide. Base-pairs above and below the intercalative proflavine molecule are twisted about 36/sup 0/. The altered magnitude of this angular twist reflects the sugar puckering pattern that is observed. We propose a proflavine-DNA and an acridine orange-DNA binding model. We will describe these models in detail in this paper.« less

Visible light-driven NADH regeneration sensitized by proflavine for biocatalysis.

PubMed

Nam, Dong Heon; Park, Chan Beum

2012-06-18

Harvest time: Proflavine drives the reduction of NAD(+) in the presence of a Rh-based electron mediator. Photoregenerated NADH was enzymatically active for oxidation by NADH-dependent L-glutamate dehydrogenase for the synthesis of L-glutamate. This work suggests that proflavine has the potential to become an efficient light-harvesting component in biocatalytic photosynthesis driven by solar energy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immunologic and Genetic Selection of Adenovirus Vaccine Strains: Synthesis and Characterization of Adenovirus Antigens.

DTIC Science & Technology

1984-08-01

exhibited strikingly different chromatographic characteristics. 2. Effect of proflavine on the synthesis of adenovirus, type 5, and associated soluble...antigens. The synthesis of type 5 adenovirus in HeLa cells was suppressed to a considerable extent by low concentrations of proflavine , an acridine dye...chemical. Addition of proflavine to infected cells at different times during the virus growth cycle revealed that the processes leading to the synthesis

A C-di-GMP-proflavine-hemin supramolecular complex has peroxidase activity--implication for a simple colorimetric detection.

PubMed

Nakayama, Shizuka; Roelofs, Kevin; Lee, Vincent T; Sintim, Herman O

2012-03-01

Herein, we demonstrate that the bacterial signaling molecule, c-di-GMP, can enhance the peroxidation of hemin when proflavine is present. The c-di-GMP-proflavine-hemin nucleotidezyme can oxidize the colorless compound 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, to the colored radical cation ABTS˙(+) and hence provides simple colorimetric detection of c-di-GMP at low micromolar concentrations.

RESISTANCE AND CROSS-RESISTANCE OF ESCHERICHIA COLI S MUTANTS TO THE RADIOMIMETIC AGENT PROFLAVINE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woody-Karrer, P.; Greenberg, J.

1964-03-01

All 50 of the first-step mutants of Escherichia coli S selected for resistance to proflavine were resistant to uv light and each of five different radiomimetic chemicals. The mutants were classified into eight types on the basis of their relative resistance to six different radiomimetic drugs and on the basis of the shape of their uv survival curves. Three of these types are identical to types previously isolated with other radiomimetic drugs; five of the types are new. A high proportion of the clones surviving proflavine treatment were phenotypically but not genetically resistant, and no strains were isolated which weremore » resistant to proflavine but were not resistant to radiation. (auth)« less

A molecular model for proflavine-DNA intercalation.

PubMed Central

Neidle, S; Pearl, L H; Herzyk, P; Berman, H M

1988-01-01

A molecular model has been derived for the intercalation of proflavine into the CpG site of the decamer duplex of d(GATACGATAC). The starting geometry of the intercalation site was taken from previous crystallographic studies on the d(CpG)-proflavine complex, and molecular mechanics used to obtain a stereochemically acceptable structure. This has widened grooves compared to standard A- or B- double helices, as well as distinct conformational, roll, twist and tilt features. PMID:3174439

Selective protection of cultured human cells from the toxic effects of ultraviolet light by proflavine pretreatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, J.R.; Little, J.B.

1977-10-01

Pretreatment of LICH human cells by nontoxic doses (0.1 to 5.0 ..mu..g/ml) of proflavine protects them from inactivation by ultraviolet light. The protection is acquired rapidly after exposure of cells to proflavine, with 50 percent of maximum protection being afforded within 5 min and cells being maximally protected by 20 min. Loss of protection follows similar kinetics upon removal of proflavine from the culture medium. Protection is selective and cannot be explained on the basis of proflavine absorption of uv light. Cellular survival curves after ultraviolet light for cells protected by 1, 2, 3, 4, or 5 ..mu..g/ml of proflavinemore » show that protection alters only the slope of the survival curve, not altering the quasi-threshold dose, D/sub q/. The D/sub 0/ varies from 4.8 J/m/sup 2/ for untreated cells to 10.5 J/m/sup 2/ for cells pretreated with 5 ..mu..g/ml. These data suggest the D/sub 0/ and D/sub q/ do not represent parameters of a single underlying process, manifested in a random stochastic manner, but may reflect different cellular mechanisms or responses to different DNA damage. Proflavine is selective in mitigating only those which predominate at uv doses greater than the D/sub q/.« less

Studies on transcription termination and splicing of the rRNA precursor in vivo in the presence of proflavine.

PubMed Central

Nielsen, O F; Carin, M; Westergaard, O

1984-01-01

In isolated nucleoli from Tetrahymena thermophila, low concentrations of the intercalating agent proflavine inhibit both transcription termination and splicing of the rRNA precursor. Proflavine also exerts an in vivo effect on the process of transcription termination under conditions, where the growth rate is only slightly reduced. Thus, approximately 40% of the rRNA precursor molecules, accumulated in nucleoli during 60 min of treatment with the drug, are longer than the normal 35S rRNA precursor. R-Loop mapping of these longer precursor molecules isolated after 30 and 60 min of incubation demonstrates that the RNA polymerases have a 50 fold lower elongation rate in the spacer region than in the coding region. Proflavine in the given concentration is found to have no significant effect on the splicing of properly terminated precursor molecules. In contrast, none of the longer non-terminated molecules are found to be spliced. These results indicate that proflavine primarily affects the process of transcription termination and that the splicing event is inhibited due to the improper termination of the precursor molecule. Images PMID:6694912

Electroanalytical study of proflavine intercalation in 5-methyl or inosine-containing amplicons.

PubMed

Alexiadou, Despina K; Ioannou, Andrea K; Kouidou-Andreou, Sofia A; Voulgaropoulos, Anastasios N; Girousi, Stella Th

2008-10-01

Amplicons corresponding to the GC-rich p53 exon 5 and its analogues, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, or 60% of guanosine by inosine and GC-poor p53 exon 6 were synthesized and investigated electrochemically, in the presence and absence of proflavine, by differential pulse voltammetry (DPV). Incorporation of base analogues and the thermal stability of the resulting amplicons were tested in the presence of a fluorescent probe (Sybr-Green). Peak current at 1.0 V was lower for methylated than for unmethylated PCR amplicons and was similarly affected by proflavine intercalation. In contrast, considerable peak current differences were observed in the presence of proflavine for unmodified exon 5 v.s. exon 6 or inosine-containing amplicons. Thermal analysis verified the expected shifts in melting temperature (T (m)) due to the base analogue incorporation and GC-content variations. In conclusion, methylated and unmethylated PCR amplicons could be distinguished in model DNA systems using differential pulse voltammetry (DPV) and use of proflavine could serve as an electrochemical probe for identifying different DNA conformations.

Interaction of Ionizing Radiation, Genetically Active Chemicals, and Radiofrequency Radiation in Human and Rodent Cells

DTIC Science & Technology

1990-12-01

proflavin , a drug known to intercalate with DNA. Again, when cells were exposed simultaneously to RFR SAR = 40.8- + 13.4 (SD) W/kg or 40 W/kg at power...densities of 87 or 65 mW/cm ), no effect of the RFR on the proflavin induced mutagenicity was observed (Meltz et al., 1990). SCE Induction Previously...Meltz ML, Eagan P, and Erwin DN (1990). Proflavin and Microwave Radiation: Absence of a Mutagenic Interaction. Bioelectromagnetics 11:149-157. Ciaravino

Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects".

PubMed

Antonini, E; Ascenzi, P; Bolognesi, M; Menegatti, E; Guarneri, M

1983-04-25

The formation of the bovine beta-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time (Antonini, E., Ascenzi, P., Menegatti, E., and Guarneri, M. (1983) Biopolymers 22, 363-375). These data indicated that the bovine beta-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine beta-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine beta-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine beta-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contact area.

Optical spectrum of proflavine and its ions

NASA Astrophysics Data System (ADS)

Bonaca, A.; Bilalbegović, G.

2010-06-01

Motivated by possible astrophysical and biological applications we calculate visible and near UV spectral lines of proflavine (C13H11N3, 3,6-diaminoacridine) in vacuum, as well as its anion, cation, and dication. The pseudopotential density functional and time-dependent density functional methods are used. We find a good agreement in spectral line positions calculated by two real-time propagation methods and the Lanczos chain method. Spectra of proflavine and its ions show characteristic UV lines which are good candidates for a detection of these molecules in interstellar space and various biological processes.

Sequence specificity of mutagen-nucleic acid complexes in solution: intercalation and mutagen-base pair overlap geometries for proflavine binding to dC-dC-dG-dG and dG-dG-dC-dC self-complementary duplexes.

PubMed

Patel, D J; Canuel, L L

1977-07-01

The complex formed between the mutagen proflavine and the dC-dC-dG-dG and dG-dG-dC-dC self-complementary tetranucleotide duplexes has been monitored by proton high resolution nuclear magnetic resonance spectroscopy in 0.1 M phosphate solution at high nucleotide/drug ratios. The large upfield shifts (0.5 to 0.85 ppm) observed at all the proflavine ring nonexchangeable protons on complex formation are consistent with intercalation of the mutagen between base pairs of the tetranucleotide duplex. We have proposed an approximate overlap geometry between the proflavine ring and nearest neighbor base pairs at the intercalation site from a comparison between experimental shifts and those calculated for various stacking orientations. We have compared the binding of actinomycin D, propidium diiodide, and proflavine to self-complementary tetranucleotide sequences dC-dC-dG-dG and dG-dG-dC-dC by UV absorbance changes in the drug bands between 400 and 500 nm. Actinomycin D exhibits a pronounced specificity for sequences with dG-dC sites (dG-dG-dC-dC), while propidium diiodide and proflavine exhibit a specificity for sequences with dC-dG sites (dC-dC-dG-dG). Actinomycin D binds more strongly than propidium diiodide and proflavine to dC-dG-dC-dG (contains dC-dG and dG-dC binding sites), indicative of the additional stabilization from hydrogen bonding and hydrophobic interactions between the pentapeptide lactone rings of actinomycin D and the base pair edges and sugar-phosphate backbone of the tetranucleotide duplex.

Sequence specificity of mutagen-nucleic acid complexes in solution: Intercalation and mutagen-base pair overlap geometries for proflavine binding to dC-dC-dG-dG and dG-dG-dC-dC self-complementary duplexes

PubMed Central

Patel, Dinshaw J.; Canuel, Lita L.

1977-01-01

The complex formed between the mutagen proflavine and the dC-dC-dG-dG and dG-dG-dC-dC self-complementary tetranucleotide duplexes has been monitored by proton high resolution nuclear magnetic resonance spectroscopy in 0.1 M phosphate solution at high nucleotide/drug ratios. The large upfield shifts (0.5 to 0.85 ppm) observed at all the proflavine ring nonexchangeable protons on complex formation are consistent with intercalation of the mutagen between base pairs of the tetranucleotide duplex. We have proposed an approximate overlap geometry between the proflavine ring and nearest neighbor base pairs at the intercalation site from a comparison between experimental shifts and those calculated for various stacking orientations. We have compared the binding of actinomycin D, propidium diiodide, and proflavine to self-complementary tetranucleotide sequences dC-dC-dG-dG and dG-dG-dC-dC by UV absorbance changes in the drug bands between 400 and 500 nm. Actinomycin D exhibits a pronounced specificity for sequences with dG-dC sites (dG-dG-dC-dC), while propidium diiodide and proflavine exhibit a specificity for sequences with dC-dG sites (dC-dC-dG-dG). Actinomycin D binds more strongly than propidium diiodide and proflavine to dC-dG-dC-dG (contains dC-dG and dG-dC binding sites), indicative of the additional stabilization from hydrogen bonding and hydrophobic interactions between the pentapeptide lactone rings of actinomycin D and the base pair edges and sugar-phosphate backbone of the tetranucleotide duplex. PMID:268613

Comparison of the treatment of herpes genitalis in men with proflavine photoinactivation, idoxuridine ointment, and normal saline.

PubMed Central

Taylor, P K; Doherty, N R

1975-01-01

36 male patients with genital infection by HSV confirmed by culture were each allocated to one of three treatment groups: (1) Proflavine photoinactivation, (2) 0.5 per cent. idoxuridine ointment (IDU), (3) Normal saline. They were assessed objectively at each attendance by measurement of the lesions with an operating microscope fitted with a measuring grid in one eyepiece. Material for culture for HSV was taken at each visit; the presence of symptoms (pain, discomfort, and irritation) was noted. The areas of lesions in the proflavine photoinactivation group remained larger significantly longer than in the other groups, the healing time was slower, and HSV could be isolated for longer. It is concluded that proflavine photoinactivation is of no greater value than 0.5 per cent. IDU or normal saline in the treatment of genital infection by HSV in the male. PMID:1093634

Tulane/Xavier Vaccine Development/Engineering Project

DTIC Science & Technology

2009-02-01

spectroscopic studies with polar dyes (e.g. proflavine ) have verified these compounds’ ability to encapsulate and solvate small polar dye molecules in...systems. Fluorescent microscopy studies verify that they significantly enhance the transport of polar small molecules ( proflavin dye) through

Proflavine derivatives as fluorescent imaging agents of amyloid deposits.

PubMed

Garin, Dominique; Oukhatar, Fatima; Mahon, Andrew B; Try, Andrew C; Dubois-Dauphin, Michel; Laferla, Frank M; Demeunynck, Martine; Sallanon, Marcelle Moulin; Chierici, Sabine

2011-04-15

A series of proflavine derivatives for use to further image Aβ amyloid deposits were synthesized and characterized. Aged 3xTg-AD (23 months old) mice hippocampus sections incubated with these derivatives revealed preferential labeling of amyloid plaques. Furthermore an in vitro binding study showed an inhibitory effect, although moderate, of these compounds on Aβ(40) fibril formation. This study highlights the potential of proflavine as a molecular scaffold for designing new Aβ imaging agents, its native fluorescence allowing in vitro neuropathological staining in AD damaged brain sections. Copyright © 2011 Elsevier Ltd. All rights reserved.

PHOTOSENSITIZATION OF ARBOVIRUSES BY ACRIDINE DYES

DTIC Science & Technology

This paper describes the photodynamic action of proflavine and acridine orange on eastern equine encephalitis (EEE) and Venezuelan equine...encephalitis (VEE) viruses. The viruses were more sensitive to the photodynamic action of acridine orange than to that of proflavine . VEE virus was slightly more

Kir Channel Blockages by Proflavine Derivatives via Multiple Modes of Interaction.

PubMed

Inanobe, Atsushi; Itamochi, Hideaki; Kurachi, Yoshihisa

2018-06-01

Many compounds inhibit tetrameric and pseudo-tetrameric cation channels by associating with the central cavity located in the middle of the membrane plane. They traverse the ion conduction pathway from the intracellular side and through access to the cavity. Previously, we reported that the bacteriostatic agent, proflavine, preferentially blocked a subset of inward rectifier K + (Kir) channels. However, the development of the inhibition of Kir1.1 by the compound was obviously different from that operating in Kir3.2 as a pore blocker. To gain mechanistic insights into the compound-channel interaction, we analyzed its chemical specificity, subunit selectivity, and voltage dependency using 13 different combinations of Kir-channel family members and 11 proflavine derivatives. The Kir-channel family members were classified into three groups: 1) Kir2.2, Kir3.x, Kir4.2, and Kir6.2Δ36, which exhibited Kir3.2-type inhibition (slow onset and recovery, irreversible, and voltage-dependent blockage); 2) Kir1.1 and Kir4.1/Kir5.1 (prompt onset and recovery, reversible, and voltage-independent blockage); and 3) Kir2.1, Kir2.3, Kir4.1, and Kir7.1 (no response). The degree of current inhibition depended on the combination of compounds and channels. Chimera between proflavine-sensitive Kir1.1 and -insensitive Kir4.1 revealed that the extracellular portion of Kir1.1 is crucial for the recognition of the proflavine derivative acrinol. In conclusion, preferential blockage of Kir-channel family members by proflavine derivatives is based on multiple modes of action. This raises the possibility of designing subunit-specific inhibitors. Copyright © 2018 by The Author(s).

Existence of a new emitting singlet state of proflavine: femtosecond dynamics of the excited state processes and quantum chemical studies in different solvents.

PubMed

Kumar, Karuppannan Senthil; Selvaraju, Chellappan; Malar, Ezekiel Joy Padma; Natarajan, Paramasivam

2012-01-12

Proflavine (3,6-diaminoacridine) shows fluorescence emission with lifetime, 4.6 ± 0.2 ns, in all the solvents irrespective of the solvent polarity. To understand this unusual photophysical property, investigations were carried out using steady state and time-resolved fluorescence spectroscopy in the pico- and femtosecond time domain. Molecular geometries in the ground and low-lying excited states of proflavine were examined by complete structural optimization using ab initio quantum chemical computations at HF/6-311++G** and CIS/6-311++G** levels. Time dependent density functional theory (TDDFT) calculations were performed to study the excitation energies in the low-lying excited states. The steady state absorption and emission spectral details of proflavine are found to be influenced by solvents. The femtosecond fluorescence decay of the proflavine in all the solvents follows triexponential function with two ultrafast decay components (τ(1) and τ(2)) in addition to the nanosecond component. The ultrafast decay component, τ(1), is attributed to the solvation dynamics of the particular solvent used. The second ultrafast decay component, τ(2), is found to vary from 50 to 215 ps depending upon the solvent. The amplitudes of the ultrafast decay components vary with the wavelength and show time dependent spectral shift in the emission maximum. The observation is interpreted that the time dependent spectral shift is not only due to solvation dynamics but also due to the existence of more than one emitting state of proflavine in the solvent used. Time resolved area normalized emission spectral (TRANES) analysis shows an isoemissive point, indicating the presence of two emitting states in homogeneous solution. Detailed femtosecond fluorescence decay analysis allows us to isolate the two independent emitting components of the close lying singlet states. The CIS and TDDFT calculations also support the existence of the close lying emitting states. The near constant lifetime observed for proflavine in different solvents is suggested to be due to the similar dipole moments of the ground and the evolved emitting singlet state of the dye from the Franck-Condon excited state.

Spectroscopic study of proflavine adsorption on the carbon nanotube surface.

PubMed

Buchelnikov, Anatoly S; Dovbeshko, Galina I; Voronin, Dmitry P; Trachevsky, Vladimir V; Kostjukov, Viktor V; Evstigneev, Maxim P

2014-01-01

Despite the fact that non-covalent interactions between various aromatic compounds and carbon nanotubes are being extensively investigated now, there is still a lack of understanding about the nature of such interactions. The present paper sheds light on one of the possible mechanisms of interaction between the typical aromatic dye proflavine and the carbon nanotube surface, namely, π-stacking between aromatic rings of these compounds. To investigate such a complexation, a qualitative analysis was performed by means of ultraviolet visible, infrared, and nuclear magnetic resonance spectroscopy. The data obtained suggest that π-stacking brings the major contribution to the stabilization of the complex between proflavine and the carbon nanotube.

Time-resolved and steady-state fluorescence studies of excited-state proton-transfer reactions of proflavine

NASA Astrophysics Data System (ADS)

De Silvestri, S.; Laporta, P.

1984-01-01

Time-resolved and steady-state fluorescence studies of proflavine in aqueous solution are presented. The observation of a monoexponential fluorescence decay with a time constant decreasing with increasing pH and the presence of an anomalous red-shift in the fluorescence spectrum as a function of pH indicate the existence of a complex proton-transfer mechanism in the excited state. A reaction scheme is proposed and the corresponding proton-transfer rates are evaluated. An excited-state pK value of 12.85 is obtained for the equilibrium between the cationic form of proflavine and the same form dissociated at an amino group.

Attempt to develop live attenuated bacterial vaccines by selecting resistance to gossypol, proflavine hemisulfate, novobiocin, or ciprofloxacin

USDA-ARS?s Scientific Manuscript database

In an attempt to develop attenuated bacteria as potential live vaccines, four chemicals (gossypol, proflavine hemisulfate, novobiocin, and ciprofloxacin) were used to modify the following four genera of bacteria through chemical-resistance strategy: 1) Aeromonas hydrophila (9 isolates); 2) Edwardsie...

Attempt to develop live attenuated bacterial vaccines by selecting resistance to gossypol, proflavine hemisulfate, novobiocin, or ciprofloxacin

USDA-ARS?s Scientific Manuscript database

In an attempt to develop attenuated bacteria as potential live vaccines, four chemicals (gossypol, proflavine hemisulfate, novobiocin, and ciprofloxacin) were used to modify the following four genera of bacteria through chemical-resistance strategy: (1) Aeromonas hydrophila (9 isolates); (2) Edwards...

INFLUENCE OF CHEMICAL AND PHYSICAL MUTAGENIC FACTORS ON THE VIRUS of tick-BORNE ENCEPHALITIS

DTIC Science & Technology

In experiments the chemicals azouridine, azouracil, 5-bromuracil, formaldehyde, urethan and proflavine were used as mutagenic agents. The influence of...with passaging (tests with proflavine ). Subject to further study is the problem of the reversibility of these properties, and also the fixing of

Interaction of proflavin with aromatic amines in homogeneous and micellar media: Photoinduced electron transfer probed by magnetic field effect

NASA Astrophysics Data System (ADS)

Chakraborty, Brotati; Basu, Samita

2010-02-01

Photoinduced electron transfer (PET) between proflavin (PF +) and two aromatic amines viz., dimethylaniline (DMA) and 4,4'-bis(dimethylamino)diphenylmethane (DMDPM) is studied in homogeneous and heterogeneous media using steady-state as well as time-resolved fluorescence spectroscopy and laser flash photolysis with an associated magnetic field. Ionic micelles have been used to study the effect of charge of proflavin on PET with amines. Magnetic field effect on PET reactions reveals that the parent spin-state of precursors of PET for DMA-PF + system is singlet while for DMDPM-PF + system is triplet, implying that the dynamics of PET is influenced by the structure of the donor.

Enantiospecific recognition of DNA sequences by a proflavine Tröger base.

PubMed

Bailly, C; Laine, W; Demeunynck, M; Lhomme, J

2000-07-05

The DNA interaction of a chiral Tröger base derived from proflavine was investigated by DNA melting temperature measurements and complementary biochemical assays. DNase I footprinting experiments demonstrate that the binding of the proflavine-based Tröger base is both enantio- and sequence-specific. The (+)-isomer poorly interacts with DNA in a non-sequence-selective fashion. In sharp contrast, the corresponding (-)-isomer recognizes preferentially certain DNA sequences containing both A. T and G. C base pairs, such as the motifs 5'-GTT. AAC and 5'-ATGA. TCAT. This is the first experimental demonstration that acridine-type Tröger bases can be used for enantiospecific recognition of DNA sequences. Copyright 2000 Academic Press.

Photoinactivation of Latent Herpes Simplex Virus in Rabbit Kidney Cells

PubMed Central

Kelleher, J. J.; Varani, J.

1976-01-01

The photoinactivation of actively and nonactively growing herpes simplex virus by neutral red and proflavine was studied in rabbit kidney cells. Active virus growth was inhibited by both dyes under conditions which did not destroy the cells. Neutral red caused a much greater inhibition than proflavine. Neutral red also caused a reduction in the reactivation rate of latent virus when the infected cells were treated during the latent period. In the treated cultures that did reactivate virus, the average length of the latent period was increased over the control value. Proflavine treatment did not reduce the rate of reactivation of latent virus and did not increase the average latent period of the treated cultures. PMID:185948

Relaxation kinetics of the interaction between RNA and metal-intercalators: the Poly(A).Poly(U)/platinum-proflavine system.

PubMed

Biver, Tarita; Secco, Fernando; Venturini, Marcella

2005-05-15

The interactions of Poly(A).Poly(U) with the cis-platinum derivative of proflavine [{PtCl(tmen)}(2){HNC(13)H(7)(NHCH(2)CH(2))(2)}](+) (PRPt) and proflavine (PR) are investigated by spectrophotometry, spectrofluorimetry and T-jump relaxation at I=0.2M, pH 7.0, and T=25 degrees C. Base-dye interactions prevail at high RNA/dye ratio and binding isotherms analysis reveals that both dyes bind to Poly(A).Poly(U) according to the excluded site model (n=2). Only one relaxation effect is observed for the Poly(A).Poly(U)/PRPt system, whereas two effects are observed with Poly(A).Poly(U)/PR. The results agree with the sequence D+S <==> D, S <==> DS(I) <==> DS(II), where D,S is an external complex, DS(I) is a partially intercalated species, and DS(II) is the fully intercalated complex. Formation of DS(II) could be observed in the case of proflavine only. This result is interpreted by assuming that the platinum-containing residue of PRPt hinders the full intercalation of the acridine residue.

Electrochemical and spectroscopic studies of the interaction of proflavine with DNA.

PubMed

Aslanoglu, Mehmet

2006-03-01

The interaction of proflavine with herring sperm DNA has been investigated by cyclic voltammetry and UV-Vis spectroscopy as well as viscosity measurements. Shifts in the peak potentials in cyclic voltammetry, spectral changes in UV absorption titration, an increase in viscosity of DNA and the results of the effect of ionic strength on the binding constant strongly support the intercalation of proflavine into the DNA double helix. The binding constant for the interaction between proflavine and DNA was K = 2.32 (+/- 0.41) x 10(4) M(-1) and the binding site size was 2.07 (+/- 0.1) base pairs, estimated in voltammetric measurements. The value of the binding site size was determined to be closer to that expected for a planar intercalating agent. The standard Gibbs free-energy change is ca. -24.90 kJ/mol at 25 degrees C, indicating the spontaneity of the binding interaction. The binding constant determined by UV absorption measurements was K = 2.20 (+/- 0.48) x 10(4) M(-1), which is very close to the value determined by cyclic voltammetry assuming that the binding equilibrium is static.

A 1:2 crystalline complex of ApA:proflavine: a model for binding to single-stranded regions in RNA.

PubMed Central

Neidle, S; Taylor, G; Sanderson, M

1978-01-01

The structure of a 1"2 complex of adenylyl-(3',5')-adenosine phosphate and proflavine hemisulfate has been determined using the methods of x-ray crystallography. Since the ApA does not form a mini double helix, it may serve as a model for the interaction of planar molecules with single stranded nucleic acids. The dinucleotide adopts an extended conformation with the adenines in adjacent molecules forming base pairs. A most unusual feature of the molecule is that it does not obey the "rigid nucleotide" concept although none of the torsion angles occur in energetically unfavourable regions. This is most probably due to the strong interactions between the proflavine and the oligonucleotide. PMID:724521

Synthesis and DNA interaction of a mixed proflavine-phenanthroline Tröger base.

PubMed

Baldeyrou, Brigitte; Tardy, Christelle; Bailly, Christian; Colson, Pierre; Houssier, Claude; Charmantray, Franck; Demeunynck, Martine

2002-04-01

We report the synthesis of an asymmetric Tröger base containing the two well characterised DNA binding chromophores, proflavine and phenanthroline. The mode of interaction of the hybrid molecule was investigated by circular and linear dichroism experiments and a biochemical assay using DNA topoisomerase I. The data are compatible with a model in which the proflavine moiety intercalates between DNA base pairs and the phenanthroline ring occupies the DNA groove. DNase I cleavage experiments were carried out to investigate the sequence preference of the hybrid ligand and a well resolved footprint was detected at a site encompassing two adjacent 5'-GTC.5-GAC triplets. The sequence preference of the asymmetric molecule is compared to that of the symmetric analogues.

Interaction of small molecules with double-stranded RNA: spectroscopic, viscometric, and calorimetric study of hoechst and proflavine binding to PolyCG structures.

PubMed

Sinha, Rangana; Hossain, Maidul; Kumar, Gopinatha Suresh

2009-04-01

Design and synthesis of new small molecules binding to double-stranded RNA necessitate complete understanding of the molecular aspects of the binding of many existing molecules. Toward this goal, in this work we evaluated the biophysical aspects of the interaction of a DNA intercalator (proflavine) and a minor groove binder (hoechst 33258) with two polymorphic forms of polyCG, namely, the right-handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form, by absorption, fluorescence, and viscometry experiments. The energetics of the interaction of these molecules with the RNA structures has also been elucidated by isothermal titration calorimetry (ITC). Results suggest that proflavine strongly intercalates in both forms of polyCG, whereas hoechst shows mainly groove-binding modes. The binding of both drugs to both forms of RNA resulted in significant conformational change to the RNA structure with the bound molecules being placed in the chiral RNA helix. ITC profiles for both proflavine and hoechst show two binding sites. Binding of proflavine to both forms of RNA is endothermic and entropy driven in the first site and exothermic and enthalpy driven in the second site, whereas hoechst binding to both forms of RNA is exothermic and enthalpy driven in the first site and endothermic and entropy driven in the second site. This study suggests that the binding affinity characteristics and energetics of interaction of these DNA binding molecules with the RNA conformations are significantly different and may serve as data for future development of effective structure-selective RNA-based drugs.

Resonance Raman investigation of the photoreduction of methylviologen with Ru(bpy) 2+3 and proflavine as sensitizers

NASA Astrophysics Data System (ADS)

Forster, Martin; Hester, Ronald E.

1982-01-01

Reduced methylviologen (MV +) is detected by conventional resonance Raman spectroscopy in photoreactions of Ru(bpy) 2+3 or proflavine (PFH +) with MV 2+ Using apparatus for modulated excitation resonance Raman (MERR) spectroscopy, the irreversible MV + production with PFH + as sensitizer is traced back to triplet-triplet annihilation with simultaneous destruction of PFH +.

Mechanism(s) of Molecular Responses in Mammalian Cells Exposed to Physical Agents (Alone and in Combination).

DTIC Science & Technology

1998-04-30

Mutagenesis. 13, 29-4-303. Cvtogenic studies in human lymphocytes exposed to 2450AHz RFR 757 MELTZ. M1. L.. EGAN. P. and ERWIN. D. N.. 1990. Proflavin ...Causes of death following occu- 22. M. L. Meltz. P. Eagan and D. N. Erwin. Proflavin and microwave pational exposure to microwave radiation (radar

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

PubMed

Bruylants, Gilles; Wintjens, René; Looze, Yvan; Redfield, Christina; Bartik, Kristin

2007-12-01

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), DeltaH(obs)(0) ). 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 alpha-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 alpha-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.

Flow cytometric analysis of leukocytes and reticulocytes stained with proflavine.

PubMed

Sagawa, H; Tatsumi, N

1997-12-01

Proflavine, an acridine analog for industrial use, was used to stain blood cells. A drop of blood treated with ethylenediaminetetraacetic acid-2K was mixed with a 0.00001% solution of the dye and observed immediately by fluorescence microscopy with a green filter. Leukocytes, platelets, and reticulocytes were stained but mature red blood cells were not. Chromatin in the nuclei of all leukocytes and nucleoli of lymphocytes and monocytes had greenish-yellow fluorescence, and the kind of cell could be identified by the tone and intensity of this color. Granules in granulocytes were in green. Reticular fine-granular or granulofibrous structures in the reticulocytes were brownish. The proflavine could be used routinely in clinical laboratories because this single stain makes possible simultaneous differentiation of leukocytes and counting of reticulocytes.

Evidence for the role of double-helical structures in the maturation of simian virus-40 messenger RNA.

PubMed Central

Chiu, N H; Bruszewski, W B; Salzman, N P

1980-01-01

Simian Virus-40 infected BSC-1 cells were pretreated with glucosamine and briefly pulsed with [3H]-uridine. The labeling can be halted instantaneously by the addition of cold uridine and glucosamine. Under these pulse-chase conditions, the inhibitory effects of the intercalating agent proflavine on the processing of prelabeled nuclear RNA precursors were examined in vivo. Proflavine inhibits the cleavage of viral nuclear RNA precursors. However, turnover of the mature viral mRNAs in the cytoplasm is not inhibited. The effect of proflavine on processing is not a secondary consequence of its inhibition of protein synthesis. The data suggest that base-paired secondary structures in the primary transcripts are important processing signals in the generation of viral mRNA molecules. Images PMID:6243778

Attempt to develop live attenuated bacterial vaccines by selecting resistance to gossypol, proflavine hemisulfate, novobiocin, or ciprofloxacin.

PubMed

Pridgeon, Julia W; Klesius, Phillip H; Yildirim-Aksoy, Mediha

2013-04-26

In an attempt to develop attenuated bacteria as potential live vaccines, four chemicals (gossypol, proflavine hemisulfate, novobiocin, and ciprofloxacin) were used to modify the following four genera of bacteria through chemical-resistance strategy: (1) Aeromonas hydrophila (9 isolates); (2) Edwardsiella tarda (9 isolates); (3) Streptococcus iniae (9 isolates); and (4) S. agalactiae (11 isolates). All bacteria used in this study were able to develop high resistance to gossypol. However, only some bacteria were able to develop resistance to proflavine hemisulfate, novobiocin, or ciprofloxacin. When the virulence of resistant bacteria was tested in tilapia or catfish, none of the gossypol-resistant isolate was attenuated, whereas majority of the proflavine hemisulfate-resistant isolates were attenuated. However, all proflavine hemisulfate-attenuated bacteria failed to provide significant protection to fish. Eight novobiocin- or ciprofloxacin-resistant Gram-positive bacteria (S. agalactiae and S. inaie) were found to be attenuated. However, none of them offered protection higher than 70%. Of seven attenuated novobiocin- or ciprofloxacin-resistant Gram-negative isolates (A. hydrophila and E. tarda), only one (novobiocin-resistant E. tarda 30305) was found to safe and highly efficacious. When E. tarda 30305-novo vaccinated Nile tilapia were challenged by its virulent E. tarda 30305, relative percent of survival of vaccinated fish at 14- and 28-days post vaccination (dpv) was 100% and 92%, respectively. Similarly, E. tarda 30305-novo offered 100% protection to channel catfish against challenges with virulent parent isolate E. tarda 30305 at both 14- and 28-dpv. Our results suggest that the development of live attenuated bacterial vaccines that are safe and efficacious is challenging, although it is feasible. Published by Elsevier Ltd.

Ethidium and proflavine binding to a 2',5'-linked RNA duplex.

PubMed

Horowitz, Eric D; Hud, Nicholas V

2006-12-06

Despite over 40 years of physical investigations, fundamental questions persist regarding the energetics of RNA and DNA intercalation. The dramatic unwinding of a nucleic acid duplex upon intercalation immediately suggests that the nucleic acid backbone should play a significant role in dictating the free energy of intercalation. However, the contribution of the backbone to intercalation free energy is difficult to appreciate given the intertwined energetics associated with intercalation (e.g., pi-pi stacking and solvent effects). Fluorescence titrations were used to determine the association constants of two known intercalators, proflavine and ethidium, for duplex 2',5'-linked RNA. Proflavine was found to bind 2',5' RNA with an association constant 25-fold greater than that measured for standard, 3',5'-linked RNA. In contrast, ethidium binds 2',5' RNA less favorably than standard RNA.

High resolution microendoscopy with structured illumination and Lugol's iodine staining for evaluation of breast cancer architecture

NASA Astrophysics Data System (ADS)

Dobbs, Jessica; Kyrish, Matthew; Krishnamurthy, Savitri; Grant, Benjamin; Kuerer, Henry; Yang, Wei; Tkaczyk, Tomasz; Richards-Kortum, Rebecca

2016-03-01

Intraoperative margin assessment to evaluate resected tissue margins for neoplastic tissue is performed to prevent reoperations following breast-conserving surgery. High resolution microendoscopy (HRME) can rapidly acquire images of fresh tissue specimens, but is limited by low image contrast in tissues with high optical scattering. In this study we evaluated two techniques to reduce out-of-focus light: HRME image acquisition with structured illumination (SI-HRME) and topical application of Lugol's Iodine. Fresh breast tissue specimens from 19 patients were stained with proflavine alone or Lugol's Iodine and proflavine. Images of tissue specimens were acquired using a confocal microscope and an HRME system with and without structured illumination. Images were evaluated based on visual and quantitative assessment of image contrast. The highest mean contrast was measured in confocal images stained with proflavine. Contrast was significantly lower in HRME images stained with proflavine; however, incorporation of structured illumination significantly increased contrast in HRME images to levels comparable to that in confocal images. The addition of Lugol's Iodine did not increase mean contrast significantly for HRME or SI-HRME images. These findings suggest that structured illumination could potentially be used to increase contrast in HRME images of breast tissue for rapid image acquisition.

X-ray-structure of a cytidylyl-3',5'-adenosine-proflavine complex: a self-paired parallel-chain double helical dimer with an intercalated acridine dye.

PubMed Central

Westhof, E; Sundaralingam, M

1980-01-01

The non-self-complementary dinucleoside monophosphate cytidylyl-3',5'-adenosine (CpA) forms a base-paired parallel-chain dimer with an intercalated proflavine. The dimer complex possesses a right-handed helical twist. The dimer helix has an irregular girth with a neutral adenine-adenine (A-A) pair, hydrogen-bonded through the N6 and N7 sites (C1'...C1' separation of 10.97 A), and a triply hydrogen-bonded protonated cytosine-cytosine (C-C) pair with a proton shared between the base N3 sites (Cl'...Cl' separation of 9.59 A). The torsion angles of the sugar-phosphate backbone are within their most preferred ranges and the sugar puckering sequence (5' leads to 3') is C3'-endo, C2'-endo. There is also a second proflavine molecule sandwiched between CpA dimers on the 21-axis. Both proflavines are necessarily disordered, being on dyad axis, and this suggests possible insights into the dynamics of intercalation of planar drugs. This structure shows that intercalation of planar drugs in nucleic acids may not be restricted to antiparallel complementary Watson-Crick pairing regions and provides additional mechanisms for acridine mutagenesis. PMID:6929524

Comparison of the effect of UV laser radiation and of a radiomimetic substance on chromatin

NASA Astrophysics Data System (ADS)

Radulescu, Irina; Radu, Liliana; Serbanescu, Ruxandra; Nelea, V. D.; Martin, C.; Mihailescu, Ion N.

1998-07-01

The damages of the complex of deoxyribonucleic acid (DNA) and proteins from chromatin, produced by the UV laser radiation and/or by treatment with a radiomimetic substance, bleomycin, were compared. The laser radiation and bleomycin effects on chromatin structure were determined by the static and dynamic fluorimetry of chromatin complexes with the DNA specific ligand-- proflavine and by the analysis of tryptophan chromatin intrinsic fluorescence. Time resolved spectroscopy is a sensitive technique which allows to determine the excited state lifetimes of chromatin--proflavine complexes. Also, the percentage contributions to the fluorescence of proflavine, bound and unbound to chromatin DNA, were evaluated. The damages produced by the UV laser radiation on chromatin are similar with those of radiomimetic substance action and consists in DNA and proteins destruction. The DNA damage degree has been determined. The obtained results may constitute some indications in the laser utilization in radiochimiotherapy.

In vitro and in vivo photosensitized inactivation of dermatophyte fungi by heterotricyclic dyes.

PubMed Central

Propst, C; Lubin, L

1978-01-01

The ability of three heterotricyclic dyes to photosensitize dermatophyte fungi was studied with Trichophyton mentagrophytes and Microsporum gypseum. In vitro studies showed that methylene blue, neutral red, and proflavine were capable of killing these fungi when used in conjunction with broad-spectrum light. Proflavine, however, killed both fungi most rapidly and was used for further studies. Fungal killing by proflavine plus light was dependent on dye concentration, pH, light wavelength, and light intensity. Based on the in vitro studies, a treatment regimen was developed for in vivo use on experimentally infected animals. When treatment of guinea pigs inoculated with T. mentagrophytes was begun during fungal invasion, lesion formation at inoculated sites was either prevented or substantially reduced. When treatment was begun after lesion formation, however, light-plus-dyed treated sites showed only slightly faster curing than untreated sites. PMID:669788

Small molecule-mediated duplex formation of nucleic acids with 'incompatible' backbones.

PubMed

Cafferty, Brian J; Musetti, Caterina; Kim, Keunsoo; Horowitz, Eric D; Krishnamurthy, Ramanarayanan; Hud, Nicholas V

2016-04-07

Proflavine, a known intercalator of DNA and RNA, promotes duplex formation by nucleic acids with natural and non-natural backbones that otherwise form duplexes with low thermal stability, and even some that show no sign of duplex formation in the absence of proflavine. These findings demonstrate the potential for intercalators to be used as cofactors for the assembly of rationally designed nucleic acid structures, and could provide fundamental insights regarding intercalation of natural nucleic acid duplexes.

DNA sensing by a Eu-binding peptide containing a proflavine unit.

PubMed

Ancel, Laetitia; Gateau, Christelle; Lebrun, Colette; Delangle, Pascale

2013-01-18

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

Activation of cGAS-dependent antiviral responses by DNA intercalating agents

PubMed Central

Pépin, Geneviève; Nejad, Charlotte; Thomas, Belinda J.; Ferrand, Jonathan; McArthur, Kate; Bardin, Philip G.; Williams, Bryan R.G.; Gantier, Michael P.

2017-01-01

Acridine dyes, including proflavine and acriflavine, were commonly used as antiseptics before the advent of penicillins in the mid-1940s. While their mode of action on pathogens was originally attributed to their DNA intercalating activity, work in the early 1970s suggested involvement of the host immune responses, characterized by induction of interferon (IFN)-like activities through an unknown mechanism. We demonstrate here that sub-toxic concentrations of a mixture of acriflavine and proflavine instigate a cyclic-GMP-AMP (cGAMP) synthase (cGAS)-dependent type-I IFN antiviral response. This pertains to the capacity of these compounds to induce low level DNA damage and cytoplasmic DNA leakage, resulting in cGAS-dependent cGAMP-like activity. Critically, acriflavine:proflavine pre-treatment of human primary bronchial epithelial cells significantly reduced rhinovirus infection. Collectively, our findings constitute the first evidence that non-toxic DNA binding agents have the capacity to act as indirect agonists of cGAS, to exert potent antiviral effects in mammalian cells. PMID:27694309

Dependence of erythroid differentiation on cell replication in dimethyl sulfoxide-treated friend leukemia-virus-infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiens, A.W.; McClintock, P.R.; Papaconstantinou, J.

1976-01-01

The dimethyl sulfoxide (Me/sub 2/SO)-mediated induction of hemoglobin synthesis in Friend leukemia cells (a murine erythroblastoid cell line) is coupled with the number of cell replications occurring in the presence of inducer. Varying concentrations of proflavine increase the generation time of these cells from 24 hours to over 50 hours, and in each case the induction of hemoglobin synthesis follows the completion of two cell doublings. Once the induction is initiated, the rate of hemoglobin accumulation is not affected by proflavine. These data indicate that proflavine does not affect the transcription or translation of globin mRNA and that the delaymore » in induction of hemoglobin synthesis is due to its effect on the rate of cellular replication. In experiments using high concentrations of thymidine to block replication, hemoglobin accumulation is prevented only if the cells are blocked prior to 36 hours after Me/sub 2/SO addition. If the cells have completed two generations in the presence of Me/sub 2/SO, there is no effect upon their ability to synthesize hemoglobin even though their growth is arrested. Thus, the inhibition of hemoglobin synthesis by proflavine is not merely the result of a toxic effect on newly subcultured cells but is due to its effect on cellular replication. These experiments confirm that, after addition of Me/sub 2/SO, Friend leukemia cells require more than one complete cell cycle in order to synthesize hemoglobin.« less

Stereochemical model for proflavin intercalation in A-DNA.

PubMed Central

Alden, C J; Arnott, S

1977-01-01

Linked-atom molecular modelling was employed to determine the steric and torsional requirements for intercalation of proflavine into a double-stranded region of DNA compatible with adjacent regions of cohelical A-DNA. The optimum intercalation conformation is characterized by the dihedral angles xi and psi becoming trans, with all sugars retaining the characteristics C3'-endo pucker. This extended conformation results in virtually no helical unwinding, suggesting it may be an appropriate model for an intercalative intermediary in mutagenesis by virtue of its similarity to standard helical DNA. PMID:593890

Nucleic acid binding drugs. Part XIII. Molecular motion in a drug-nucleic acid model system: thermal motion analysis of a proflavine-dinucleoside crystal structure.

PubMed Central

Aggarwal, A K; Neidle, S

1985-01-01

The high-resolution crystal structure of the intercalation complex between proflavine and cytidylyl-3',5'-guanosine (CpG) has been studied by thermalmotion analysis. This has provided information on the translational and librational motions of individual groups in the complex. Many of these motions are similar to, though of larger magnitude than in uncomplexed dinucleosides. Pronounced librational effects were observed along the base pairs and in the plane of the drug chromophore. PMID:4034394

Correlation of Resistance to Proflavine and Penicillin in Escherichia coli

PubMed Central

McKellar, Robin C.; McKenzie, Colin N.; Kushner, Donn J.

1976-01-01

A number of proflavine (PF)-resistant mutants of Escherichia coli B were also resistant to penicillin and cephalothin. Mutants resistant to 1.0 mM PF were 10 times more penicillin resistant than were the PF-susceptible, wild-type cells. Single-step mutants selected for resistance to either PF or penicillin were also resistant to the other drug. None of the resistant mutants tested possessed β-lactamase activity. These results suggest that resistance to PF and penicillin in E. coli B may be due to permeability changes in the cell envelope. PMID:791110

Activation of cGAS-dependent antiviral responses by DNA intercalating agents.

PubMed

Pépin, Geneviève; Nejad, Charlotte; Thomas, Belinda J; Ferrand, Jonathan; McArthur, Kate; Bardin, Philip G; Williams, Bryan R G; Gantier, Michael P

2017-01-09

Acridine dyes, including proflavine and acriflavine, were commonly used as antiseptics before the advent of penicillins in the mid-1940s. While their mode of action on pathogens was originally attributed to their DNA intercalating activity, work in the early 1970s suggested involvement of the host immune responses, characterized by induction of interferon (IFN)-like activities through an unknown mechanism. We demonstrate here that sub-toxic concentrations of a mixture of acriflavine and proflavine instigate a cyclic-GMP-AMP (cGAMP) synthase (cGAS)-dependent type-I IFN antiviral response. This pertains to the capacity of these compounds to induce low level DNA damage and cytoplasmic DNA leakage, resulting in cGAS-dependent cGAMP-like activity. Critically, acriflavine:proflavine pre-treatment of human primary bronchial epithelial cells significantly reduced rhinovirus infection. Collectively, our findings constitute the first evidence that non-toxic DNA binding agents have the capacity to act as indirect agonists of cGAS, to exert potent antiviral effects in mammalian cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Electronic absorption spectra of hydrogenated protonated naphthalene and proflavine

NASA Astrophysics Data System (ADS)

Bonaca, A.; Bilalbegović, G.

2011-09-01

We study hydrogenated cations of two polycyclic hydrocarbon molecules as models of hydrogenated organic species that form in the interstellar medium. Optical spectra of the hydrogenated naphthalene cation Hn-C10H+8 for n= 1, 2 and 10, as well as the astrobiologically interesting hydrogenated proflavine cation Hn-C13H11N+3 for n= 1 and 14, are calculated. The pseudopotential time-dependent density functional theory is used. It is found that the fully hydrogenated proflavine cation H14-C13H11N+3 shows a broad spectrum in which the positions of individual lines are almost lost. The positions, shapes and intensities of lines change in hydronaphthalene and hydroproflavine cations, showing that hydrogen additions induce substantially different optical spectra in comparison with base polycyclic hydrocarbon cations. One calculated line in the visible spectrum of H10-C10H+8 and one in the visible spectrum of H-C13H11N+3 are close to the measured diffuse interstellar bands. We also present the positions of near-ultraviolet lines.

Compensation Effect in the Electrical Conduction Process in Some Nucleic Acid Base Complexes with Proflavine Dye

NASA Astrophysics Data System (ADS)

Sarkar, D.; Misra, T. N.

1988-11-01

Compensation behaviour has been found in electrical conduction process in proflavine complexes with nucleic acid bases, guanine, adenine, uracil and thymine. At low dye concentrations these semiconducting complexes follow a three constant compensation equation σ(T){=}σ0'\\exp (E/2kT0)\\exp (-E/2kT), σ0' and T0 being constants for a specific base. The other notations have their usual meaning. Consistent values of these constants have been obtained by different experimental methods of evaluation. These results suggest that compensation effect has a physical origin.

Grand canonical ensemble Monte Carlo simulation of the dCpG/proflavine crystal hydrate.

PubMed

Resat, H; Mezei, M

1996-09-01

The grand canonical ensemble Monte Carlo molecular simulation method is used to investigate hydration patterns in the crystal hydrate structure of the dCpG/proflavine intercalated complex. The objective of this study is to show by example that the recently advocated grand canonical ensemble simulation is a computationally efficient method for determining the positions of the hydrating water molecules in protein and nucleic acid structures. A detailed molecular simulation convergence analysis and an analogous comparison of the theoretical results with experiments clearly show that the grand ensemble simulations can be far more advantageous than the comparable canonical ensemble simulations.

Comparison of the effect of acridine derivatives and similar substances on the dimerization of thymine in mammalian DNA in situ and isolated (in Russian)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klimek, M.; Shevchikova, P.

1973-01-01

From international conference on the bases of the biological effects of ultraviolet radiation; Brno, Czechoslovakia (2 Oct If the cells were exposed to the effect of varying concentrations of proflavine, acridine orange, riboflavine, and methyl green before uv irradiatlon, the most effective of these substances was proflavine, which reduced the yield of dimerization in vivo by 50%. The other substances were much less effective and accounted for a maximum 20% decrease of the dimer yield. The different results in the thymidine dimerization rate, obtained with isolated DNA and DNA in situ, are discussed. (auth)

Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

NASA Astrophysics Data System (ADS)

de Paula D'Almeida, Camila; Fortunato, Thereza Cury; Teixeira Rosa, Ramon Gabriel; Romano, Renan Arnon; Moriyama, Lilian Tan; Pratavieira, Sebastião.

2018-02-01

Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

The structure of drug-deoxydinucleoside phosphate complex; generalized conformational behavior of intercalation complexes with RNA and DNA fragments.

PubMed Central

Shieh, H S; Berman, H M; Dabrow, M; Neidle, S

1980-01-01

A 2:2 complex of proflavine and deoxycytidylyl-3', 5'-guanosine has been crystallized and its structure determined by x-ray crystallography. The two dinucleoside phosphate strands form self complementary duplexes with Watson Crick hydrogen bonds. One proflavin is asymmetrically intercalated between the base pairs and the other is stacked above them. The conformations of the nucleotides are unusual in that one strand has C3',C2'endomixed sugar puckering and the other has C3',C3' endo deoxyribose sugars. These results show that the conformation of the 3'sugar is of secondary importance to the intercalated geometry. PMID:7355129

Grand canonical ensemble Monte Carlo simulation of the dCpG/proflavine crystal hydrate.

PubMed Central

Resat, H; Mezei, M

1996-01-01

The grand canonical ensemble Monte Carlo molecular simulation method is used to investigate hydration patterns in the crystal hydrate structure of the dCpG/proflavine intercalated complex. The objective of this study is to show by example that the recently advocated grand canonical ensemble simulation is a computationally efficient method for determining the positions of the hydrating water molecules in protein and nucleic acid structures. A detailed molecular simulation convergence analysis and an analogous comparison of the theoretical results with experiments clearly show that the grand ensemble simulations can be far more advantageous than the comparable canonical ensemble simulations. Images FIGURE 5 FIGURE 7 PMID:8873992

Fluorimetric study on the interaction between Norfloxacin and Proflavine hemisulphate.

PubMed

More, Vishalkumar R; Anbhule, Prashant V; Lee, Sang H; Patil, Shivajirao R; Kolekar, Govind B

2011-07-01

The interaction between Norfloxacin (NF) and Proflavine hemisulphate (PF) was investigated by spectroscopic tools like UV-VIS absorption and Fluorescence spectroscopy. It was proved that fluorescence quenching of NF by PF is due to the formation of NF-PF complex which was supported by UV-VIS absorption study. The study of thermodynamic parameters suggested that the key interacting forces are hydrogen bond and van der Waal's interactions and the binding interaction was spontaneous. The distance r between NF and PF was obtained according to the Förster's theory of non-radiative energy transfer. The fluorescence quenching mechanism was applied to estimate PF directly from pharmaceutical samples. © Springer Science+Business Media, LLC 2011

Spectroscopic and thermodynamic insights into the interaction between proflavine and human telomeric G-quadruplex DNA.

PubMed

Kumar, Vivek; Sengupta, Abhigyan; Gavvala, Krishna; Koninti, Raj Kumar; Hazra, Partha

2014-09-25

The G-quadruplex (GQ-DNA), an alternative structure motif of DNA, has emerged as a novel and exciting target for anticancer drug discovery. GQ-DNA formed in the presence of monovalent cations (Na(+)/K(+)) by human telomeric DNA is a point of interest due to their direct relevance for cellular aging and abnormal cell growths. Small molecules that selectively target and stabilize G-quadruplex structures are considered to be potential therapeutic anticancer agents. Herein, we probe G-quadruplex and proflavine (a well-known DNA intercalator, hence acting as an anticarcinogen) association through steady state and time-resolved fluorescence spectroscopy to explore the effect of stabilization of GQ-DNA by this well-known DNA intercalator. The structural modifications of G-quadruplex upon binding are highlighted through circular dichroism (CD) spectra. Moreover, a detailed insight into the thermodynamics of this interaction has been provided though isothermal titration calorimetry (ITC) studies. The thermodynamic parameters obtained from ITC help to gain knowledge about the nature as well as the driving forces of binding. This present study shows that proflavine (PF) can act as a stabilizer of telomeric GQ-DNA through an entropically as well as enthalpically feasible process with high binding affinity and thereby can be considered as a potential telomerase inhibitor.

Photoabsorption of acridine yellow and proflavin bound to human serum albumin studied by means of quantum mechanics/molecular dynamics.

PubMed

Aidas, Kęstutis; Olsen, Jógvan Magnus H; Kongsted, Jacob; Ågren, Hans

2013-02-21

Attempting to unravel mechanisms in optical probing of proteins, we have performed pilot calculations of two cationic chromophores-acridine yellow and proflavin-located at different binding sites within human serum albumin, including the two primary drug binding sites as well as a heme binding site. The computational scheme adopted involves classical molecular dynamics simulations of the ligands bound to the protein and subsequent linear response polarizable embedding density functional theory calculations of the excitation energies. A polarizable embedding potential consisting of point charges fitted to reproduce the electrostatic potential and isotropic atomic polarizabilities computed individually for every residue of the protein was used in the linear response calculations. Comparing the calculated aqueous solution-to-protein shifts of maximum absorption energies to available experimental data, we concluded that the cationic proflavin chromophore is likely not to bind albumin at its drug binding site 1 nor at its heme binding site. Although agreement with experimental data could only be obtained in qualitative terms, our results clearly indicate that the difference in optical response of the two probes is due to deprotonation, and not, as earlier suggested, to different binding sites. The ramifications of this finding for design of molecular probes targeting albumin or other proteins is briefly discussed.

Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A).

PubMed

Ciatto, C; D'Amico, M L; Natile, G; Secco, F; Venturini, M

1999-11-01

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.

Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A)

PubMed Central

Ciatto, C; D'Amico, ML; Natile, G; Secco, F; Venturini, M

1999-01-01

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine. PMID:10545371

Synthesis and antileishmanial activity of 6-mono-substituted and 3,6-di-substituted acridines obtained by acylation of proflavine.

PubMed

Di Giorgio, Carole; Shimi, Kamal; Boyer, Gérard; Delmas, Florence; Galy, Jean-Pierre

2007-10-01

Two new series of diaminoacridinic derivatives obtained from proflavine and N-(6-amino-3-acridinyl)acetamide were synthesised and assessed for their cytotoxic and antileishmanial activities. Two compounds, N-[6-(acetylamino)-3-acridinyl]acetamide and N-[6-(benzoylamino)-3-acridinyl]benzamide demonstrated highly specific antileishmanial properties against the intracellular amastigote form of the parasite. Structure-activity relationships established that the antiproliferative activity against human cells was greatly enhanced by the presence of a benzoylamino group in 6-mono-substituted acridines, while the presence of two acetylamino or benzoylamino groups in 3,6-di-substituted acridines strongly increased the specificity of the molecules for Leishmania parasite, suggesting that symmetric conformations could preferentially interfere with Leishmania metabolism.

Isolation of proflavine as a blocker of G protein-gated inward rectifier potassium channels by a cell growth-based screening system.

PubMed

Kawada, Hitoshi; Inanobe, Atsushi; Kurachi, Yoshihisa

2016-10-01

The overexpression of Kir3.2, a subunit of the G protein-gated inwardly rectifying K(+) channel, is implicated in some of the neurological phenotypes of Down syndrome (DS). Chemical compounds that block Kir3.2 are expected to improve the symptoms of DS. The purpose of this study is to develop a cell-based screening system to identify Kir3.2 blockers and then investigate the mode of action of the blocker. Chemical screening was carried out using a K(+) transporter-deficient yeast strain that expressed a constitutively active Kir3.2 mutant. The mode of action of an effective blocker was electrophysiologically analyzed using Kir channels expressed in Xenopus oocytes. Proflavine was identified to inhibit the growth of Kir3.2-transformant cells and Kir3.2 activity in a concentration-dependent manner. The current inhibition was strong when membrane potentials (Vm) was above equilibrium potential of K(+) (EK). When Vm was below EK, the blockage apparently depended on the difference between Vm and [K(+)]. Furthermore, the inhibition became stronger by lowering extracellular [K(+)]. These results indicated that the yeast strain serves as a screening system to isolate Kir3.2 blockers and proflavine is a prototype of a pore blocker of Kir3.2. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular mechanism of direct proflavine-DNA intercalation: evidence for drug-induced minimum base-stacking penalty pathway.

PubMed

Sasikala, Wilbee D; Mukherjee, Arnab

2012-10-11

DNA intercalation, a biophysical process of enormous clinical significance, has surprisingly eluded molecular understanding for several decades. With appropriate configurational restraint (to prevent dissociation) in all-atom metadynamics simulations, we capture the free energy surface of direct intercalation from minor groove-bound state for the first time using an anticancer agent proflavine. Mechanism along the minimum free energy path reveals that intercalation happens through a minimum base stacking penalty pathway where nonstacking parameters (Twist→Slide/Shift) change first, followed by base stacking parameters (Buckle/Roll→Rise). This mechanism defies the natural fluctuation hypothesis and provides molecular evidence for the drug-induced cavity formation hypothesis. The thermodynamic origin of the barrier is found to be a combination of entropy and desolvation energy.

[Studies by means of 1H NMR spectroscopy of complex formation of aromatic biologically active compounds with antibiotic topotecan].

PubMed

Mosunov, A A; Kostiukov, V V; Evstigneev, M P

2012-01-01

The analysis of heteroassociation of antibiotic topotecan (TPT) with aromatic biologically active compounds (BAC): caffeine, mutagens ethidium bromide and proflavine, antibiotic daunomycin, vitamins flavin-mononucleotide and nicotinamide, has been carried out in the work using 1H NMR spectroscopy data. The equilibrium constants of heteroassociation and induced chemical shifts of the protons have been obtained in the complexes with BAC. It is found that the complex formation TPT-BAC has the nature of stacking of the chromophores, additionally stabilized in the case of proflavine by intermolecular hydrogen bond. Calculation of the basic components of the Gibbs free energy of the complexation reactions is carried out, and the factors which stabilize and destabilize the heterocomplexes of molecules are revealed.

Stacking interactions and DNA intercalation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Dr. Shen; Cooper, Valentino R; Thonhauser, Prof. Timo

2009-01-01

The relationship between stacking interactions and the intercalation of proflavine and ellipticine within DNA is investigated using a nonempirical van der Waals density functional for the correlation energy. Our results, employing a binary stack model, highlight fundamental, qualitative differences between base-pair base-pair interactions and that of the stacked intercalator base pair system. Most notable result is the paucity of torque which so distinctively defines the Twist of DNA. Surprisingly, this model, when combined with a constraint on the twist of the surrounding base-pair steps to match the observed unwinding of the sugar-phosphate backbone, was sufficient for explaining the experimentally observedmore » proflavine intercalator configuration. Our extensive mapping of the potential energy surface of base-pair intercalator interactions can provide valuable information for future nonempirical studies of DNA intercalation dynamics.« less

HOST-PARASITE INTERACTIONS AND GENETICS OF ACTINOPHAGES

DTIC Science & Technology

A very brief summary is given of work involving the photoinactivation of actinophages by proflavin and methylene blue, and the synergistic toxicity of bacterial endotoxin and the antitumor antibiotic pactamycin.

Role of the third strand in the binding of proflavine and pt-proflavine to poly(rA).2poly(rU): a thermodynamic and kinetic study.

PubMed

García, Begoña; Leal, José M; Paiotta, Vittorio; Ruiz, Rebeca; Secco, Fernando; Venturini, Marcella

2008-06-12

The interactions of triple strands of poly(rA).2poly(rU) with proflavine (PR) and the proflavine cis-platinum derivative [{PtCl (tmen)} 2{NC 13H 7(NCH 2CH 2) 2}] (+) (PRPt) are examined at pH 7.0, T = 25 degrees C, and 0.2 M ionic strength by spectrophotometry, spectrofluorometry, circular dichroism, viscosimetry, stopped-flow, and T-jump relaxation techniques. The melting experiments demonstrate that both drugs tend to destabilize the triplex structure, although the PRPt effect is more relevant. By contrast, both drugs tend to slightly stabilize the duplex structure. The viscosity and circular dichroism measurements show that, at a low dye-to-polymer ratio ( C D/ C P), the binding is intercalative, whereas at high C D/ C P values, the external binding dominates. The binding kinetics and equilibria have been investigated over the C D/ C P region, where intercalation is operative. Both drugs bind to the RNA triplex according to the excluded site model. With PR, two kinetic effects have been observed, whereas with PRPt, only one has been observed. The results are interpreted according to the reaction schemes D + S right arrow over left arrow DS I, with PRPt, and D + S right arrow over left arrow DS I right arrow over left arrow DS II, with PR. The electrostatic contribution to the formation activation energy for DS I is similar (40%) for both systems. The results suggest that DS I is a partially intercalated species. Absence of the second step with PRPt is put down to groove interaction of the Pt-containing moiety, which prevents the PR residue from further penetration through the base pairs to form the fully intercalated complex, DS II. Comparison with the binding of the same drugs to the duplex reveals that the occupation of the major groove in poly(rA).2poly(rU) by the third strand plays a critical role in the kinetic behavior.

Micellar-mediated binding interaction between proflavine hemisulfate and salicylic acid: spectroscopic insights and its analytical application.

PubMed

Patil, Dhanshri T; Mokashi, Vidya V; Kolekar, Govind B; Patil, Shivajirao R

2013-01-01

The molecular interactions between salicylic acid (SA) and proflavin hemisulfate (PF) were investigated using fluorescence and UV-VIS absorption spectroscopy in an aqueous micellar environment. Changes in the absorption spectra of SA in the presence of PF indicate a ground state interaction between salicylate and proflavine hemisulfate ions to form a complex. The excitation bands of SA monitored at its emission wavelength reveal a red spectral shift of 8390.54 and 2037.75 cm(-1) when compared with absorption bands. The intensity of both excitation bands decreased in the presence of increasing amounts of PF. The absence of excitation bands of PF rules out the possibility of its direct excitation and suggests energy transfer from excited SA to PF, resulting in quenching of the SA fluorescence. The fluorescence quenching results were found to fit the well-known Stern-Volmer (S-V) relation. S-V plots at different temperatures were used to further evaluate thermodynamic parameters such as ∆G, ∆H and ΔS. The thermodynamic and kinetic data obtained from the quenching results were used to investigate the possible mechanism of binding, the nature of the binding force and the distance between SA and PF molecules. The linear relation between SA fluorescence quenching and PF concentration used to develop an analytical method for the determination of PF from Lorexane (a veterinary cream) using a fluorescence quenching method. Copyright © 2012 John Wiley & Sons, Ltd.

Optical Imaging with a High Resolution Microendoscope to Identify Cholesteatoma of the Middle Ear

PubMed Central

Levy, Lauren L.; Jiang, Nancy; Smouha, Eric; Richards-Kortum, Rebecca; Sikora, Andrew G.

2013-01-01

Objective High resolution optical imaging is an imaging modality which allows visualization of structural changes in epithelial tissue in real time. Our prior studies using contrast-enhanced microendoscopy to image squamous cell carcinoma in the head and neck demonstrated that the contrast agent, proflavine, has high affinity for keratinized tissue. Thus, high-resolution microendoscopy with proflavine provides a potential mechanism to identify ectopic keratin production, such as that associated with cholesteatoma formation and distinguish between uninvolved mucosa and residual keratin at the time of surgery. Study Design Ex vivo imaging of histopathologically-confirmed samples of cholesteatoma and uninvolved middle-ear epithelium. Methods Seven separate specimens collected from patients who underwent surgical treatment for cholesteatoma were imaged ex vivo with the fiberoptic endoscope after surface staining with proflavine. Following imaging, the specimens were submitted for hematoxylin &eosin staining to allow histopathological correlation. Results Cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Keratin-bearing areas of cholesteatoma lack nuclei and appear as confluent hyperfluorescence, while nuclei are easily visualized in specimens containing normal middle ear epithelium. Hyperfluorescence and loss of cellular detail is the imaging hallmark of keratin allowing for discrimination of cholesteatoma from normal middle ear epithelium. Conclusions This study demonstrates the feasibility of high-resolution optical imaging to discriminate cholesteatoma from uninvolved middle ear mucosa, based on the unique staining properties of keratin. Use of real-time imaging may facilitate more complete extirpation of cholesteatoma by identifying areas of residual disease. PMID:23299781

Molecular mechanical studies of proflavine and acridine orange intercalation.

PubMed Central

Dearing, A; Weiner, P; Kollman, P A

1981-01-01

Previous workers have reported that proflavine and acridine orange form various structurally different complexes with the dinucleoside phosphates rCpG and dCpG, with uniform C3'-endo and mixed C3'-endo (3'-5') C2'-endo sugar puckers being observed. We present theoretical calculations, based on the method of molecular mechanics, which support the experimental observations. The results suggest that the mixed C3'-edo (3'-5') C2'-endo pucker conformation isi intrinsically more stable than the uniform C3'-endo conformation, but that the additional stabilisation gained from specific, hydrogen bonding, interactions between nucleic acid and solvent, or intramolecularly within the nucleic acid, can lead to the adoption of the latter conformation, or of variants between the two. The role played by hydrogen bonding between amino-groups and nucleic acid phosphate appears more subtle than previously supposed. PMID:7232221

Photocarcinogenesis by methoxypsoralen, neutral red, proflavine, and long UV radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santamaria, L.; Bianchi, A.; Arnaboldi, A.

1985-01-01

A study of the photosensitizing effects of 8-methoxypsoralen (MOP), neutral red (NR), and proflavine (PF) on the skin of female Swiss albino mice, strain 955, was carried out using fractionated exposure to long ultraviolet light (300-400 nm) and visible light (tungsten emission). The results (1) confirmed MOP photocarcinogenicity, (2) demonstrated that both NR and PF are photocarcinogens, and, further, (3) showed that the above UV light with 2.6% of fluence at 313 nm is a long-term carcinogenic agent even though the total dose of 313 nm was 100 times less than the minimal UV tumorigenic dose in mice. The tumorsmore » were mammary adenocarcinomas, carcinomas of skin appendages, carcino-mixo-sarcomas, lymphomas, and one case of thyroid adenocarcinoma. The implications of the above data regarding the controversy about oncogenic risks in photochemotherapy are discussed.« less

Optical imaging with a high-resolution microendoscope to identify cholesteatoma of the middle ear.

PubMed

Levy, Lauren L; Jiang, Nancy; Smouha, Eric; Richards-Kortum, Rebecca; Sikora, Andrew G

2013-04-01

High-resolution optical imaging is an imaging modality that allows visualization of structural changes in epithelial tissue in real time. Our prior studies using contrast-enhanced microendoscopy to image squamous cell carcinoma in the head and neck demonstrated that the contrast agent, proflavine, has high affinity for keratinized tissue. Thus, high-resolution microendoscopy with proflavine provides a potential mechanism to identify ectopic keratin production, such as that associated with cholesteatoma formation, and distinguish between uninvolved mucosa and residual keratin at the time of surgery. Ex vivo imaging of histopathologically confirmed samples of cholesteatoma and uninvolved middle ear epithelium. Seven separate specimens collected from patients who underwent surgical treatment for cholesteatoma were imaged ex vivo with the fiberoptic endoscope after surface staining with proflavine. Following imaging, the specimens were submitted for hematoxylin and eosin staining to allow histopathological correlation. Cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Keratin-bearing areas of cholesteatoma lack nuclei and appear as confluent hyperfluorescence, whereas nuclei are easily visualized in specimens containing normal middle ear epithelium. Hyperfluorescence and loss of cellular detail is the imaging hallmark of keratin, allowing for discrimination of cholesteatoma from normal middle ear epithelium. This study demonstrates the feasibility of high-resolution optical imaging to discriminate cholesteatoma from uninvolved middle ear mucosa based on the unique staining properties of keratin. Use of real-time imaging may facilitate more complete extirpation of cholesteatoma by identifying areas of residual disease. Laryngoscope, 2012. Copyright © 2013 The American Laryngological, Rhinological, and Otological Society, Inc.

Intercalation and de-intercalation pathway of proflavine through the minor and major grooves of DNA: roles of water and entropy.

PubMed

Sasikala, Wilbee D; Mukherjee, Arnab

2013-05-07

DNA intercalation is a clinically relevant biophysical process due to its potential to inhibit the growth and survival of tumor cells and microbes through the arrest of the transcription and replication processes. Extensive kinetic and thermodynamic studies have followed since the discovery of the intercalative binding mode. However, the molecular mechanism and the origin of the thermodynamic and kinetic profile of the process are still not clear. Here we have constructed the free energy landscape of intercalation, de-intercalation and dissociation from both the major and minor grooves of DNA using extensive all-atom metadynamics simulations, capturing both the free energy barriers and stability in close agreement with fluorescence kinetic experiments. In the intercalated state, an alternate orientation of proflavine is found with an almost equal stability compared to the crystal orientation, however, separated by a 5.0 kcal mol(-1) barrier that decreases as the drug approaches the groove edges. This study provides a comprehensive picture in comparison with experiments, which indicates that the intercalation and de-intercalation of proflavine happen through the major groove side, although the effective intercalation barrier increases because the path of intercalation goes through the stable (abortive) minor groove bound state, making the process a millisecond long one in excellent agreement with the experiments. The molecular origin of the higher barrier for the intercalation from the minor groove side is attributed to the desolvation energy of DNA and the loss of entropy, while the barrier from the major groove, in the absence of desolvation energy, is primarily entropic.

Developing live bacterial vaccines by selecting resistance to antibacterials

USDA-ARS?s Scientific Manuscript database

Four chemicals were used in this study to modify bacterial isolates through chemical-resistance strategy. All bacteria were able to develop high resistance to gossypol. However, none of the gossypol-resistant isolate was attenuated. Although majority of the proflavine hemisulfate-resistant isolates ...

Vital-dye enhanced fluorescence imaging of gastrointestinal mucosa: metaplasia, neoplasia, inflammation

PubMed Central

Muldoon, Timothy J; Polydorides, Alexandros D; Maru, Dipen M; Harpaz, Noam; Harris, Michael T; Hofstettor, Wayne; Hiotis, Spiros P; Kim, Sanghyun A; Ky, Alex J; Anandasabapathy, Sharmila; Richards-Kortum, Rebecca

2012-01-01

Background Confocal endomicroscopy has revolutionized endoscopy by offering sub-cellular images of gastrointestinal epithelium; however, field-of-view is limited. There is a need for multi-scale endoscopy platforms that use widefield imaging to better direct placement of high-resolution probes. Design Feasibility Study Objective This study evaluates the feasibility of a single agent, proflavine hemisulfate, as a contrast medium during both widefield and high resolution imaging to characterize morphologic changes associated with a variety of gastrointestinal conditions. Setting U.T. M.D. Anderson Cancer Center (Houston, TX) and Mount Sinai Medical Center (New York, NY) Patients, Interventions, and Main Outcome Measurements Surgical specimens were obtained from 15 patients undergoing esophagectomy/colectomy. Proflavine, a vital fluorescent dye, was applied topically. Specimens were imaged with a widefield multispectral microscope and a high-resolution microendoscope. Images were compared to histopathology. Results Widefield-fluorescence imaging enhanced visualization of morphology, including the presence and spatial distribution of glands, glandular distortion, atrophy and crowding. High-resolution imaging of widefield-abnormal areas revealed that neoplastic progression corresponded to glandular heterogeneity and nuclear crowding in dysplasia, with glandular effacement in carcinoma. These widefield and high-resolution image features correlated well with histopathology. Limitations This imaging approach must be validated in vivo with a larger sample size. Conclusions Multi-scale proflavine-enhanced fluorescence imaging can delineate epithelial changes in a variety of gastrointestinal conditions. Distorted glandular features seen with widefield imaging could serve as a critical ‘bridge’ to high-resolution probe placement. An endoscopic platform combining the two modalities with a single vital-dye may facilitate point-of-care decision-making by providing real-time, in vivo diagnoses. PMID:22301343

Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation.

PubMed

Thekkek, Nadhi; Muldoon, Timothy; Polydorides, Alexandros D; Maru, Dipen M; Harpaz, Noam; Harris, Michael T; Hofstettor, Wayne; Hiotis, Spiros P; Kim, Sanghyun A; Ky, Alex Jenny; Anandasabapathy, Sharmila; Richards-Kortum, Rebecca

2012-04-01

Confocal endomicroscopy has revolutionized endoscopy by offering subcellular images of the GI epithelium; however, the field of view is limited. Multiscale endoscopy platforms that use widefield imaging are needed to better direct the placement of high-resolution probes. Feasibility study. This study evaluated the feasibility of a single agent, proflavine hemisulfate, as a contrast medium during both widefield and high-resolution imaging to characterize the morphologic changes associated with a variety of GI conditions. The University of Texas MD Anderson Cancer Center, Houston, Texas, and Mount Sinai Medical Center, New York, New York. PATIENTS, INTERVENTIONS, AND MAIN OUTCOME MEASUREMENTS: Resected specimens were obtained from 15 patients undergoing EMR, esophagectomy, or colectomy. Proflavine hemisulfate, a vital fluorescent dye, was applied topically. The specimens were imaged with a widefield multispectral microscope and a high-resolution microendoscope. The images were compared with histopathologic examination. Widefield fluorescence imaging enhanced visualization of morphology, including the presence and spatial distribution of glands, glandular distortion, atrophy, and crowding. High-resolution imaging of widefield abnormal areas revealed that neoplastic progression corresponded to glandular heterogeneity and nuclear crowding in dysplasia, with glandular effacement in carcinoma. These widefield and high-resolution image features correlated well with the histopathologic features. This imaging approach must be validated in vivo with a larger sample size. Multiscale proflavine-enhanced fluorescence imaging can delineate epithelial changes in a variety of GI conditions. Distorted glandular features seen with widefield imaging could serve as a critical bridge to high-resolution probe placement. An endoscopic platform combining the two modalities with a single vital dye may facilitate point-of-care decision making by providing real-time, in vivo diagnoses. Copyright © 2012 American Society for Gastrointestinal Endoscopy. Published by Mosby, Inc. All rights reserved.

Large-scale symmetry-adapted perturbation theory computations via density fitting and Laplace transformation techniques: investigating the fundamental forces of DNA-intercalator interactions.

PubMed

Hohenstein, Edward G; Parrish, Robert M; Sherrill, C David; Turney, Justin M; Schaefer, Henry F

2011-11-07

Symmetry-adapted perturbation theory (SAPT) provides a means of probing the fundamental nature of intermolecular interactions. Low-orders of SAPT (here, SAPT0) are especially attractive since they provide qualitative (sometimes quantitative) results while remaining tractable for large systems. The application of density fitting and Laplace transformation techniques to SAPT0 can significantly reduce the expense associated with these computations and make even larger systems accessible. We present new factorizations of the SAPT0 equations with density-fitted two-electron integrals and the first application of Laplace transformations of energy denominators to SAPT. The improved scalability of the DF-SAPT0 implementation allows it to be applied to systems with more than 200 atoms and 2800 basis functions. The Laplace-transformed energy denominators are compared to analogous partial Cholesky decompositions of the energy denominator tensor. Application of our new DF-SAPT0 program to the intercalation of DNA by proflavine has allowed us to determine the nature of the proflavine-DNA interaction. Overall, the proflavine-DNA interaction contains important contributions from both electrostatics and dispersion. The energetics of the intercalator interaction are are dominated by the stacking interactions (two-thirds of the total), but contain important contributions from the intercalator-backbone interactions. It is hypothesized that the geometry of the complex will be determined by the interactions of the intercalator with the backbone, because by shifting toward one side of the backbone, the intercalator can form two long hydrogen-bonding type interactions. The long-range interactions between the intercalator and the next-nearest base pairs appear to be negligible, justifying the use of truncated DNA models in computational studies of intercalation interaction energies.

Large-scale symmetry-adapted perturbation theory computations via density fitting and Laplace transformation techniques: Investigating the fundamental forces of DNA-intercalator interactions

NASA Astrophysics Data System (ADS)

Hohenstein, Edward G.; Parrish, Robert M.; Sherrill, C. David; Turney, Justin M.; Schaefer, Henry F.

2011-11-01

Symmetry-adapted perturbation theory (SAPT) provides a means of probing the fundamental nature of intermolecular interactions. Low-orders of SAPT (here, SAPT0) are especially attractive since they provide qualitative (sometimes quantitative) results while remaining tractable for large systems. The application of density fitting and Laplace transformation techniques to SAPT0 can significantly reduce the expense associated with these computations and make even larger systems accessible. We present new factorizations of the SAPT0 equations with density-fitted two-electron integrals and the first application of Laplace transformations of energy denominators to SAPT. The improved scalability of the DF-SAPT0 implementation allows it to be applied to systems with more than 200 atoms and 2800 basis functions. The Laplace-transformed energy denominators are compared to analogous partial Cholesky decompositions of the energy denominator tensor. Application of our new DF-SAPT0 program to the intercalation of DNA by proflavine has allowed us to determine the nature of the proflavine-DNA interaction. Overall, the proflavine-DNA interaction contains important contributions from both electrostatics and dispersion. The energetics of the intercalator interaction are are dominated by the stacking interactions (two-thirds of the total), but contain important contributions from the intercalator-backbone interactions. It is hypothesized that the geometry of the complex will be determined by the interactions of the intercalator with the backbone, because by shifting toward one side of the backbone, the intercalator can form two long hydrogen-bonding type interactions. The long-range interactions between the intercalator and the next-nearest base pairs appear to be negligible, justifying the use of truncated DNA models in computational studies of intercalation interaction energies.

Photolysis of water for H2 production with the use of biological and artificial catalysts

NASA Astrophysics Data System (ADS)

Hall, D. O.; Adams, M. W. W.; Morris, P.; Rao, K. K.

1980-02-01

An aqueous mixture of chloroplasts, hydrogenase and electron transfer catalyst on illumination liberates H2, the source of the H atoms being water. The rate and duration of H2 production from such a system depends on the stability of chloroplast and hydrogenase activities in light and oxygen. Both chloroplasts and hydrogenases can be stabilized to a certain degree by immobilization in gels or by incubation in bovine serum albumin. Natural electron carriers of hydrogenases are ferredoxin, cytochrome c3 and NAD. Viologen dyes and synthetic iron-sulphur particles (Jeevanu) can substitute for the biological carriers. Methyl viologen, photoreduced in the presence of chloroplasts, can liberate H2 in combination with Pt (Adam's catalyst). An aqueous solution of proflavine can be photoreduced in the presence of organic electron donors such as EDTA, cysteine, dithiothreitol, etc.; the reduced proflavine can subsequently liberate H2 with MV-Pt, MV-hydrogenase, ferredoxin-hydrogenase or cytochrome-hydrogenase systems.

A biochemical logic gate using an enzyme and its inhibitor. Part II: The logic gate.

PubMed

Sivan, Sarit; Tuchman, Samuel; Lotan, Noah

2003-06-01

Enzyme-Based Logic Gates (ENLOGs) are key components in bio-molecular systems for information processing. This report and the previous one in this series address the characterization of two bio-molecular switching elements, namely the alpha-chymotrypsin (alphaCT) derivative p-phenylazobenzoyl-alpha-chymotrypsin (PABalphaCT) and its inhibitor (proflavine), as well as their assembly into a logic gate. The experimental output of the proposed system is expressed in terms of enzymic activity and this was translated into logic output (i.e. "1" or "0") relative to a predetermined threshold value. We have found that an univalent link exists between the dominant isomers of PABalphaCT (cis or trans), the dominant form of either acridine (proflavine) or acridan and the logic output of the system. Thus, of all possible combinations, only the trans-PABalphaCT and the acridan lead to an enzymic activity that can be defined as logic output "1". The system operates under the rules of Boolean algebra and performs as an "AND" logic gate.

Liquid chromatographic determination of acriflavine and proflavine residues in channel catfish muscle.

PubMed

Plakas, S M; el Said, K R; Jester, E L; Bencsath, F A; Hayton, W L

1997-01-01

A liquid chromatographic (LC) method was developed for determination of acriflavine (ACR) and proflavine (PRO) residues in channel catfish muscle. Residues were extracted with acidified methanol solution, and extracts were cleaned up with C18 solid-phase extraction columns. Residue concentrations were determined on an LC cyano column, with spectrophotometric detection at 454 nm. Catfish muscle was individually fortified with ACR (purified from commercial product) and PRO at concentrations of 5, 10, 20, 40, and 80 ppb (5 replicates per level). Mean recoveries from fortified muscle at each level ranged from 86 to 95%, with relative standard deviations (RSDs) of 2.5 to 5.7%. The method was applied to incurred residues of ACR and PRO in muscle after waterborne exposure of channel catfish to commercial acriflavine (10 ppm total dye for 4 h). RSDs for incurred residues of ACR and PRO were in the same range as those for fortified muscle. Low residue concentrations (< 1% of exposure water concentration) suggested poor absorption of ACR and PRO in catfish.

Fluorescence of acridinic dyes in anionic surfactant solution

NASA Astrophysics Data System (ADS)

Pereira, Robson Valentim; Gehlen, Marcelo Henrique

2005-10-01

The interaction of the cationic dyes acridine, 9-aminoacridine (9AA), and proflavine, with sodium dodecyl sulfate (SDS) was studied by electronic absorption, steady-state and time-resolved fluorescence spectroscopies. The dyes interact with SDS in the pre-micellar region leading in two cases to dimerization in dye-surfactant aggregates, but with distinct molecular arrangements. For proflavine, the observed red shift of the electronic absorption band indicates the presence of J-aggregate, which are nonfluorescent. In the case of 9AA, the aggregates were characterized as nonspecific (neither J- nor H-type is spectroscopically observed). The time-resolved emission spectra gives evidences of the presence of weakly bound dimers by the recovery of three defined decay times by global analysis: dye monomer ( τ1 = 16.4 ns), dimer ( τ2 = 7.1 ns), and a faster component ( τ3 = 2.1 ns) ascribed to intracluster energy migration between monomer and dimer. Acridine has a weak interaction with SDS forming only an ion pair without further self-aggregation of the dye.

Fluorescence of acridinic dyes in anionic surfactant solution.

PubMed

Pereira, Robson Valentim; Gehlen, Marcelo Henrique

2005-10-01

The interaction of the cationic dyes acridine, 9-aminoacridine (9AA), and proflavine, with sodium dodecyl sulfate (SDS) was studied by electronic absorption, steady-state and time-resolved fluorescence spectroscopies. The dyes interact with SDS in the pre-micellar region leading in two cases to dimerization in dye-surfactant aggregates, but with distinct molecular arrangements. For proflavine, the observed red shift of the electronic absorption band indicates the presence of J-aggregate, which are nonfluorescent. In the case of 9AA, the aggregates were characterized as nonspecific (neither J- nor H-type is spectroscopically observed). The time-resolved emission spectra gives evidences of the presence of weakly bound dimers by the recovery of three defined decay times by global analysis: dye monomer (tau1 = 16.4 ns), dimer (tau2 = 7.1 ns), and a faster component (tau3 = 2.1 ns) ascribed to intracluster energy migration between monomer and dimer. Acridine has a weak interaction with SDS forming only an ion pair without further self-aggregation of the dye.

Time-Delayed Two-Step Selective Laser Photodamage of Dye-Biomolecule Complexes

NASA Astrophysics Data System (ADS)

Andreoni, A.; Cubeddu, R.; de Silvestri, S.; Laporta, P.; Svelto, O.

1980-08-01

A scheme is proposed for laser-selective photodamage of biological molecules, based on time-delayed two-step photoionization of a dye molecule bound to the biomolecule. The validity of the scheme is experimentally demonstrated in the case of the dye Proflavine, bound to synthetic polynucleotides.

Differences in multiplication of virulent and vaccine strains of poliovirus type I, II, and III in laboratory animals.

PubMed

Koroleva, G A; Lashkevich, V A; Voroshilova, M K

1977-01-01

Multiplication of virulent and vaccine strains of poliovirus type I, II and III in laboratory animals of different species was studied comparatively. The main criterion of virus reproduction was the production of the photoresistant virus progeny after inoculation of the animals with proflavin-photosensitized virus strains. On the whole, virulent poliovirus strains were characterized by replication in a wide range of hosts (monkeys, cotton rats, white mice, guinea pigs, rabbits, chickens, chick embryos), a low infective dose, production of the photoresistant progeny to a high titre, clinically overt disease in some animal species. The vaccine strains multiplied in a norrower range of hosts, had a high infective dose, a low titre of virus progeny, and caused no clinical symptoms of infection. These differences may serve as a marker for differentiation between virulent and attenuated strains in vivo. Administration of guanidine before inoculation of newborn cotton rats completely prevented or delayed by several days the production of photoresistant virus progeny. This fact confirms the stability of the proflavin-poliovirus complex under conditions ruling out virus replication.

Synthesis, DNA interaction, and cytotoxic activity of a novel proflavine-dithiazolidinone pharmacophore.

PubMed

Janovec, Ladislav; Sabolova, Danica; Kozurkova, Maria; Paulíkova, Helena; Kristian, Pavol; Ungvarský, Jan; Moravcíkova, Erika; Bajdichova, Maria; Podhradský, Dusan; Imrich, Jan

2007-01-01

Five novel proflavine-dithiazolidinone derivatives 4a-4e have been designed and synthesized by the reaction of dialkyl acridin-3,6-diyl dithioureas 3a-3e with methyl bromoacetate. The binding affinity of dithiazolidinone hydrochlorides 5a-5e with calf thymus DNA and plasmid (pUC19) DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence, and CD spectroscopy. The effects of 5a-5e on the thermal denaturation profiles of calf thymus DNA were also studied. From spectrophotometric and spectrofluorimetric titrations, the binding constants for the pUC19 DNA-drug complexes were determined (K = 6.2-2.2 x 104 M-1). In vitro cytotoxic activities of compounds 5a-5e toward murine leukemia cell line L1210 and human uterus carcinoma HeLa cells were also examined. 2',2' '-[(Acridin-3,6-diyl)diimino]-3',3' '-dipropyl-1,3-dithiazolidin-4-one hydrochloride (5b) showed the highest activity against these cells with IC50 values of 6.3 microM and 12.9 microM over the course of 72 h.

Studies on the interactions of 3,6-diaminoacridine derivatives with human serum albumin by fluorescence spectroscopy.

PubMed

Gökoğlu, Elmas; Kıpçak, Fulya; Seferoğlu, Zeynel

2014-11-01

This study reports the preparation and investigation of the modes of binding of the two symmetric 3,6-diaminoacridine derivatives obtained from proflavine, which are 3,6-diphenoxycarbonyl aminoacridine and 3,6-diethoxycarbonyl aminoacridine to human serum albumin (HSA). The interaction of HSA with the derivatives was investigated using fluorescence quenching and ultraviolet-visible absorption spectra at pH 7.2 and different temperatures. The results suggest that the derivatives used can interact strongly with HSA and are the formation of HSA-derivative complexes and hydrophobic interactions as the predominant intermolecular forces in stabilizing for each complex. The Stern-Volmer quenching constants, binding constants, binding sites and corresponding thermodynamic parameters ΔH, ΔS and ΔG were calculated at different temperatures. The binding distance (r) ~ 3 nm between the donor (HSA) and acceptors (3,6-diethoxycarbonyl aminoacridine, 3,6-diphenoxycarbonyl aminoacridine and proflavine) was obtained according to Förster's non-radiative energy transfer theory. Moreover, the limit of detection and limit of quantification of derivatives were calculated in the presence of albumin. Copyright © 2014 John Wiley & Sons, Ltd.

The nature of the force-induced conformation transition of dsDNA studied by using single molecule force spectroscopy.

PubMed

Liu, Ningning; Bu, Tianjia; Song, Yu; Zhang, Wei; Li, Jinjing; Zhang, Wenke; Shen, Jiacong; Li, Hongbin

2010-06-15

Single-stranded DNA binding proteins (SSB) interact with single-stranded DNA (ssDNA) specifically. Taking advantage of this character, we have employed Bacillus subtilis SSB protein to investigate the nature of force-induced conformation transition of double-stranded DNA (dsDNA) by using AFM-based single molecule force spectroscopy (SMFS) technique. Our results show that, when a dsDNA is stretched beyond its contour length, the dsDNA is partially melted, producing some ssDNA segments which can be captured by SSB proteins. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. Furthermore, the effect of proflavine, a DNA intercalator, on the SSB-DNA interactions has been investigated, and the results indicate that the proflavine-saturated dsDNA can be stabilized to the extent that the dsDNA will no longer melt into ssDNA under the mechanical force even up to 150 pN, and no SSB-DNA interactions are detectable.

Physical nature of ethidium and proflavine interactions with nucleic acid bases in the intercalation plane.

PubMed

Langner, Karol M; Kedzierski, Pawel; Sokalski, W Andrzej; Leszczynski, Jerzy

2006-05-18

On the basis of the crystallographic structures of three nucleic acid intercalation complexes involving ethidium and proflavine, we have analyzed the interaction energies between intercalator chromophores and their four nearest bases, using a hybrid variation-perturbation method at the second-order Møller-Plesset theory level (MP2) with a 6-31G(d,p) basis set. A total MP2 interaction energy minimum precisely reproduces the crystallographic position of the ethidium chromophore in the intercalation plane between UA/AU bases. The electrostatic component constitutes the same fraction of the total energy for all three studied structures. The multipole electrostatic interaction energy, calculated from cumulative atomic multipole moments (CAMMs), was found to converge only after including components above the fifth order. CAMM interaction surfaces, calculated on grids in the intercalation planes of these structures, reasonably reproduce the alignment of intercalators in crystal structures; they exhibit additional minima in the direction of the DNA grooves, however, which also need to be examined at higher theory levels if no crystallographic data are given.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walton, B.T.

A chemical impurity isolated from commercially purchased acridine causes cricket embryos to develop extra compound eyes, branched antennae, extra antennae, and extra heads. Purified acridine does not produce similar duplications of cricket heads or head structures nor do the substituted acridines proflavine, acriflavine, or acridine orange. A dose-response relation exists such that the number and severity of abnormalities increase with increasing concentration of the teratogen.

Low temperature structures of dCpG-proflavine. Conformational and hydration effects.

PubMed Central

Schneider, B; Ginell, S L; Berman, H M

1992-01-01

The structure of the complex of dCpG with proflavine was determined using x-ray data taken at -130 degrees C (low temperature) and at -2 degrees C (cold temperature) and compared with the structure of the complex determined previously at room temperature (Shieh, H. S., H. M. Berman, M. Dabrow, and S. Neidle. 1980. Nucleic Acids Res. 8:85-97). Low temperature was refined with 5,125 reflections between 8.0 and 0.93 A, Anisotropically modeled temperature factors were used for DNA/drug atoms and isotropic ones for water oxygens to R factor of 12.2% in P2(1)2(1)2; a = 32.853, b = 21.760, c = 13.296 A. Cold temperature was refined isotropically with 2,846 reflections 8.0-0.89 A to R = 15.1% in P2(1)2(1)2; a = 32.867, b = 22.356, c = 13.461 A. Both structures are very similar to the room temperature one, though some important differences were observed: one guanine sugar moiety is disordered and additional water molecules have been located that give rise to infinite polyhedral hydration networks. Images FIGURE 2 PMID:1489914

A trap potential model investigation of the optical activity induced in dye-DNA intercalation complexes

NASA Astrophysics Data System (ADS)

Kamiya, Mamoru

1988-02-01

The fundamental features of the optical activity induced in dye-DNA intercalation complexes are studied by application of the trap potential model which is useful to evaluate the induced rotational strength without reference to detailed geometrical information about the intercalation complexes. The specific effect of the potential depth upon the induced optical activity is explained in terms of the relative magnitudes of the wave-phase and helix-phase variations in the path of an electron moving on a restricted helical segment just like an exciton trapped around the dye intercalation site. The parallel and perpendicular components of the induced rotational strength well reflect basic properties of the helicity effects about the longitudinal and tangential axes of the DNA helical cylinder. The trap potential model is applied to optimize the potential parameters so as to reproduce the ionic strength effect upon the optical activity induced to proflavine-DNA intercalation complexes. From relationships between the optimized potential parameters and ionic strengths, it is inferred that increase in the ionic strength contributes to the optical activity induced by the nearest-neighbour interaction between intercalated proflavine and DNA base pairs.

Low temperature structures of dCpG-proflavine.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schneider, B.; Ginell, S. L.; Berman, H. M.

1992-12-01

The structure of the complex of dCpG with proflavine was determined using x-ray data taken at -130 degrees C (low temperature) and at -2 degrees C (cold temperature) and compared with the structure of the complex determined previously at room temperature (Shieh, H. S., H. M. Berman, M. Dabrow, and S. Neidle. 1980. Nucleic Acids Res. 8:85-97). Low temperature was refined with 5,125 reflections between 8.0 and 0.93 A, Anisotropically modeled temperature factors were used for DNA/drug atoms and isotropic ones for water oxygens to R factor of 12.2% in P2(1)2(1)2; a = 32.853, b = 21.760, c = 13.296more » A. Cold temperature was refined isotropically with 2,846 reflections 8.0-0.89 A to R = 15.1% in P2(1)2(1)2; a = 32.867, b = 22.356, c = 13.461 A. Both structures are very similar to the room temperature one, though some important differences were observed: one guanine sugar moiety is disordered and additional water molecules have been located that give rise to infinite polyhedral hydration networks.« less

Vital-dye-enhanced multimodal imaging of neoplastic progression in a mouse model of oral carcinogenesis

NASA Astrophysics Data System (ADS)

Hellebust, Anne; Rosbach, Kelsey; Wu, Jessica Keren; Nguyen, Jennifer; Gillenwater, Ann; Vigneswaran, Nadarajah; Richards-Kortum, Rebecca

2013-12-01

In this longitudinal study, a mouse model of 4-nitroquinoline 1-oxide chemically induced tongue carcinogenesis was used to assess the ability of optical imaging with exogenous and endogenous contrast to detect neoplastic lesions in a heterogeneous mucosal surface. Widefield autofluorescence and fluorescence images of intact 2-NBDG-stained and proflavine-stained tissues were acquired at multiple time points in the carcinogenesis process. Confocal fluorescence images of transverse fresh tissue slices from the same specimens were acquired to investigate how changes in tissue microarchitecture affect widefield fluorescence images of intact tissue. Widefield images were analyzed to develop and evaluate an algorithm to delineate areas of dysplasia and cancer. A classification algorithm for the presence of neoplasia based on the mean fluorescence intensity of 2-NBDG staining and the standard deviation of the fluorescence intensity of proflavine staining was found to separate moderate dysplasia, severe dysplasia, and cancer from non-neoplastic regions of interest with 91% sensitivity and specificity. Results suggest this combination of noninvasive optical imaging modalities can be used in vivo to discriminate non-neoplastic from neoplastic tissue in this model with the potential to translate this technology to the clinic.

1H NMR study of the hetero-association of flavin-mononucleotide with mutagenic dyes: ethidium bromide and proflavine

NASA Astrophysics Data System (ADS)

Evstigneev, M. P.; Mukhina, Yu. V.; Davies, D. B.

The hetero-association of the vitamin B2 derivative, flavin-mononucleotide (FMN), with a mutagenic dye, ethidium bromide (EB) or proflavine (PF), has been studied by 1D and 2D 500 MHz 1H NMR spectroscopy. The variations of proton chemical shifts of both the vitamin and dye as a function of concentration and temperature were analysed in terms of the structural and thermodynamical properties of the FMN-EB and FMN-PF complexes in solution. The structures of the complexes were also investigated by observed intermolecular ROE contacts and molecular mechanics calculations. The results show that the 1 : 1 hetero-association complexes in solution are more stable than the self-association complexes, which is consistent with formation of an intermolecular hydrogen-bond in the hetero-complexes of FMN-EB and FMN-PF. Hence it is possible that the toxicity of aromatic molecules such as EB and PF may be reduced in vitro by the presence of FMN, partly because of the known antimutagenic action of FMN and partly because it has been shown in this work that there is an effective intermolecular association between the mutagens and the vitamin.

Biophotolysis systems for hydrogen production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rao, K.K.; Adams, M.W.W.; Gisby, P.E.

1981-01-01

Model systems containing natural and synthetic catalysts were constructed for the production of H/sub 2/ from water using visible solar radiation as the energy source. The authors have investigated the use of aqueous systems with proflavine as the light activator and artificial electron donors for subsequent production of H/sub 2/ when coupled to electron mediators and hydrogenase (or Pt). The characteristics, relative merits and defects of these systems are discussed. 22 refs.

AFRRI Reports, July-September 1990

DTIC Science & Technology

1990-10-01

Geacintov, N. E. Linear dichroism characteristics of 0 ethidium- and proflavine-supercoiled DNA complexes. SR90-24: Weiss, J. F., Kumar, K. S...forming units per milliliter, based on colony count within contain 10% fetal bovine serum (HyClone, Logan, UT), plus 10% the linear part of the dilution...dps Press. Hyalomers und des Granulomers . Acta Haemat. 6, 207- BLASCHKO, H., FIREMARK, H., SMITH, A. D. & WINKLFR, 12. H. (1967) Lipids of the adrenal

Design and validation of a diffuse reflectance and spectroscopic microendoscope with poly(dimethylsioxane)-based phantoms

PubMed Central

Greening, Gage J.; Powless, Amy J.; Hutcheson, Joshua A.; Prieto, Sandra P.; Majid, Aneeka A.; Muldoon, Timothy J.

2015-01-01

Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra. PMID:25983372

Host-Guest Complexes of Carboxylated Pillar[n]arenes With Drugs.

PubMed

Wheate, Nial J; Dickson, Kristie-Ann; Kim, Ryung Rae; Nematollahi, Alireza; Macquart, René B; Kayser, Veysel; Yu, Guocan; Church, W Bret; Marsh, Deborah J

2016-12-01

Pillar[n]arenes are a new family of nanocapsules that have shown application in a number of areas, but because of their poor water solubility their biomedical applications are limited. Recently, a method of synthesizing water-soluble pillar[n]arenes was developed. In this study, carboxylated pillar[n]arenes (WP[n], n = 6 or 7) have been examined for their ability to form host-guest complexes with compounds relevant to drug delivery and biodiagnostic applications. Both pillar[n]arenes form host-guest complexes with memantine, chlorhexidine hydrochloride, and proflavine by 1 H nuclear magnetic resonance and modeling. Binding is stabilized by hydrophobic effects within the cavities, and hydrogen bonding and electrostatic interactions at the portals. Encapsulation within WP[6] results in the complete and efficient quenching of proflavine fluorescence, giving rise to "on" and "off" states that have potential in biodiagnostics. The toxicity of the pillar[n]arenes was examined using in vitro growth assays with the OVCAR-3 and HEK293 cell lines. The pillar[n]arenes are relatively nontoxic to cells except at high doses and after prolonged continuous exposure. Overall, the results show that there could be a potentially large range of medical applications for carboxylated pillar[n]arene nanocapsules. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Pharmacokinetics, tissue distribution and metabolism of acriflavine and proflavine in the channel catfish (Ictalurus punctatus).

PubMed

Plakas, S M; el Said, K R; Bencsath, F A; Musser, S M; Hayton, W L

1998-06-01

1. The disposition of proflavine (PRO) and acriflavine (ACR) were examined in channel catfish after intravascular (i.v.) dosing (1 mg/kg) or waterborne exposure (10 mg/l for 4 h). 2. After i.v. dosing, plasma concentration-time profiles of parent PRO and ACR were best described by two- and three-compartment pharmacokinetic models respectively. Terminal elimination half-lives of PRO and ACR in plasma were 8.7 and 11.4 h respectively. 3. In animals dosed with 14C-PRO or 14C-ACR, total drug equivalent concentrations were highest in the excretory organs and lowest in muscle, fat and plasma. In PRO-dosed animals, residues in the liver and trunk kidney were composed primarily of glucuronosyl and acetyl conjugates of PRO; residues in muscle were composed mostly (> 95%) of the parent drug. In ACR-dosed animals, the parent compound comprised > 90% of the total residues in all tissues examined. 4. PRO and ACR were poorly absorbed in catfish during waterborne exposure. At the end of a 4-h exposure, parent PRO and ACR concentrations in muscle were 0.064 and 0.020 microgram/g respectively. Levels in muscle declined below the limit of determination (0.005 microgram/g) within 1-2 weeks.

Design and validation of a diffuse reflectance and spectroscopic microendoscope with poly(dimethylsiloxane)-based phantoms

NASA Astrophysics Data System (ADS)

Greening, Gage J.; Powless, Amy J.; Hutcheson, Joshua A.; Prieto, Sandra P.; Majid, Aneeka A.; Muldoon, Timothy J.

2015-03-01

Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra.

c-di-GMP can form remarkably stable G-quadruplexes at physiological conditions in the presence of some planar intercalators.

PubMed

Nakayama, Shizuka; Kelsey, Ilana; Wang, Jingxin; Sintim, Herman O

2011-04-28

The ubiquitous bacterial biofilm regulator, c-di-GMP can form G-quadruplexes at physiological conditions in the presence of some aromatic compounds, such as acriflavine and proflavine. The fluorescence of these compounds is quenched upon c-di-GMP binding and some of the formed c-di-GMP G-quadruplexes are stable even at 75 °C. © The Royal Society of Chemistry 2011

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging.

PubMed

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J

2012-07-01

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ∼7  μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Tumor margin assessment of surgical tissue specimen of cancer patients using label-free hyperspectral imaging

NASA Astrophysics Data System (ADS)

Fei, Baowei; Lu, Guolan; Wang, Xu; Zhang, Hongzheng; Little, James V.; Magliocca, Kelly R.; Chen, Amy Y.

2017-02-01

We are developing label-free hyperspectral imaging (HSI) for tumor margin assessment. HSI data, hypercube (x,y,λ), consists of a series of high-resolution images of the same field of view that are acquired at different wavelengths. Every pixel on the HSI image has an optical spectrum. We developed preprocessing and classification methods for HSI data. We used spectral features from HSI data for the classification of cancer and benign tissue. We collected surgical tissue specimens from 16 human patients who underwent head and neck (H&N) cancer surgery. We acquired both HSI, autofluorescence images, and fluorescence images with 2-NBDG and proflavine from the specimens. Digitized histologic slides were examined by an H&N pathologist. The hyperspectral imaging and classification method was able to distinguish between cancer and normal tissue from oral cavity with an average accuracy of 90+/-8%, sensitivity of 89+/-9%, and specificity of 91+/-6%. For tissue specimens from the thyroid, the method achieved an average accuracy of 94+/-6%, sensitivity of 94+/-6%, and specificity of 95+/-6%. Hyperspectral imaging outperformed autofluorescence imaging or fluorescence imaging with vital dye (2-NBDG or proflavine). This study suggests that label-free hyperspectral imaging has great potential for tumor margin assessment in surgical tissue specimens of H&N cancer patients. Further development of the hyperspectral imaging technology is warranted for its application in image-guided surgery.

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs.

PubMed

Alonso, A; Almendral, M J; Curto, Y; Criado, J J; Rodríguez, E; Manzano, J L

2006-08-15

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

Combined optical resolution photoacoustic and fluorescence micro-endoscopy

NASA Astrophysics Data System (ADS)

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J.

2012-02-01

We present a new micro-endoscopy system combining real-time C-scan optical-resolution photoacoustic micro-endoscopy (OR-PAME), and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the OR-PAM sub-system is capable of imaging with a resolution of ~ 7μm. The fluorescence sub-system consists of a diode laser with 445 nm-centered emissions as the light source, an objective lens and a CCD camera. Proflavine, a FDA approved drug for human use, is used as the fluorescent contrast agent by topical application. The fluorescence system does not require any mechanical scanning. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single mode fibers. The absorption of Proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural and functional information given by OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping researchers and clinicians visualize angiogenesis, effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Needle endomicroscope with a plastic, achromatic objective to perform optical biopsies of breast tissue

NASA Astrophysics Data System (ADS)

Kyrish, Matthew; Dobbs, Jessica; Richards-Kortum, Rebecca; Tkaczyk, Tomasz

2013-03-01

In order to diagnose cancer in breast tissue, a sample must be removed, prepared, and examined under a microscope. To provide an alternative to conventional biopsies, an endomicroscope intended to perform optical biopsies is demonstrated. The system provides high resolution, high contrast images in real-time which could allow a diagnosis to be made during surgery without the need for tissue removal. Optical sectioning is achieved via structured illumination to reject out of focus light. An image is relayed between the sample plane and the imaging system by a coherent fiber bundle with an achromatized objective lens at the distal tip of the fiber bundle which is the diameter of a biopsy needle. The custom, plastic objective provides correction for both the excitation and emission wavelengths of proflavine (452 nm and 515 nm, respectively). It also magnifies the object onto the distal tip of the fiber bundle to increase lateral resolution. The lenses are composed of the optical plastics Zeonex E48R, PMMA, and polystyrene. The lenses are fabricated via single point diamond turning and assembled using a zero alignment technique. The lateral resolution and chromatic focal shift were measured and in vitro images of breast carcinoma cells stained with proflavine were captured. The optical biopsy system is able to achieve optical sectioning and to resolve smaller features than the current high resolution microendoscope.

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging

NASA Astrophysics Data System (ADS)

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J.

2012-07-01

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ~7 μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

DNA binding of a proflavine derivative bearing a platinum hanging residue.

PubMed

Biagini, Silvia; Bianchi, Antonio; Biver, Tarita; Boggioni, Alessia; Nikolayenko, Igor V; Secco, Fernando; Venturini, Marcella

2011-04-01

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I=0.1M and 25°C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides. Copyright © 2010 Elsevier Inc. All rights reserved.

Biochemical transformation of mouse cells by herpes simplex virus type 2: enhancement by means of low-level photodynamic treatment.

PubMed Central

Verwoerd, D W; Rapp, F

1978-01-01

The biochemical transformation of thymidine kinase-deficient cells by UV-inactivated herpes simplex virus is enhanced by low-level photodynamic treatment of the infected cells. At the concentration of proflavine used, the virus was not inactivated and both virus and cellular DNA syntheses were only marginally inhibited. The observed enhancement of the transfer of a virus gene to the cell genome suggests a possible cocarcinogenic role for photodynamically active dyes at very low concentrations. PMID:206727

Binding of 3O2 and 1O2 to dyes used in photodynamic therapy in gas phase and aqueous media

NASA Astrophysics Data System (ADS)

Kushwaha, P. S.; Mishra, P. C.

Density functional theory (DFT) was employed at the B3LYP/6-31+G* level to study complexes of 1O2 and 3O2 with the dye molecules proflavine, methylene blue, and acridine orange, which are useful in photodynamic therapy. It was found that the most stable complex between 1O2 and proflavine are formed when 1O2 is located above the central ring, while the most stable complex between 1O2 and methylene blue is formed when 1O2 is located above the molecular plane, but not above any of the rings, near the sulfur atom. 1O2 can make a stable complex with acridine orange, as it is located above the outer ring of the dye. The binding energies of the complexes of 1O2 with all three dyes are enhanced considerably in going from gas phase to aqueous media. The complexes of 3O2 with the dyes will be unstable in all cases, while those of 1O2 with the same will be quite stable and will not be dissociated due to thermal fluctuations at room temperature. In the complexes of 1O2 and 3O2 with the dyes, charge transfer occurs from the dyes to the O2 moiety, the amount of charge transfer being much more to 1O2 than to 3O2 in each case.

COB231 targets amyloid plaques in post-mortem human brain tissue and in an Alzheimer mouse model.

PubMed

Garin, Dominique; Virgone-Carlotta, Angélique; Gözel, Bülent; Oukhatar, Fatima; Perret, Pascale; Marti-Battle, Danièle; Touret, Monique; Millet, Philippe; Dubois-Dauphin, Michel; Meyronet, David; Streichenberger, Nathalie; Laferla, Frank M; Demeunynck, Martine; Chierici, Sabine; Sallanon Moulin, Marcelle; Ghezzi, Catherine

2015-03-01

Previous works have shown the interest of naturally fluorescent proflavine derivatives to label Abeta deposits in vitro. This study aimed to further characterize the properties of the proflavine 3-acetylamino-6-[3-(propargylamino)propanoyl]aminoacridine (COB231) derivative as a probe. This compound was therefore evaluated on human post-mortem and mice brain slices and in vivo in 18-month-old triple transgenic mice APPswe, PS1M146V and tauP301L (3xTgAD) mice presenting the main characteristics of Alzheimer's disease (AD). COB231 labelled amyloid plaques on brain slices of AD patients, and 3xTgAD mice at 10 and 0.1 μM respectively. However, no labelling of the neurofibrillary tangle-rich areas was observed either at high concentration or in the brain of fronto-temporal dementia patients. The specificity of this mapping was attested in mice using Thioflavin S and IMPY as positive controls of amyloid deposits. After intravenous injection of COB231 in old 3xTgAD mice, fluorescent amyloid plaques were detected in the cortex and hippocampus, demonstrating COB231 blood–brain barrier permeability. We also controlled the cellular localization of COB231 on primary neuronal cultures and showed that COB231 accumulates into the cytoplasm and not into the nucleus. Finally, using a viability assay, we only detected a slight cytotoxic effect of COB231 (< 10%) for the highest concentration (100 μM).

Kinetic and thermodynamic hysteresis imposed by intercalation of proflavine in ferrocene-modified double-stranded DNA.

PubMed

Gebala, Magdalena; La Mantia, Fabio; Schuhmann, Wolfgang

2013-07-22

Surface-confined immobilized redox species often do not show the expected zero peak separation in slow-scan cyclic voltammograms. This phenomenon is frequently associated to experimental drawbacks and hence neglected. However, a nonzero peak separation, which is common to many electrochemical systems with high structural flexibility, can be rationally assigned to a thermodynamic hysteresis. To study this phenomenon, a surface-confined redox species was used. Specifically, a DNA strand which is tagged with ferrocene (Fc) moieties at its 5' end and its complementary capture probe is thiolated at the 3' end was self-assembled in a monolayer at a Au electrode with the Fc moieties being located at the bottom plane of the double-stranded DNA (dsDNA). The DNA-bound Fc undergoes rapid electron transfer with the electrode surface as evaluated by fast scan cyclic voltammetry. The electron transfer is sensitive to the ion transport along the DNA strands, a phenomenon which is modulated upon specific intercalation of proflavine into surface-bound dsDNA. The electron transfer rate of the Fc(0/+) redox process is influenced by the cationic permselectivity of the DNA monolayer. In addition to the kinetic hindrance, a thermodynamic effect correlated with changes in the activity coefficients of the Fc(0/+) moieties near the gold-dsDNA interface is observed and discussed as source of the observed hysteresis causing the non-zero peak separation in the voltammograms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA binding by a new metallointercalator that contains a proflavine group bearing a hanging chelating unit.

PubMed

Bazzicalupi, Carla; Bencini, Andrea; Bianchi, Antonio; Biver, Tarita; Boggioni, Alessia; Bonacchi, Sara; Danesi, Andrea; Giorgi, Claudia; Gratteri, Paola; Ingraín, Antonio Marchal; Secco, Fernando; Sissi, Claudia; Valtancoli, Barbara; Venturini, Marcella

2008-01-01

The new bifunctional molecule 3,6-diamine-9-[6,6-bis(2-aminoethyl)-1,6-diaminohexyl]acridine (D), which is characterised by both an aromatic moiety and a separate metal-complexing polyamine centre, has been synthesised. The characteristics of D and its ZnII complex ([ZnD]) (protonation and metal-complexing constants, optical properties and self-aggregation phenomena) have been analysed by means of NMR spectroscopy, potentiometric, spectrophotometric and spectrofluorimetric techniques. The equilibria and kinetics of the binding process of D and [ZnD] to calf thymus DNA have been investigated at I=0.11 M (NaCl) and 298.1 K by using spectroscopic methods and the stopped-flow technique. Static measurements show biphasic behaviour for both D-DNA and [ZnD]-DNA systems; this reveals the occurrence of two different binding processes depending on the polymer-to-dye molar ratio (P/D). The binding mode that occurs at low P/D values is interpreted in terms of external binding with a notable contribution from the polyamine residue. The binding mode at high P/D values corresponds to intercalation of the proflavine residue. Stopped-flow, circular dichroism and supercoiled-DNA unwinding experiments corroborate the proposed mechanism. Molecular-modelling studies support the intercalative process and evidence the influence of NH+...O interactions between the protonated acridine nitrogen atom and the oxygen atoms of the polyanion; these interactions play a key role in determining the conformation of DNA adducts.

Comparison of high-resolution microendoscope images and histopathological sections in ex vivo middle ear cholesteatomas and surrounding tissue

NASA Astrophysics Data System (ADS)

Bradley, James; Levy, Lauren; Richards-Kortum, Rebecca; Sikora, Andrew G.; Smouha, Eric

2013-03-01

Objective: To investigate the concordance between optical images obtained with high-resolution microendoscopy (HRME) and conventional histopathology for ex vivo cholesteatoma specimens and surrounding middle ear epithelium. Methods: After resection of cholesteatoma and surrounding middle ear epithelium from surgical patients, tissues were stained with a contrast agent, proflavine, and the HRME fiberoptic scope was placed directly on each tissue specimen. 4- 10 short movie clips were recorded for both the cholesteatoma and surrounding middle ear epithelium specimens. The imaged areas were sent for standard histopathology, and the stained specimens were correlated with the HRME images. IRB approval was obtained, and each patient was consented for the study. Results: Ten cholesteatoma specimens and 9 middle ear specimens were collected from 10 patients. In each case, cholesteatoma was easily discriminated from normal middle ear epithelium by its hyperfluorescence and loss of cellular detail. Qualitative analysis for concordance between HRME images and histological images from the same surgical specimen yielded a strong correlation between imaging modalities. Conclusions: Keratinizing cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Loss of cellular detail and hyperfluorescence with proflavine are the hallmark characteristics of cholesteatoma which allow for differentiation from normal middle ear epithelium. Real-time optical imaging can potentially improve the results of otologic surgery by allowing for extirpation of cholesteatomas while eliminating residual disease. We anticipate performing an in vivo study to test this hypothesis.

Dynamics of DNA/intercalator complexes

NASA Astrophysics Data System (ADS)

Schurr, J. M.; Wu, Pengguang; Fujimoto, Bryant S.

1990-05-01

Complexes of linear and supercoiled DNAs with different intercalating dyes are studied by time-resolved fluorescence polarization anisotropy using intercalated ethidium as the probe. Existing theory is generalized to take account of excitation transfer between intercalated ethidiums, and Forster theory is shown to be valid in this context. The effects of intercalated ethidium, 9-aminoacridine, and proflavine on the torsional rigidity of linear and supercoiled DNAs are studied up to rather high binding ratios. Evidence is presented that metastable secondary structure persists in dye-relaxed supercoiled DNAs, which contradicts the standard model of supercoiled DNAs.

Photodynamic treatment of herpes simplex virus during its replicative cycle.

PubMed Central

Khan, N C; Melnick, J L; Biswal, N

1977-01-01

Photodynamic treatment of herpes simplex virus type 1-infected hamster embryo fibroblasts (LSH strain) with a low concentration of proflavine (0.08 mug/10(5) cells per ml), a 3-9-diamine acridine dye, inhibited production not only of infectious progeny but also of virion particles. However, there was no appreciable inhibition of viral or cellular DNA synthesis, even when the infected cells were repeatedly exposed to this low concentration of dye and light during the replication cycle of the virus. It thus appears that photodynamic treatment of infected cells interferes with the processes involved in virus maturation. PMID:189063

Rotational strength of dye-helix complexes as studied by a potential model theory

NASA Astrophysics Data System (ADS)

Kamiya, Mamoru

1988-03-01

The fundamental features of the induced optical activity in dye-helix complexes are clarified by the trap potential model. The effect of the potential depth on the induced rotational strength is explained in terms of the relative magnitudes of the wave-phase and helix-phase variations in the path of an electron moving along a restricted helix segment just like an exciton trapped around a dye intercalation site. The potential parameters have been optimized so as to reproduce the ionic strength effect upon the rotational strengths induced in proflavine-DNA intercalation complexes.

Ionic strength and temperature induced conformational changes in mononucleosomes and oligonucleosomes. [Chromatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmitz, K.S.; Kent, J.C.; Parthasarathy, N.

1980-10-01

Chromatin is a nucleohistone complex which exhibits a repeat unit structure as inferred from nuclease digestion studies. The repeat unit, or nucleosome, is defined as approx. 200 base pairs of DNA wrapped about the surface of an octameric histone complex (two copies each of the histones H2A, H2B, H3, and H4). We report in this communication preliminary studies on the conformation of chromatin mononucleosomes and oligonucleosomes as a function of temperature and ionic strength. The methods used were conductivity, fluorescence of bound proflavine, and quasielastic light scattering.

A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J.

2015-09-01

Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min-1 with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels-1.

Micro-anatomical quantitative optical imaging: toward automated assessment of breast tissues.

PubMed

Dobbs, Jessica L; Mueller, Jenna L; Krishnamurthy, Savitri; Shin, Dongsuk; Kuerer, Henry; Yang, Wei; Ramanujam, Nirmala; Richards-Kortum, Rebecca

2015-08-20

Pathologists currently diagnose breast lesions through histologic assessment, which requires fixation and tissue preparation. The diagnostic criteria used to classify breast lesions are qualitative and subjective, and inter-observer discordance has been shown to be a significant challenge in the diagnosis of selected breast lesions, particularly for borderline proliferative lesions. Thus, there is an opportunity to develop tools to rapidly visualize and quantitatively interpret breast tissue morphology for a variety of clinical applications. Toward this end, we acquired images of freshly excised breast tissue specimens from a total of 34 patients using confocal fluorescence microscopy and proflavine as a topical stain. We developed computerized algorithms to segment and quantify nuclear and ductal parameters that characterize breast architectural features. A total of 33 parameters were evaluated and used as input to develop a decision tree model to classify benign and malignant breast tissue. Benign features were classified in tissue specimens acquired from 30 patients and malignant features were classified in specimens from 22 patients. The decision tree model that achieved the highest accuracy for distinguishing between benign and malignant breast features used the following parameters: standard deviation of inter-nuclear distance and number of duct lumens. The model achieved 81 % sensitivity and 93 % specificity, corresponding to an area under the curve of 0.93 and an overall accuracy of 90 %. The model classified IDC and DCIS with 92 % and 96 % accuracy, respectively. The cross-validated model achieved 75 % sensitivity and 93 % specificity and an overall accuracy of 88 %. These results suggest that proflavine staining and confocal fluorescence microscopy combined with image analysis strategies to segment morphological features could potentially be used to quantitatively diagnose freshly obtained breast tissue at the point of care without the need for tissue preparation.

Label-free reflectance hyperspectral imaging for tumor margin assessment: a pilot study on surgical specimens of cancer patients

NASA Astrophysics Data System (ADS)

Fei, Baowei; Lu, Guolan; Wang, Xu; Zhang, Hongzheng; Little, James V.; Patel, Mihir R.; Griffith, Christopher C.; El-Diery, Mark W.; Chen, Amy Y.

2017-08-01

A label-free, hyperspectral imaging (HSI) approach has been proposed for tumor margin assessment. HSI data, i.e., hypercube (x,y,λ), consist of a series of high-resolution images of the same field of view that are acquired at different wavelengths. Every pixel on an HSI image has an optical spectrum. In this pilot clinical study, a pipeline of a machine-learning-based quantification method for HSI data was implemented and evaluated in patient specimens. Spectral features from HSI data were used for the classification of cancer and normal tissue. Surgical tissue specimens were collected from 16 human patients who underwent head and neck (H&N) cancer surgery. HSI, autofluorescence images, and fluorescence images with 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) and proflavine were acquired from each specimen. Digitized histologic slides were examined by an H&N pathologist. The HSI and classification method were able to distinguish between cancer and normal tissue from the oral cavity with an average accuracy of 90%±8%, sensitivity of 89%±9%, and specificity of 91%±6%. For tissue specimens from the thyroid, the method achieved an average accuracy of 94%±6%, sensitivity of 94%±6%, and specificity of 95%±6%. HSI outperformed autofluorescence imaging or fluorescence imaging with vital dye (2-NBDG or proflavine). This study demonstrated the feasibility of label-free, HSI for tumor margin assessment in surgical tissue specimens of H&N cancer patients. Further development of the HSI technology is warranted for its application in image-guided surgery.

A fiber-optic fluorescence microscope using a consumer-grade digital camera for in vivo cellular imaging.

PubMed

Shin, Dongsuk; Pierce, Mark C; Gillenwater, Ann M; Williams, Michelle D; Richards-Kortum, Rebecca R

2010-06-23

Early detection is an essential component of cancer management. Unfortunately, visual examination can often be unreliable, and many settings lack the financial capital and infrastructure to operate PET, CT, and MRI systems. Moreover, the infrastructure and expense associated with surgical biopsy and microscopy are a challenge to establishing cancer screening/early detection programs in low-resource settings. Improvements in performance and declining costs have led to the availability of optoelectronic components, which can be used to develop low-cost diagnostic imaging devices for use at the point-of-care. Here, we demonstrate a fiber-optic fluorescence microscope using a consumer-grade camera for in vivo cellular imaging. The fiber-optic fluorescence microscope includes an LED light, an objective lens, a fiber-optic bundle, and a consumer-grade digital camera. The system was used to image an oral cancer cell line labeled with 0.01% proflavine. A human tissue specimen was imaged following surgical resection, enabling dysplastic and cancerous regions to be evaluated. The oral mucosa of a healthy human subject was imaged in vivo, following topical application of 0.01% proflavine. The fiber-optic microscope resolved individual nuclei in all specimens and tissues imaged. This capability allowed qualitative and quantitative differences between normal and precancerous or cancerous tissues to be identified. The optical efficiency of the system permitted imaging of the human oral mucosa in real time. Our results indicate this device as a useful tool to assist in the identification of early neoplastic changes in epithelial tissues. This portable, inexpensive unit may be particularly appropriate for use at the point-of-care in low-resource settings.

A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.

Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated aftermore » imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min{sup −1} with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels{sup −1}.« less

Structural and thermodynamic analysis of the hetero-association of theophylline with aromatic drug molecules

NASA Astrophysics Data System (ADS)

Andrejuk, D. D.; Hernandez Santiago, A. A.; Khomich, V. V.; Voronov, V. K.; Davies, D. B.; Evstigneev, M. P.

2008-10-01

The hetero-association of theophylline (THP) with other biologically-active aromatic molecules ( e.g. the anti-cancer drugs daunomycin and novantrone, the antibiotic norfloxacin, the vitamin flavin-mononucleotide and two mutagens ethidium bromide and proflavine) has been studied by NMR in aqueous-salt solution (0.1 M Na-phosphate buffer, p D 7.1). It was found that THP shows an essentially similar hetero-association ability as caffeine (CAF) towards aromatic drugs, except for novantrone (NOV), which has much less affinity to THP than CAF as a result of energetically unfavourable orthogonal orientation of the chromophores of THP and NOV in the hetero-complex.

Biophotolysis systems for hydrogen production

NASA Astrophysics Data System (ADS)

Rao, K. K.; Adams, M. W. W.; Morris, P.; Hall, D. O.; Gisby, P. E.

Model systems containing natural and sythetic catalysts were constructed for the production of H2 from water using visible solar radiation as the energy source. Chloroplast membranes were used for light absorption and photodecomposition of water, ferredoxin, flavodoxin, cytochrome, viologen dyes, 'Jeevanu' particles or synthetic clusters containing Fe-Mo-S centers were used as electron transfer catalysts, and hydrogenase or PtO2 served as the proton activator. We have also investigated the use of aqueous systems with proflavine as the light activator and artificial electron donors for subsequent production of H2 when coupled to electron mediators and hydrogenase (or Pt). The characteristics, relative merits and defects of these systems are discussed.

Ultraviolet-induced sister chromatid exchanges in V-79 cells with normal and BrdUrd-substituted DNA and the influence of intercalating substances and cysteine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Speit, G.; Mehnert, K.; Wolf, M.

1982-06-01

The influence of intercalating substances (proflavine, ethidium bromide) and of an SH compound (L-cysteine) on uv-induced sister chromatid exchanges (SCEs) was investigated in V-79 cells with normal and BrdUrd-substituted DNA. The results are discussed in relation to the primary damages leading to SCE induction produced by uv irradiation. The data indicate that neither the pyrimidine dimers nor DNA single-strand breaks are the primary cause of SCE induction, and that the damages leading to SCEs by uv irradiation differ from those which cause chromosome aberrations.

High-resolution microendoscope for the detection of cervical neoplasia.

PubMed

Grant, Benjamin D; Schwarz, Richard A; Quang, Timothy; Schmeler, Kathleen M; Richards-Kortum, Rebecca

2015-01-01

Cervical cancer causes 275,000 deaths each year with 85 % of these deaths occurring in the developing world. One of the primary reasons for the concentration of deaths in developing countries is a lack of effective screening methods suited for the infrastructure of these countries. In order to address this need, we have developed a high-resolution microendoscope (HRME). The HRME is a fiber-based fluorescence microscope with subcellular resolution. Using the vital stain proflavine, we are able to image cell nuclei in vivo and evaluate metrics such as nuclear-to-cytoplasmic ratio, critical to identifying precancerous epithelial regions. In this chapter, we detail the materials and methods necessary to build this system from commercially available parts.

The role of the AT pairs in the acid denaturation of DNA.

PubMed Central

Hermann, P; Fredericq, E

1977-01-01

It has been determined previously that the protonation of the GC pairs induces a DNA conformation change which leads to a "metastable" structure. The role of the AT pairs, however, is no well known because the protonation does not modify their spectral properties. By means of an indirect method based on the binding of proflavine, it has been determined that the AT pairs are protonated before the acid-induced denaturation and that they seem to be unable to assume a conformation change when protonated. These results would indicate that the protonated AT pairs may be responsible for the induction of the acid denaturation and not the GC pairs as it was thought previously. PMID:20604

On the origin of the decrease in stability of the DNA hairpin d(GCGAAGC) on complexation with aromatic drugs.

PubMed

Kostjukov, V V; Lantushenko, A O; Davies, D B; Evstigneev, M P

2007-08-01

Molecular dynamics simulations of drug-DNA complexes have been carried out in order to explain the experimentally observed decrease in thermal stability of the DNA hairpin d(GCGAAGC) on binding the aromatic drug molecules, daunomycin, ethidium bromide, novantrone and proflavine. This complexation behavior is in contrast to the stabilizing effect of the same aromatic drug molecules on DNA duplexes. Analysis of the energy parameters and the hydration properties of the complexes shows that the main factor correlating with the decrease in melting temperatures of the drug-hairpin complexes is the number of water bridges, with a reduction of at least 40% on ligand binding.

A single-sweep, nanosecond time resolution laser temperature-jump apparatus

NASA Astrophysics Data System (ADS)

Ballew, R. M.; Sabelko, J.; Reiner, C.; Gruebele, M.

1996-10-01

We describe a fast temperature-jump (T-jump) apparatus capable of acquiring kinetic relaxation transients via real-time fluorescence detection over a time interval from nanoseconds to milliseconds in a single sweep. The method is suitable for aqueous solutions, relying upon the direct absorption of laser light by the bulk water. This obviates the need for additives (serving as optical or conductive heaters) that may interact with the sample under investigation. The longitudinal temperature profile is made uniform by counterpropagating heating pulses. Dead time is limited to one period of the probe laser (16 ns). The apparatus response is tested with aqueous tryptophan and the diffusion-controlled dimerization of proflavine.

Role of glucocorticoids in increased muscle glutamine production in starvation

NASA Technical Reports Server (NTRS)

Tischler, Marc E.; Henriksen, Erik J.; Cook, Paul H.

1988-01-01

The role of glucocorticoids in the synthesis of muscle glutamine during starvation was investigated in adrenalectomized fasted rats injected with cortisol (1 mg/100 g body weight). It was found that administration of cortisol in vivo increased (compared to nontreated starved adrenalectomized controls) the glutamine/glutamate ratio and the activity of glutamine synthetase in the diaphragm and the extensor digitorum muscles, and that these effects were abolished by prior treatment with actinomycin D or proflavine. The results obtained in in vitro experiments, using fresh-frozen soleus, extensor digitorum longus, and diaphragm muscle preparations, supported the in vivo indications of the cortisol-enhanced glutamine synthesis and protein turnover in starved adrenalectomized animals.

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine.

PubMed

Shafirovich, V; Dourandin, A; Luneva, N P; Singh, C; Kirigin, F; Geacintov, N E

1999-03-01

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.

Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

PubMed

Hassan, Karl A; Liu, Qi; Henderson, Peter J F; Paulsen, Ian T

2015-02-10

Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug efflux systems which are very common in Proteobacteria: the proteobacterial antimicrobial compound efflux (PACE) family. This is the first new family of multidrug efflux pumps to be described in 15 years. Copyright © 2015 Hassan et al.

Photodynamic inactivation of antigenic determinants of single-stranded DNA bacteriophage phiX174

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khan, N.C.; Poddar, R.K.

1974-05-01

Bacteriophage phi X174 when photodynamically inactivated (i.e., when rendered unable to produce plaques as a result of exposure to visible light in air in the presence of proflavine) progressively lost their capacity to bind efficiently with homologous antiserum. Such loss of serum-blocking power was evident with heat-inactivated but not with uv-irradiated phage. The ability of the phages to adsorb to host cells, however, remained practically unaltered even after photodynamic inactivation. It thus appears that photodynamic damages in the so-called ''jacket'' component of the phi X174 coat proteins are partly responsible for the loss of plaque-forming ability, whereas the ''spikes'' aremore » either poor antigens or insensitive to photodynamic treatment. (auth)« less

1H NMR study of the complexation of aromatic drugs with dimethylxanthine derivatives

NASA Astrophysics Data System (ADS)

Hernandez Santiago, A. A.; Gonzalez Flores, M.; Rosas Castilla, S. A.; Cervantes Tavera, A. M.; Gutierrez Perez, R.; Khomich, V. V.; Ovchinnikov, D. V.; Parkes, H. G.; Evstigneev, M. P.

2012-02-01

With an aim of searching efficient interceptors of aromatic drugs, the self- and hetero-association of dimethylxanthine derivatives with different structures, selected according to Strategy 1 (variation of the position of methyl groups) and Strategy 2 (variation of the length of sbnd (CH2)nsbnd COOH group), with aromatic drug molecules: Ethidium Bromide, Proflavine and Daunomycin, were studied using 1H NMR spectroscopy. It was found that the association proceeds in a form of stacking-type complexation and its energetics is relatively independent on the structure of the dimethylxanthines. However, on average, the dimethylxanthines possess higher hetero-association constant and, hence, higher interceptor ability as compared to the trimethylxanthine, Caffeine, used during the past two decades as a typical interceptor molecule.

Preliminary morphological and X-ray diffraction studies of the crystals of the DNA cetyltrimethylammonium salt.

PubMed

Osica, V D; Pyatigorskaya, T L; Polyvtsev, O F; Dembo, A T; Kliya, M O; Vasilchenko, V N; Verkin, B I; Sukharevskya, B Y

1977-04-01

Double-stranded DNA molecules (molecular weight 2.5 X 10(5) - 5 X 10(5) daltons) have been crystallized from water-salt solutions as cetyltrimethylammonium salts (CTA-DNA). Variation of crystallization conditions results in a production of different types of CTA-DNA crystals: spherulits, dendrites, needle-shaped and faceted rhombic crystals, the latter beeing up to 0.3 mm on a side. X-ray diffraction data indicate that DNA molecules in the crystals form a hexagonal lattice which parameters vary slightly with the morphological type of the crystal. Comparison of the melting curves of the DNA preparation before and after crystallization suggests that DNA molecules are partially fractionated in the course of crystallization. Crystals of the CTA-DNA-proflavine complex have also been obtained.

Preliminary morphological and X-ray diffraction studies of the crystals of the DNA cetyltrimethylammonium salt.

PubMed Central

Osica, V D; Pyatigorskaya, T L; Polyvtsev, O F; Dembo, A T; Kliya, M O; Vasilchenko, V N; Verkin, B I; Sukharevskya, B Y

1977-01-01

Double-stranded DNA molecules (molecular weight 2.5 X 10(5) - 5 X 10(5) daltons) have been crystallized from water-salt solutions as cetyltrimethylammonium salts (CTA-DNA). Variation of crystallization conditions results in a production of different types of CTA-DNA crystals: spherulits, dendrites, needle-shaped and faceted rhombic crystals, the latter beeing up to 0.3 mm on a side. X-ray diffraction data indicate that DNA molecules in the crystals form a hexagonal lattice which parameters vary slightly with the morphological type of the crystal. Comparison of the melting curves of the DNA preparation before and after crystallization suggests that DNA molecules are partially fractionated in the course of crystallization. Crystals of the CTA-DNA-proflavine complex have also been obtained. Images PMID:866188

[Comparative toxicity of photosensitizers in varying destruction].

PubMed

Sinitsina, O O; Zholdakova, Z I; Poliakova, E E; Golovach, E N; Sycheva, L P; Beliaeva, N N; Kuznetsova, N A

2007-01-01

The toxicity of the photosensitizers proflavine acetate (PA) versus methylene blue (MB) was evaluated during their varying destruction. Under the influence of visible light, a partial (25%) transformation of the photosensitizers was shown to be attended by their enhanced toxicity and 100% destruction of the parent substances caused a reduction in their hazard. PA and its phototransformation products mainly affect the antiperoxide protection system and the structural and functional states of the liver, kidney, and duodenum. The maximum noneffective dose is 0.002 mg/kg. The possibility of using PA for water disinfection depends on the ratio of safe and effective concentrations. A partial (25%) MB destruction products cause mutagenic effects; the permissible dose of the mutagen is 0.00025 mg/kg. MB is not recommended for disinfection of all types of waters.

Intermolecular hydrogen bonds in hetero-complexes of biologically active aromatic molecules probed by the methods of vibrational spectroscopy

NASA Astrophysics Data System (ADS)

Semenov, M. A.; Blyzniuk, Iu. N.; Bolbukh, T. V.; Shestopalova, A. V.; Evstigneev, M. P.; Maleev, V. Ya.

2012-09-01

By the methods of vibrational spectroscopy (Infrared and Raman) the investigation of the hetero-association of biologically active aromatic compounds: flavin-mononucleotide (FMN), ethidium bromide (EB) and proflavine (PRF) was performed in aqueous solutions. It was shown that between the functional groups (Cdbnd O and NH2) the intermolecular hydrogen bonds are formed in the hetero-complexes FMN-EB and FMN-PRF, additionally stabilizing these structures. An estimation of the enthalpy of Н-bonding obtained from experimental shifts of carbonyl vibrational frequencies has shown that the H-bonds do not dominate in the magnitude of experimentally measured total enthalpy of the hetero-association reactions. The main stabilization is likely due to intermolecular interactions of the molecules in these complexes and their interaction with water environment.

Intermolecular hydrogen bonds in hetero-complexes of biologically active aromatic molecules probed by the methods of vibrational spectroscopy.

PubMed

Semenov, M A; Blyzniuk, Iu N; Bolbukh, T V; Shestopalova, A V; Evstigneev, M P; Maleev, V Ya

2012-09-01

By the methods of vibrational spectroscopy (Infrared and Raman) the investigation of the hetero-association of biologically active aromatic compounds: flavin-mononucleotide (FMN), ethidium bromide (EB) and proflavine (PRF) was performed in aqueous solutions. It was shown that between the functional groups (CO and NH(2)) the intermolecular hydrogen bonds are formed in the hetero-complexes FMN-EB and FMN-PRF, additionally stabilizing these structures. An estimation of the enthalpy of Н-bonding obtained from experimental shifts of carbonyl vibrational frequencies has shown that the H-bonds do not dominate in the magnitude of experimentally measured total enthalpy of the hetero-association reactions. The main stabilization is likely due to intermolecular interactions of the molecules in these complexes and their interaction with water environment. Copyright © 2012 Elsevier B.V. All rights reserved.

[Energetics of complex formation of the DNA hairpin structure d(GCGAAGC) with aromatic ligands].

PubMed

Kostiukov, V V

2011-01-01

The energy contributions of various physical interactions to the total Gibbs energy of complex formation of the biologically important DNA hairpin d(GCGAAGC) with aromatic antitumor antibiotics daunomycin and novantron and the mutagens ethidium and proflavine have been calculated. It has been shown that the relatively small value of the total energy of binding of the ligands to the hairpin is the sum of components great in absolute value and different in sign. The contributions of van der Waals interactions and both intra- and intermolecular hydrogen bonds and bonds with aqueous environment have been studied. According to the calculations, the hydrophobic and van der Waals components are energetically favorable in complex formation of the ligands with the DNA pairpin d(GCGAAGC), whereas the electrostatic (with consideration of hydrogen bonds) and entropic components are unfavorable.

Excision of thymine dimers in vitro by extracts of bacteriophage-infected Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedberg, E.C.; Minton, K.; Pawl, G.

1974-05-01

Extracts of DNA polymerase I defective Escherichia coli infected with phage T4 contain an exonuclease activity that removes thymine dimers from uv- irradiated DNA previously nicked with T4 uv endonuclease. This activity is not expressed if cells are infected in the presence of chloramphenicol. The enzyme has a requirement for divalent cation and is not affected by caffeine, but excision is inhibited in the presence of proflavine. The enzyme is present in all phage T4 mutants thus far examined, including 25 uv-sensitive mutants isolated during the course of the experiments, all of which are defective in the v gene. Amore » similar activity can be detected in cells infected with phages T2, T3, and T6, but not in cells infected with phage T7. (auth)« less

Triple dye plus rubbing alcohol versus triple dye alone for umbilical cord care.

PubMed

Suliman, Alawia K; Watts, Heidi; Beiler, Jessica; King, Tonya S; Khan, Sana; Carnuccio, Marybeth; Paul, Ian M

2010-01-01

Current practices for umbilical cord care vary across centers, but the evidence regarding these practices and their impact on cord separation, complications, and health care use are limited. The objective of this study was to compare the effect of triple dye alone (brilliant green, crystal violet, and proflavine hemisulfate) versus triple dye plus rubbing alcohol (isopropyl alcohol) twice daily on time to umbilical cord separation, complications, and health care use. For the 90 newborns who completed the study, there were no significant differences between treatment groups for time to cord separation, cord-related morbidities, or cord-related urgent care. Based on these study results, there does not appear to be significant benefit to the addition of twice daily applications of rubbing alcohol to neonatal umbilical cords following triple dye treatment after birth.

Cytotoxic activity of proflavine diureas: synthesis, antitumor, evaluation and DNA binding properties of 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas.

PubMed

Kozurková, Mária; Sabolová, Danica; Janovec, Ladislav; Mikes, Jaromír; Koval', Ján; Ungvarský, Ján; Stefanisinová, Miroslava; Fedorocko, Peter; Kristian, Pavol; Imrich, Ján

2008-04-01

The synthesis of novel 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas was reported. Their biological activity to inhibit cell proliferation was assessed by a MTT assay on two cell lines, HeLa and HCT-116, at micromolar concentration. 1',1''-(Acridin-3,6-diyl)-3',3''-dihexyldiurea hydrochloride was active on a HCT-116 cell line with an IC(50) value of 3.1 microM. The interaction of these compounds with calf thymus DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence and CD spectroscopy. From spectrofluorimetric titrations, binding constants for the DNA-drug complexes were determined (K=0.9-4.2x10(5) M(-1)). Antiproliferative activity of synthesized derivatives might be related to their intercalation into DNA.

Design and evaluation of an ultra-slim objective for in-vivo deep optical biopsy

PubMed Central

Landau, Sara M.; Liang, Chen; Kester, Robert T.; Tkaczyk, Tomasz S.; Descour, Michael R.

2010-01-01

An estimated 1.6 million breast biopsies are performed in the US each year. In order to provide real-time, in-vivo imaging with sub-cellular resolution for optical biopsies, we have designed an ultra-slim objective to fit inside the 1-mm-diameter hypodermic needles currently used for breast biopsies to image tissue stained by the fluorescent probe proflavine. To ensure high-quality imaging performance, experimental tests were performed to characterize fiber bundle’s light-coupling efficiency and simulations were performed to evaluate the impact of candidate lens materials’ autofluorescence. A prototype of NA = 0.4, 250-µm field of view, ultra-slim objective optics was built and tested, yielding diffraction-limited performance and estimated resolution of 0.9 µm. When used in conjunction with a commercial coherent fiber bundle to relay the image formed by the objective, the measured resolution was 2.5 µm. PMID:20389489

Portable fluorescence microendoscope system for smartphones and its applications

NASA Astrophysics Data System (ADS)

Gómez García, Pablo Aurelio; Teixeira Rosa, Ramon Gabriel; Pratavieira, Sebastião.; Kurachi, Cristina

2015-06-01

A portable microscope/microendoscope will be presented in this article. The system was specially designed for Smartphones and taking into account its simplicity, will be able to bring this technology to almost every doctor's office. It is worth mentioning its flexibility of use, that allows several modes since all the components are interchangeable (the illumination LED, the lens, the optic filters, etc) resulting in different applications, from medical applications until other areas (for example, the inspection of non-accessible pieces of plane engines). In addition, the system has a double platform, working as a conventional microscope or as a fiberoptic microendoscope. In situ and cell smear interrogation of oral mucosa, using a proflavine as dye will be presented. The price of the system does not exceed US 350, plus the price of the fiber bundle (around US 500) turning it onto a high resolution affordable system.

Rapid CE-UV binding tests of environmentally hazardous compounds with polymer-modified magnetic nanoparticles.

PubMed

Iqbal, Zafar; Alsudir, Samar; Miah, Musharraf; Lai, Edward P C

2011-08-01

Hazardous compounds and bacteria in water have an adverse impact on human health and environmental ecology. Polydopamine (or polypyrrole)-coated magnetic nanoparticles and polymethacrylic acid-co-ethylene glycol dimethacrylate submicron particles were investigated for their fast binding kinetics with bisphenol A, proflavine, naphthalene acetic acid, and Escherichia coli. A new method was developed for the rapid determination of % binding by sequential injection of particles first and compounds (or E. coli) next into a fused-silica capillary for overlap binding during electrophoretic migration. Only nanolitre volumes of compounds and particles were sufficient to complete a rapid binding test. After heterogeneous binding, separation of the compounds from the particles was afforded by capillary electrophoresis. % binding was influenced by applied voltage but not current flow. In-capillary coating of particles affected the % binding of compounds. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Subcellular localization of proflavine derivative and induction of oxidative stress--in vitro studies.

PubMed

Ipóthová, Z; Paulíková, H; Cižeková, L; Hunáková, L; Labudová, M; Grolmusová, A; Janovec, L; Imrich, J

2013-11-01

Acridines have been studied for several decades because of their numerous biological effects, especially anticancer activity. Recently, cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was confirmed for leukemic cell lines [Bioorg. Med. Chem.2011, 19, 1790]. The mechanism of action of the most cytotoxic hexyl-AcrDIM was studied in this paper focusing attention on a subcellular distribution of the drug. Accumulation of hexyl-AcrDIM in mitochondria was confirmed after labeling mitochondria with MitoRED using ImageStream Imaging Flow Cytometer. The derivative significantly decreased intracellular ATP level (reduction of ATP level was decreased by vitamin E), and induced oxidative stress (ROS production detected by DHE assay) as well as cell cycle arrest in the S-phase (flow cytometry analysis) already after short-time incubation and induction of apoptosis. Cytotoxicity of hexyl-AcrDIM is closely connected with induction of oxidative stress in cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dynamic perturbation effects upon the circular dichroism intensity induced in an aggregate of dye chromophores bound to biopolymers

NASA Astrophysics Data System (ADS)

Kamiya, Mamoru

1980-11-01

The dynamic perturbation effects of polarizable monomer perturbers upon the circular dichroism intensity arising from absorption transitions of an arbitrary aggregate of dye chromophores bound to a large host polymer are formulated using the linear response theory in the decorrelation approximation, where the interchromophoric retardation phase factors are eliminated by a first-order Taylor expansion which is compatible with the use of the retarded helix selection rules in the long-wavelength approximation. A space-averaged and closed-form formulation of the non-conservative circular dichroism intensity which is perturbed by intensity with the outside perturber transitions is derived in the limit of the weak dynamic perturbation where perturber—perturber interactions are negligible. The relevant formulation is applied in order to investigate the intercalation model dependence of the non-conservative circular dichroism intensity induced at the visible absorption band of proflavine molecules intercalated in either poly(A—T) or poly(G—C).

A general nuclear magnetic resonance analysis of hetero-association of aromatic molecules in aqueous solution

NASA Astrophysics Data System (ADS)

Veselkov, Alexei N.; Evstigneev, Maxim P.; Veselkov, Dennis A.; Davies, David B.

2001-08-01

A general nuclear magnetic resonance analysis of a statistical-thermodynamical model of hetero-association of aromatic molecules in solution has been developed to take "edge effects" into consideration, i.e., the dependence of proton chemical shifts on the position of the molecule situated inside or at the edge of the aggregate. This generalized approach is compared with a previously published model, where an average contribution to proton shielding is considered irrespective of the position of the molecule in the stack. Association parameters have been determined from experimental concentration and temperature dependences of 500 MHz proton chemical shifts of the hetero-association of the acridine dye, proflavine, and the phenanthridinium dye, ethidium bromide, in aqueous solution. Differences in the parameters in the range 10%-30% calculated using the basic and generalized approaches have been found to depend substantially on the magnitude of the equilibrium hetero-association constant Khet—the larger the value of Khet, the higher the discrepancy between the two methods.

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye‐Sensitized TiO2

PubMed Central

Ainsworth, Emma V.; Lockwood, Colin W. J.; White, Gaye F.; Hwang, Ee Taek; Sakai, Tsubasa; Gross, Manuela A.; Richardson, David J.; Clarke, Thomas A.

2016-01-01

Abstract The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar‐assisted chemical synthesis. In Shewanella oneidensis MR‐1 (MR‐1), trans‐outer‐membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer‐membrane‐spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR‐1. We show photoreduction of MtrC and OmcA adsorbed on RuII‐dye‐sensitized TiO2 nanoparticles and that these protein‐coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer‐membrane‐associated cytochromes of MR‐1 for solar‐driven microbial synthesis in natural and engineered bacteria. PMID:27685371

Wide-field and high-resolution optical imaging for early detection of oral neoplasia

NASA Astrophysics Data System (ADS)

Pierce, Mark C.; Schwarz, Richard A.; Rosbach, Kelsey; Roblyer, Darren; Muldoon, Tim; Williams, Michelle D.; El-Naggar, Adel K.; Gillenwater, Ann M.; Richards-Kortum, Rebecca

2010-02-01

Current procedures for oral cancer screening typically involve visual inspection of the entire tissue surface at risk under white light illumination. However, pre-cancerous lesions can be difficult to distinguish from many benign conditions when viewed under these conditions. We have developed wide-field (macroscopic) imaging system which additionally images in cross-polarized white light, narrowband reflectance, and fluorescence imaging modes to reduce specular glare, enhance vascular contrast, and detect disease-related alterations in tissue autofluorescence. We have also developed a portable system to enable high-resolution (microscopic) evaluation of cellular features within the oral mucosa in situ. This system is a wide-field epi-fluorescence microscope coupled to a 1 mm diameter, flexible fiber-optic imaging bundle. Proflavine solution was used to specifically label cell nuclei, enabling the characteristic differences in N/C ratio and nuclear distribution between normal, dysplastic, and cancerous oral mucosa to be quantified. This paper discusses the technical design and performance characteristics of these complementary imaging systems. We will also present data from ongoing clinical studies aimed at evaluating diagnostic performance of these systems for detection of oral neoplasia.

Ozonation of mutagenic and carcinogenic polyaromatic amines and polyaromatic hydrocarbons in water

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burleson, G.R.; Caulfield, M.J.; Pollard, M.

1979-06-01

The Salmonella-microsome assay for mutagenesis was used to determine the effect of ozone on the mutagenesis of selected carcinogens and mutagens in water. Short periods of ozonation were shown to completely inactivate the mutagenicity of several polyaromatic amine mutagens including acriflavine, proflavine, and beta-naphthylamine. Selected polyaromatic hydrocarbons were also sensitive to ozonation. Kinetic studies revealed that the mutagenicity of benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz(a)anthracene was destroyed after short periods of ozonation. To correlate loss of mutagenicity with loss of carcinogenicity, two polyaromatic hydrocarbons were treated with ozone, extracted from water with hexane, and tested for carcinogenicity in mice. When 7,12-dimethyl-benz(a)anthracene andmore » 3-methyl-cholanthrene were treated with ozone, there was a substantial reduction in carcinogenicity compared to control groups treated with oxygen alone. However, a small number of tumors developed in the group of animals receiving a hexane extract of ozonated 7,12-dimethylbenz(a)anthracene. This activity may be due to breakdown products of 7,12-dimethylbenz(a)anthracene that are not mutagenic.« less

Photo-inducible cytotoxic and clastogenic activities of 3,6-di-substituted acridines obtained by acylation of proflavine.

PubMed

Benchabane, Yohann; Di Giorgio, Carole; Boyer, Gérard; Sabatier, Anne-Sophie; Allegro, Diane; Peyrot, Vincent; De Méo, Michel

2009-06-01

The cytotoxicity and photo-enhanced cytotoxicity of a series of 18 3,6-di-substituted acridines were evaluated on both tumour CHO cells and human normal keratinocytes, and compared to their corresponding clastogenicity as assessed by the micronucleus assay. Compounds 2f tert-butyl N-[(6-tert-butoxycarbonylamino)acridin-3-yl]carbamate and 2d N-[6-(pivalamino)acridin-3-yl]pivalamide displayed a specific cytotoxicity on CHO cells. These results suggested that the two derivatives could be considered as interesting candidates for anticancer chemotherapy and hypothesized that the presence of 1,1-dimethylethyl substituents was responsible for a strong nonclastogenic cytotoxicity. Compounds 2b and 2c, on the contrary, displayed a strong clastogenicity. They indicated that the presence of nonbranched aliphatic chains on positions 3 and 6 of the acridine rings tended to induce a significant clastogenic effect. Finally, they established that most of the acridine compounds could be photo-activated by UVA-visible rays and focussed on the significant role of light irradiation on their biological properties.

Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function. [UV radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minor, P.D.; Dimmock, N.J.

1977-05-15

Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and ..cap alpha..-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at highmore » concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent.« less

Solution structure and thermodynamics of 2',5' RNA intercalation.

PubMed

Horowitz, Eric D; Lilavivat, Seth; Holladay, Benjamin W; Germann, Markus W; Hud, Nicholas V

2009-04-29

As a means to explore the influence of the nucleic acid backbone on the intercalative binding of ligands to DNA and RNA, we have determined the solution structure of a proflavine-bound 2',5'-linked octamer duplex with the sequence GCCGCGGC. This structure represents the first NMR structure of an intercalated RNA duplex, of either backbone structural isomer. By comparison with X-ray crystal structures, we have identified similarities and differences between intercalated 3',5' and 2',5'-linked RNA duplexes. First, the two forms of RNA have different sugar pucker geometries at the intercalated nucleotide steps, yet have the same interphosphate distances. Second, as in intercalated 3',5' RNA, the phosphate backbone angle zeta at the 2',5' RNA intercalation site prefers to be in the trans conformation, whereas unintercalated 2',5' and 3',5' RNA prefer the -gauche conformation. These observations provide new insights regarding the transitions required for intercalation of a phosphodiester-ribose backbone and suggest a possible contribution of the backbone to the origin of the nearest-neighbor exclusion principle. Thermodynamic studies presented for intercalation of both structural RNA isomers also reveal a surprising sensitivity of intercalator binding enthalpy and entropy to the details of RNA backbone structure.

Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay: A Biophysical Chemistry Laboratory Experiment

NASA Astrophysics Data System (ADS)

Shafirovich, Vladimir; Singh, Carolyn; Geacintov, Nicholas E.

2003-11-01

Oxidative damage of DNA molecules associated with electron-transfer reactions is an important phenomenon in living cells, which can lead to mutations and contribute to carcinogenesis and the aging processes. This article describes the design of several simple experiments to explore DNA damage initiated by photoinduced electron-transfer reactions sensitized by the acridine derivative, proflavine (PF). A supercoiled DNA agarose gel nicking assay is employed as a sensitive probe of DNA strand cleavage. A low-cost experimental and computer-interfaced imaging apparatus is described allowing for the digital recording and analysis of the gel electrophoresis results. The first experiment describes the formation of direct strand breaks in double-stranded DNA induced by photoexcitation of the intercalated PF molecules. The second experiment demonstrates that the addition of the well-known electron acceptor, methylviologen, gives rise to a significant enhancement of the photochemical DNA strand cleavage effect. This occurs by an electron transfer step to methylviologen that renders the inital photoinduced charge separation between photoexcited PF and DNA irreversible. The third experiment demonstrates that the action spectrum of the DNA photocleavage matches the absorption spectrum of DNA-bound, intercalated PF molecules, which differs from that of free PF molecules. This result demonstrates that the photoinduced DNA strand cleavage is initiated by intercalated rather than free PF molecules.

Hydration of nucleic acid fragments: comparison of theory and experiment for high-resolution crystal structures of RNA, DNA, and DNA-drug complexes.

PubMed Central

Hummer, G; García, A E; Soumpasis, D M

1995-01-01

A computationally efficient method to describe the organization of water around solvated biomolecules is presented. It is based on a statistical mechanical expression for the water-density distribution in terms of particle correlation functions. The method is applied to analyze the hydration of small nucleic acid molecules in the crystal environment, for which high-resolution x-ray crystal structures have been reported. Results for RNA [r(ApU).r(ApU)] and DNA [d(CpG).d(CpG) in Z form and with parallel strand orientation] and for DNA-drug complexes [d(CpG).d(CpG) with the drug proflavine intercalated] are described. A detailed comparison of theoretical and experimental data shows positional agreement for the experimentally observed water sites. The presented method can be used for refinement of the water structure in x-ray crystallography, hydration analysis of nuclear magnetic resonance structures, and theoretical modeling of biological macromolecules such as molecular docking studies. The speed of the computations allows hydration analyses of molecules of almost arbitrary size (tRNA, protein-nucleic acid complexes, etc.) in the crystal environment and in aqueous solution. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 6 FIGURE 9 FIGURE 12 FIGURE 13 PMID:7542034

Quantitative treatment of the solvent effects on the electronic absorption and fluorescence spectra of acridines and phenazines. The ground and first excited singlet-state dipole moments

NASA Astrophysics Data System (ADS)

Aaron, Jean Jacques; Maafi, Mounir; Párkányi, Cyril; Boniface, Christian

1995-04-01

Electronic absorption and fluorescence excitation and emission spectra of four acridines (acridine, Acridine Yellow, 9-aminoacridine and proflavine) and three phenazines (phenazine, neutral Red and safranine) are determined at room temperature (298 K) in several solvents of various polarities (dioxane, chloroform, ethyl ether, ethyl acetate, 1-butanol, 2-propanol, ethanol, methanol, dimethylformamide, acetonitrile and dimethyl sulfoxide). The effect of the solvent upon the spectral characteristics of the above compounds, is studied. In combination with the ground-state dipole moments of these compounds, the spectral data are used to evaluate their first excited singlet-state dipole moments by means of the solvatochromic shift method (Bakhshiev's and Kawski-Chamma-Viallet's correlations). The theoretical ground and excited singlet-state dipole moments for acridines and phenazines are also calculated as a vector sum of the π-component (obtained by the PPP method) and the σ-component (obtained from σ-bond moments). For most acridines and phenazines under study, the experimental excited singlet-state dipole moments are found to be higher than their ground state counterpart. The application of the Kamlet-Abboud-Taft solvatochromic parameters to the solvent effect on spectral properties of acridine and phenazine derivatives is discussed.

Dye-induced aggregation of single stranded RNA: a mechanistic approach.

PubMed

Biver, Tarita; Ciatto, Carlo; Secco, Fernando; Venturini, Marcella

2006-08-15

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 degrees C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PD(I) and an internal complex PD(II) form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (C(P)/C(D)). For C(P)/C(D)<2.5, cooperative formation of stacks external to polymer strands prevails (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g=1), equilibrium constant for the nucleation step (K( *)=(1.4+/-0.6)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(7)M(-1)s(-1), k(d)=1.1 x 10(2)s(-1)). Stopped-flow experiments, performed at low ionic strength (I=0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that C(P)/C(D)<2.5.

Allosteric Inhibitory Molecular Recognition of a Photochromic Dye by a Digestive Enzyme: Dihydroindolizine makes α-chymotrypsin Photo-responsive

NASA Astrophysics Data System (ADS)

Bagchi, Damayanti; Ghosh, Abhijit; Singh, Priya; Dutta, Shreyasi; Polley, Nabarun; Althagafi, Ismail. I.; Jassas, Rabab S.; Ahmed, Saleh A.; Pal, Samir Kumar

2016-09-01

The structural-functional regulation of enzymes by the administration of an external stimulus such as light could create photo-switches that exhibit unique biotechnological applications. However, molecular recognition of small ligands is a central phenomenon involved in all biological processes. We demonstrate herein that the molecular recognition of a photochromic ligand, dihydroindolizine (DHI), by serine protease α-chymotrypsin (CHT) leads to the photo-control of enzymatic activity. We synthesized and optically characterized the photochromic DHI. Light-induced reversible pyrroline ring opening and a consequent thermal back reaction via 1,5-electrocyclization are responsible for the photochromic behavior. Furthermore, DHI inhibits the enzymatic activity of CHT in a photo-controlled manner. Simultaneous binding of the well-known inhibitors 4-nitrophenyl anthranilate (NPA) or proflavin (PF) in the presence of DHI displays spectral overlap between the emission of CHT-NPA or CHT-PF with the respective absorption of cis or trans DHI. The results suggest an opportunity to explore the binding site of DHI using Förster resonance energy transfer (FRET). Moreover, to more specifically evaluate the DHI binding interactions, we employed molecular docking calculations, which suggested binding near the hydrophobic site of Cys-1-Cys-122 residues. Variations in the electrostatic interactions of the two conformers of DHI adopt unfavorable conformations, leading to the allosteric inhibition of enzymatic activity.

Confocal fluorescence microscopy to evaluate changes in adipocytes in the tumor microenvironment associated with invasive ductal carcinoma and ductal carcinoma in situ.

PubMed

Dobbs, Jessica L; Shin, Dongsuk; Krishnamurthy, Savitri; Kuerer, Henry; Yang, Wei; Richards-Kortum, Rebecca

2016-09-01

Adipose tissue is a dynamic organ that provides endocrine, inflammatory and angiogenic factors, which can assist breast carcinoma cells with invasion and metastasis. Previous studies have shown that adipocytes adjacent to carcinoma, known as cancer-associated adipocytes, undergo extensive changes that correspond to an "activated phenotype," such as reduced size relative to adipocytes in non-neoplastic breast tissue. Optical imaging provides a tool that can be used to characterize adipocyte morphology and other features of the tumor microenvironment. In this study, we used confocal fluorescence microscopy to acquire images of freshly excised breast tissue stained topically with proflavine. We developed a computerized algorithm to identify and quantitatively measure phenotypic properties of adipocytes located adjacent to and far from normal collagen, ductal carcinoma in situ and invasive ductal carcinoma. Adipocytes were measured in confocal fluorescence images of fresh breast tissue collected from 22 patients. Results show that adipocytes adjacent to neoplastic tissue margins have significantly smaller area compared to adipocytes far from the margins of neoplastic lesions and compared to adipocytes adjacent to non-neoplastic collagenous stroma. These findings suggest that confocal microscopic images can be utilized to evaluate phenotypic properties of adipocytes in breast stroma which may be useful in defining alterations in microenvironment that may aid in the development and progression of neoplastic lesions. © 2016 UICC.

Confocal fluorescence microscopy for rapid evaluation of invasive tumor cellularity of inflammatory breast carcinoma core needle biopsies.

PubMed

Dobbs, Jessica; Krishnamurthy, Savitri; Kyrish, Matthew; Benveniste, Ana Paula; Yang, Wei; Richards-Kortum, Rebecca

2015-01-01

Tissue sampling is a problematic issue for inflammatory breast carcinoma, and immediate evaluation following core needle biopsy is needed to evaluate specimen adequacy. We sought to determine if confocal fluorescence microscopy provides sufficient resolution to evaluate specimen adequacy by comparing invasive tumor cellularity estimated from standard histologic images to invasive tumor cellularity estimated from confocal images of breast core needle biopsy specimens. Grayscale confocal fluorescence images of breast core needle biopsy specimens were acquired following proflavine application. A breast-dedicated pathologist evaluated invasive tumor cellularity in histologic images with hematoxylin and eosin staining and in grayscale and false-colored confocal images of cores. Agreement between cellularity estimates was quantified using a kappa coefficient. 23 cores from 23 patients with suspected inflammatory breast carcinoma were imaged. Confocal images were acquired in an average of less than 2 min per core. Invasive tumor cellularity estimated from histologic and grayscale confocal images showed moderate agreement by kappa coefficient: κ = 0.48 ± 0.09 (p < 0.001). Grayscale confocal images require less than 2 min for acquisition and allow for evaluation of invasive tumor cellularity in breast core needle biopsy specimens with moderate agreement to histologic images. We show that confocal fluorescence microscopy can be performed immediately following specimen acquisition and could indicate the need for additional biopsies at the initial visit.

High-resolution microendoscope images of middle ear cholesteatoma and surrounding tissue: evaluation of interobserver concordance.

PubMed

Bradley, James; Jiang, Nancy; Levy, Lauren; Richards-Kortum, Rebecca; Sikora, Andrew; Smouha, Eric

2014-04-01

Investigate how accurately otolaryngologists could differentiate between images obtained with high-resolution microendoscopy (HRME) of ex vivo cholesteatoma specimens and surrounding middle ear epithelium. HRME images of surgically resected cholesteatoma and middle ear epithelium were obtained and otolaryngologists classified these images. Tertiary medical center. Resected cholesteatoma and middle ear epithelium were stained with a contrast agent, proflavine, and HRME images were captured. Specimens were sent for standard histopathology and compared with HRME images. Quality-controlled images were used to assemble a training set. After viewing training images, otolaryngologists without prior cholesteatoma HRME experience reviewed and classified test images. Ten cholesteatoma and 9 middle ear specimens were collected, of which 17 representative cholesteatoma and 19 middle ear epithelium images were extracted for a testing set. Qualitative analysis for concordance between HRME images and histological images yielded a strong correlation between modalities. The mean accuracy of all reviewers in correctly identifying images was 95% (95% confidence interval [CI], 92%-98%). The sensitivity to correctly detect cholesteatoma images was 98% (95% CI, 93%-100%), and the specificity was 92% (95% CI, 87%-97%). The Fleiss kappa interrater reliability score was 0.83, (95% CI, 0.77-0.89). Medical professionals can quickly be trained to accurately distinguish between HRME images of cholesteatoma and normal middle ear epithelium, both of which have distinct imaging characteristics. Real-time HRME optical imaging can potentially improve the results of otologic surgery by allowing for extirpation of cholesteatomas while eliminating residual disease.

Progressing single biomolecule force spectroscopy measurements for the screening of DNA binding agents

NASA Astrophysics Data System (ADS)

Zhang, Wenke; Barbagallo, Romina; Madden, Claire; Roberts, Clive J.; Woolford, Alison; Allen, Stephanie

2005-10-01

Recent studies have indicated that the force-extension properties of single molecules of double stranded (ds) DNA are sensitive to the presence of small molecule DNA binding agents, and also to their mode of binding. These observations raise the possibility of using this approach as a highly sensitive tool for the screening of such agents. However, particularly for studies employing the atomic force microscope (AFM), several non-trivial barriers hinder the progress of this approach to the non-specialist arena and hence also the full realization of this possibility. In this paper, we therefore address a series of key reproducibility and metrological issues associated with this type of measurement. Specifically, we present an improved immobilization method that covalently anchors one end (5' end) of a dual labelled (5'-thiol, 3'-biotin) p53 DNA molecule onto a gold substrate via gold-thiol chemistry, whilst the biotinylated 3' end is available for 'pick-up' using a streptavidin modified AFM tip. We also show that co-surface immobilization of DNA with 6-mercapto-1-hexanol (MCH) can also lead to a further increase the measured contour length. We demonstrate the impact of these improved protocols through the observation of the cooperative transition plateau in a DNA fragment of approximately 118 bp, a significantly smaller fragment than previously investigated. The results of a comparative study of the effects of a model minor groove binder (Hoechst 33258) and an intercalating drug (proflavine), alone, as a mixture and under different buffer conditions, are also presented.

Time dependence of triplet-singlet excitation transfer from compact poly rA to bound dye at 77 K.

PubMed Central

Pearlstein, R M; Van Nostrand, F; Nairn, J A

1979-01-01

The nonexponential phosphorescence decay of a highly folded form of poly-riboadenylic acid (poly rA) with noncovalently bound dye is explained by a novel application of a well-known theory of electronic excitation transfer based on the Förster mechanism. This theory, originally used to describe singlet-singlet energy transfer from donor molecules to an acceptor in a solution, is here applied to the transfer of triplet excitation from the adenine (in poly rA) to the singlet manifold of either of the bound dyes, ethidium bromide or proflavine. New experimental data are presented that allow straight-forward theoretical interpretation. These data fit the form predicted by the theory, U(t) exp(-Bt1/2), where U(t) is the decay of the poly rA phosphorescence in the absence of dye, for a range of relative concentrations of either dye. The self-consistency of these theoretical fits is demonstrated by the proportionality of B to the square root of the Förster triplet-singlet overlap integrals for transfer from poly rA to each of the dyes, as demanded by the theory. From these self-consistent values of B, the theory enables one to deduce the mean packing density of nucleotides in this folded poly rA, which we estimate to be approximately 1 nm-3. We conclude that some variations of the method described here may be useful for deducing packing densities of nucleotides in other compact nucleic acid structures. PMID:262411

Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery

PubMed Central

Yildiz, Ibrahim; Lee, Karin L.; Chen, Kevin; Shukla, Sourabh; Steinmetz, Nicole F.

2013-01-01

This work is focused on the development of a plant virus-based carrier system for cargo delivery, specifically 30 nm-sized cowpea mosaic virus (CPMV). Whereas previous reports described the engineering of CPMV through genetic or chemical modification, we report a non-covalent infusion technique that facilitates efficient cargo loading. Infusion and retention of 130–155 fluorescent dye molecules per CPMV using DAPI (4’,6-diamidino-2-phenylindole dihydrochloride), propidium iodide (3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide), and acridine orange (3,6-bis(dimethylamino)acridinium chloride), as well as 140 copies of therapeutic payload proflavine (PF, acridine-3,6-diamine hydrochloride), is reported. Loading is achieved through interaction of the cargo with the CPMV’s encapsidated RNA molecules. The loading mechanism is specific; empty RNA-free eCPMV nanoparticles could not be loaded. Cargo-infused CPMV nanoparticles remain chemically active, and surface lysine residues were covalent modified with dyes leading to the development of dual-functional CPMV carrier systems. We demonstrate cargo-delivery to a panel of cancer cells (cervical, breast, and colon): CPMV nanoparticles enter cells via the surface marker vimentin, the nanoparticles target the endolysosome, where the carrier is degraded and the cargo released allowing imaging and/or cell killing. In conclusion, we demonstrate cargo-infusion and delivery to cells; the methods discussed provide a useful means for functionalization of CPMV toward its application as drug and/or contrast agent delivery vehicle. PMID:23665254

High-throughput microfluidic line scan imaging for cytological characterization

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Powless, Amy J.; Majid, Aneeka A.; Claycomb, Adair; Fritsch, Ingrid; Balachandran, Kartik; Muldoon, Timothy J.

2015-03-01

Imaging cells in a microfluidic chamber with an area scan camera is difficult due to motion blur and data loss during frame readout causing discontinuity of data acquisition as cells move at relatively high speeds through the chamber. We have developed a method to continuously acquire high-resolution images of cells in motion through a microfluidics chamber using a high-speed line scan camera. The sensor acquires images in a line-by-line fashion in order to continuously image moving objects without motion blur. The optical setup comprises an epi-illuminated microscope with a 40X oil immersion, 1.4 NA objective and a 150 mm tube lens focused on a microfluidic channel. Samples containing suspended cells fluorescently stained with 0.01% (w/v) proflavine in saline are introduced into the microfluidics chamber via a syringe pump; illumination is provided by a blue LED (455 nm). Images were taken of samples at the focal plane using an ELiiXA+ 8k/4k monochrome line-scan camera at a line rate of up to 40 kHz. The system's line rate and fluid velocity are tightly controlled to reduce image distortion and are validated using fluorescent microspheres. Image acquisition was controlled via MATLAB's Image Acquisition toolbox. Data sets comprise discrete images of every detectable cell which may be subsequently mined for morphological statistics and definable features by a custom texture analysis algorithm. This high-throughput screening method, comparable to cell counting by flow cytometry, provided efficient examination including counting, classification, and differentiation of saliva, blood, and cultured human cancer cells.

High-resolution microendoscope imaging of inverted papilloma and normal sinonasal mucosa: evaluation of interobserver concordance.

PubMed

Parasher, Arjun K; Kidwai, Sarah M; Schorn, Victor J; Goljo, Erden; Weinberg, Alan D; Richards-Kortum, Rebecca; Sikora, Andrew G; Iloreta, Alfred Marc; Govindaraj, Satish; Miles, Brett A

2015-12-01

High-resolution microendoscopy (HRME) enables real-time imaging of epithelial tissue. The utility of this novel imaging modality for inverted papilloma has not been previously described. This study examines the ability of otolaryngologists to differentiate between images of inverted papilloma and normal sinonasal mucosa obtained with a HRME. Inverted papilloma and normal sinonasal mucosa specimens were stained with a contrast agent, proflavine. HRME images were subsequently captured. Histopathological diagnosis was obtained for each sample. Quality-controlled images were used to assemble a training set. After reviewing the training images, 6 otolaryngologists without prior HRME experience reviewed and classified test images. Five samples of inverted papilloma and 2 normal sinonasal mucosa samples were collected. Four representative images from each specimen were used for the 28-image test set. The mean accuracy among all reviewers was 89.9% (95% confidence interval [CI], 84.3% to 94.0%). The sensitivity to correctly identify inverted papilloma was 86.7% (95% CI, 79.2% to 92.2%), and the specificity was 92.9% (95% CI, 89.0% to 100.0%). The Fleiss kappa interrater reliability score was 0.80 (95% CI, 0.70 to 0.89). Inverted papilloma and normal sinonasal mucosa have distinct HRME imaging characteristics. Otolaryngologists can be successfully trained to distinguish between inverted papilloma and normal sinonasal mucosa. HRME is a feasible tool for identification of inverted papilloma. By conducting future in vivo trials, HRME potentially may enable real-time surgical margin determination during surgical excision of inverted papilloma. © 2015 ARS-AAOA, LLC.

High-resolution fiber-optic microendoscopy for in situ cellular imaging.

PubMed

Pierce, Mark; Yu, Dihua; Richards-Kortum, Rebecca

2011-01-11

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user. We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture, in excised and living animal tissues, and in human tissues in vivo. Users have reported the use of several different fluorescent contrast agents, including proflavine, benzoporphyrin-derivative monoacid ring A (BPD-MA), and fluoroscein, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

Discrimination of benign and neoplastic mucosa with a high-resolution microendoscope (HRME) in head and neck cancer.

PubMed

Vila, Peter M; Park, Chan W; Pierce, Mark C; Goldstein, Gregg H; Levy, Lauren; Gurudutt, Vivek V; Polydorides, Alexandros D; Godbold, James H; Teng, Marita S; Genden, Eric M; Miles, Brett A; Anandasabapathy, Sharmila; Gillenwater, Ann M; Richards-Kortum, Rebecca; Sikora, Andrew G

2012-10-01

The efficacy of ablative surgery for head and neck squamous cell carcinoma (HNSCC) depends critically on obtaining negative margins. Although intraoperative "frozen section" analysis of margins is a valuable adjunct, it is expensive, time-consuming, and highly dependent on pathologist expertise. Optical imaging has potential to improve the accuracy of margins by identifying cancerous tissue in real time. Our goal was to determine the accuracy and inter-rater reliability of head and neck cancer specialists using high-resolution microendoscopic (HRME) images to discriminate between cancerous and benign mucosa. Thirty-eight patients diagnosed with head and neck squamous cell carcinoma (HNSCC) were enrolled in this single-center study. HRME was used to image each specimen after application of proflavine, with concurrent standard histopathologic analysis. Images were evaluated for quality control, and a training set containing representative images of benign and neoplastic tissue was assembled. After viewing training images, seven head and neck cancer specialists with no previous HRME experience reviewed 36 test images and were asked to classify each. The mean accuracy of all reviewers in correctly diagnosing neoplastic mucosa was 97% (95% confidence interval (CI), 94-99%). The mean sensitivity and specificity were 98% (97-100%) and 92% (87-98%), respectively. The Fleiss kappa statistic for inter-rater reliability was 0.84 (0.77-0.91). Medical professionals can be quickly trained to use HRME to discriminate between benign and neoplastic mucosa in the head and neck. With further development, the HRME shows promise as a method of real-time margin determination at the point of care.

Flow cytometric detection of micronuclei by combined staining of DNA and membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wessels, J.M.; Nuesse, M.

1995-03-01

A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN frommore » debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB or DPH were comparable to the results obtained with the combination of EB and HO. 23 refs., 7 figs.« less

Time dependence of triplet--singlet excitation transfer from compact poly rA to bound dye at 77 k

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pearlstein, R.M.; Nostrand, F.V.; Nairn, J.A.

1979-04-01

The nonexponential phosphorescence decay of a highly folded form of polyriboadenylic acid (poly rA) with noncovalently bound dye is explained by a novel application of a well-known theory of electronic excitation transfer based on the Forster mechanism. This theory, originally used to describe singlet-singlet energy transfer from donor molecules to an acceptor in a solution, is here applied to the transfer of triplet excitation from the adenine (in poly rA) to the singlet manifold of either of the bound dyes, ethidium bromide or proflavine. New experimental data are presented that allow straight-forward theoretical interpretation. These data fit the form predictedmore » by the theory, U(t)exp(-Bt/sup 1/2/), where U(t) is the decay of the poly rA phosphorescence in the absence of dye, for a range of relative concentrations of either dye. The self-consistency of these theoretical fits is demonstrated by the proportionality of B to the square root of the Forster triplet-singlet overlap integrals for transfer from poly rA to each of the dyes, as demanded by the theory. From these self-consistent values of B, the theory enables one to deduce the mean packing density of nucleotides in this folded poly rA, which we estimate to be approx. 1 nm/sup -3/. We conclude that some variation of the method described here may be useful for deducing packing densities of nucleotides in other compact nucleic acid structures.« less

Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery.

PubMed

Yildiz, Ibrahim; Lee, Karin L; Chen, Kevin; Shukla, Sourabh; Steinmetz, Nicole F

2013-12-10

This work is focused on the development of a plant virus-based carrier system for cargo delivery, specifically 30nm-sized cowpea mosaic virus (CPMV). Whereas previous reports described the engineering of CPMV through genetic or chemical modification, we report a non-covalent infusion technique that facilitates efficient cargo loading. Infusion and retention of 130-155 fluorescent dye molecules per CPMV using DAPI (4',6-diamidino-2-phenylindole dihydrochloride), propidium iodide (3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide), and acridine orange (3,6-bis(dimethylamino)acridinium chloride), as well as 140 copies of therapeutic payload proflavine (PF, acridine-3,6-diamine hydrochloride), is reported. Loading is achieved through interaction of the cargo with the CPMV's encapsidated RNA molecules. The loading mechanism is specific; empty RNA-free eCPMV nanoparticles could not be loaded. Cargo-infused CPMV nanoparticles remain chemically active, and surface lysine residues were covalent modified with dyes leading to the development of dual-functional CPMV carrier systems. We demonstrate cargo-delivery to a panel of cancer cells (cervical, breast, and colon): CPMV nanoparticles enter cells via the surface marker vimentin, the nanoparticles target the endolysosome, where the carrier is degraded and the cargo is released allowing imaging and/or cell killing. In conclusion, we demonstrate cargo-infusion and delivery to cells; the methods discussed provide a useful means for functionalization of CPMV toward its application as drug and/or contrast agent delivery vehicle. Copyright © 2013 Elsevier B.V. All rights reserved.

Discrimination of Benign and Neoplastic Mucosa with a High-Resolution Microendoscope (HRME) in Head and Neck Cancer

PubMed Central

Vila, Peter M.; Park, Chan W.; Pierce, Mark C.; Goldstein, Gregg H.; Levy, Lauren; Gurudutt, Vivek V.; Polydorides, Alexandras D.; Godbold, James H.; Teng, Marita S.; Genden, Eric M.; Miles, Brett A.; Anandasabapathy, Sharmila; Gillenwater, Ann M.; Richards-Kortum, Rebecca; Sikora, Andrew G.

2012-01-01

Background The efficacy of ablative surgery for head and neck squamous cell carcinoma (HNSCC) depends critically on obtaining negative margins. While intraoperative "frozen section" analysis of margins is a valuable adjunct, it is expensive, time-consuming, and highly dependent on pathologist expertise. Optical imaging has potential to improve the accuracy of margins by identifying cancerous tissue in real time. Our aim was to determine the accuracy and inter-rater reliability of head and neck cancer specialists using high-resolution microendoscopic (HRME) images to discriminate between cancerous and benign mucosa. Methods Thirty-eight patients diagnosed with HNSCC were enrolled in this single-center study. HRME was used to image each specimen after application of proflavine, with concurrent standard histopathologic analysis. Images were evaluated for quality control, and a training set containing representative images of benign and neoplastic tissue was assembled. After viewing training images, seven head and neck cancer specialists with no prior HRME experience reviewed 37 test images and were asked to classify each. Results The mean accuracy of all reviewers in correctly diagnosing neoplastic mucosa was 97 percent (95% Cl = 94–99%). The mean sensitivity and specificity were 98 percent (97–100%) and 92 percent (87–98%), respectively. The Fleiss kappa statistic for inter-rater reliability was 0.84 (0.77–0.91). Conclusions Medical professionals can be quickly trained to use HRME to discriminate between benign and neoplastic mucosa in the head and neck. With further development, the HRME shows promise as a method of real-time margin determination at the point of care. PMID:22492225

Detection of Head and Neck Cancer in Surgical Specimens Using Quantitative Hyperspectral Imaging.

PubMed

Lu, Guolan; Little, James V; Wang, Xu; Zhang, Hongzheng; Patel, Mihir R; Griffith, Christopher C; El-Deiry, Mark W; Chen, Amy Y; Fei, Baowei

2017-09-15

Purpose: This study intends to investigate the feasibility of using hyperspectral imaging (HSI) to detect and delineate cancers in fresh, surgical specimens of patients with head and neck cancers. Experimental Design: A clinical study was conducted in order to collect and image fresh, surgical specimens from patients ( N = 36) with head and neck cancers undergoing surgical resection. A set of machine-learning tools were developed to quantify hyperspectral images of the resected tissue in order to detect and delineate cancerous regions which were validated by histopathologic diagnosis. More than two million reflectance spectral signatures were obtained by HSI and analyzed using machine-learning methods. The detection results of HSI were compared with autofluorescence imaging and fluorescence imaging of two vital-dyes of the same specimens. Results: Quantitative HSI differentiated cancerous tissue from normal tissue in ex vivo surgical specimens with a sensitivity and specificity of 91% and 91%, respectively, and which was more accurate than autofluorescence imaging ( P < 0.05) or fluorescence imaging of 2-NBDG ( P < 0.05) and proflavine ( P < 0.05). The proposed quantification tools also generated cancer probability maps with the tumor border demarcated and which could provide real-time guidance for surgeons regarding optimal tumor resection. Conclusions: This study highlights the feasibility of using quantitative HSI as a diagnostic tool to delineate the cancer boundaries in surgical specimens, and which could be translated into the clinic application with the hope of improving clinical outcomes in the future. Clin Cancer Res; 23(18); 5426-36. ©2017 AACR . ©2017 American Association for Cancer Research.

Dual-modality smartphone endoscope for cervical pre-cancer detection (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hong, Xiangqian; Yu, Bing

2017-02-01

Early detection is the key to the prevention of cervical cancer. There is an urgent need for a portable, affordable, and easy-to-use device for cervical pre-cancer detection, especially in low-resource settings. We have developed a dual-modality fiber-optic endoscope system (SmartME) that integrates high-resolution fluorescence imaging (FLI) and quantitative diffuse reflectance spectroscopy (DRS) onto a smartphone platform. The SmartME consists of a smartphone, a miniature fiber-optic endoscope, a phone attachment containing imaging optics, and a smartphone application (app). FLI is obtained by painting the tissue with a contrast agent (e.g., proflavine), illuminating the tissue and collecting its fluorescence images through an imaging bundle that is coupled to the phone camera. DRS is achieved by using a white LED, attaching additional source and detection fibers to the imaging bundle, and converting the phone camera into a spectrometer. The app collects images/spectra and transmits them to a remote server for analysis to extract the tissue parameters, including nuclear-to-cytoplasm ratio (calculated from FLI), concentrations of oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) as well as scattering (measured by DRS). These parameters can be used to detect cervical dysplasia. Our preliminary studies have demonstrated that the SmartME can clearly visualize the nuclei in living cells and in vivo biological samples, with a high spatial resolution of 3.1μm. The device can also measure tissue absorption and scattering properties with comparable accuracy to those of a benchtop DRS system. The SmartME has great potential to provide a compact, affordable, and `smart' solution for early detection of neoplastic changes in cervix.

Positive and negative ion mode ESI-MS and MS/MS for studying drug-DNA complexes

NASA Astrophysics Data System (ADS)

Rosu, Frédéric; Pirotte, Sophie; Pauw, Edwin De; Gabelica, Valérie

2006-07-01

We report systematic investigation of duplex DNA complexes with minor groove binders (Hoechsts 33258 and 33342, netropsin and DAPI) and intercalators (daunomycin, doxorubicin, actinomycin D, ethidium, cryptolepine, neocryptolepine, m-Amsacrine, proflavine, ellipticine and mitoxantrone) by ESI-MS and ESI-MS/MS in the negative ion mode and in the positive ion mode. The apparent solution phase equilibrium binding constants can be determined by measuring relative intensities in the ESI-MS spectrum. While negative ion mode gives reliable results, positive ion mode gives a systematic underestimation of the binding constants and even a complete suppression of the complexes for intercalators lacking functional groups capable of interacting in the grooves. In the second part of the paper we systematically compare MS/MS fragmentation channels and breakdown curves in the positive and the negative modes, and discuss the possible uses and caveats of MS/MS in drug-DNA complexes. In the negative mode, the drugs can be separated in three groups: (1) those that leave the complex with no net charge; (2) those that leave the complex with a negative charge; and (3) those that remain attached on the strands upon dissociation of the duplex due to their positive charge. In the positive ion mode, all complexes fragment via the loss of protonated drug. Information on the stabilization of the complex by drug-DNA noncovalent interactions can be obtained straightforwardly only in the case of neutral drug loss. In all other cases, proton affinity (in the positive ion mode), gas-phase basicity (in the negative ion mode) and coulombic repulsion are the major factors influencing the fragmentation channel and the dissociation kinetics.

Synthesis and characterization of thermally stable and/or conductive polymers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gajiwala, H.M.

1992-01-01

Eight new thermally stable polyimides were synthesized from two tricyclic heterocyclic diamines: thionine which has a phenothiazine moiety and proflavine which has an acridine unit. The polymerization reactions were optimized with respect to solvents, reaction time, reaction temperature, solid contents, etc., and their structure property relationships were studied. All these soluble polyimides have nice film forming properties. One of the polyimides containing the acridine moiety, appears to have a tendency to form a liquid crystalline state when its solution is passed through a fine capillary. All of these polyimides were thermally stable in air up to 500-550[degrees]C and up tomore » 600[degrees]C in a nitrogen atmosphere. They have refractive indices in the range of 1.65 to 1.85 and have relatively low value of permittivity. Two other thermally stable polymers, viz., polybenzimidazole and the ladder polymer having the phenazine moiety in the backbone were also synthesized. For these polymerization reactions, tetraamino derivative of phenazine was synthesized from the commercially available diamino, dinitro derivative of benzene. The polybenzimidazole was prepared via the azomethine pathway. This polymer had an intrinsic viscosity of 0.94 in methanesulfonic acid. The nice film forming polybenzimidazole polymer was found to be thermally stable up to 400[degrees]C. The ladder type of a polymer was synthesized by condensation polymerization between tetraaminophenazine and dihydroxybenzophenone in polyphosphoric acid at an elevated temperature. The completely conjugated ladder polymer was found to be semiconducting on doping with iodine. This polymer was highly crystalline as demonstrated by its X-ray diffraction pattern. A morphology study of the polymer indicated that the material has a tendency to form dendritic crystals. The polymer was thermally stable up to about 400[degrees]C in air.« less

Magnetic field effect corroborated with docking study to explore photoinduced electron transfer in drug-protein interaction.

PubMed

Chakraborty, Brotati; Roy, Atanu Singha; Dasgupta, Swagata; Basu, Samita

2010-12-30

Conventional spectroscopic tools such as absorption, fluorescence, and circular dichroism spectroscopy used in the study of photoinduced drug-protein interactions can yield useful information about ground-state and excited-state phenomena. However, photoinduced electron transfer (PET) may be a possible phenomenon in the drug-protein interaction, which may go unnoticed if only conventional spectroscopic observations are taken into account. Laser flash photolysis coupled with an external magnetic field can be utilized to confirm the occurrence of PET and authenticate the spin states of the radicals/radical ions formed. In the study of interaction of the model protein human serum albumin (HSA) with acridine derivatives, acridine yellow (AY) and proflavin (PF(+)), conventional spectroscopic tools along with docking study have been used to decipher the binding mechanism, and laser flash photolysis technique with an associated magnetic field (MF) has been used to explore PET. The results of fluorescence study indicate that fluorescence resonance energy transfer takes place from the protein to the acridine-based drugs. Docking study unveils the crucial role of Ser 232 residue of HSA in explaining the differential behavior of the two drugs towards the model protein. Laser flash photolysis experiments help to identify the radicals/radical ions formed in the due course of PET (PF(•), AY(•-), TrpH(•+), Trp(•)), and the application of an external MF has been used to characterize their initial spin-state. Owing to its distance dependence, MF effect gives an idea about the proximity of the radicals/radical ions during interaction in the system and also helps to elucidate the reaction mechanisms. A prominent MF effect is observed in homogeneous buffer medium owing to the pseudoconfinement of the radicals/radical ions provided by the complex structure of the protein.

Spatial assignment of symmetry adapted perturbation theory interaction energy components: The atomic SAPT partition

NASA Astrophysics Data System (ADS)

Parrish, Robert M.; Sherrill, C. David

2014-07-01

We develop a physically-motivated assignment of symmetry adapted perturbation theory for intermolecular interactions (SAPT) into atom-pairwise contributions (the A-SAPT partition). The basic precept of A-SAPT is that the many-body interaction energy components are computed normally under the formalism of SAPT, following which a spatially-localized two-body quasiparticle interaction is extracted from the many-body interaction terms. For electrostatics and induction source terms, the relevant quasiparticles are atoms, which are obtained in this work through the iterative stockholder analysis (ISA) procedure. For the exchange, induction response, and dispersion terms, the relevant quasiparticles are local occupied orbitals, which are obtained in this work through the Pipek-Mezey procedure. The local orbital atomic charges obtained from ISA additionally allow the terms involving local orbitals to be assigned in an atom-pairwise manner. Further summation over the atoms of one or the other monomer allows for a chemically intuitive visualization of the contribution of each atom and interaction component to the overall noncovalent interaction strength. Herein, we present the intuitive development and mathematical form for A-SAPT applied in the SAPT0 approximation (the A-SAPT0 partition). We also provide an efficient series of algorithms for the computation of the A-SAPT0 partition with essentially the same computational cost as the corresponding SAPT0 decomposition. We probe the sensitivity of the A-SAPT0 partition to the ISA grid and convergence parameter, orbital localization metric, and induction coupling treatment, and recommend a set of practical choices which closes the definition of the A-SAPT0 partition. We demonstrate the utility and computational tractability of the A-SAPT0 partition in the context of side-on cation-π interactions and the intercalation of DNA by proflavine. A-SAPT0 clearly shows the key processes in these complicated noncovalent interactions, in systems with up to 220 atoms and 2845 basis functions.

Spatial assignment of symmetry adapted perturbation theory interaction energy components: The atomic SAPT partition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parrish, Robert M.; Sherrill, C. David, E-mail: sherrill@gatech.edu

2014-07-28

We develop a physically-motivated assignment of symmetry adapted perturbation theory for intermolecular interactions (SAPT) into atom-pairwise contributions (the A-SAPT partition). The basic precept of A-SAPT is that the many-body interaction energy components are computed normally under the formalism of SAPT, following which a spatially-localized two-body quasiparticle interaction is extracted from the many-body interaction terms. For electrostatics and induction source terms, the relevant quasiparticles are atoms, which are obtained in this work through the iterative stockholder analysis (ISA) procedure. For the exchange, induction response, and dispersion terms, the relevant quasiparticles are local occupied orbitals, which are obtained in this work throughmore » the Pipek-Mezey procedure. The local orbital atomic charges obtained from ISA additionally allow the terms involving local orbitals to be assigned in an atom-pairwise manner. Further summation over the atoms of one or the other monomer allows for a chemically intuitive visualization of the contribution of each atom and interaction component to the overall noncovalent interaction strength. Herein, we present the intuitive development and mathematical form for A-SAPT applied in the SAPT0 approximation (the A-SAPT0 partition). We also provide an efficient series of algorithms for the computation of the A-SAPT0 partition with essentially the same computational cost as the corresponding SAPT0 decomposition. We probe the sensitivity of the A-SAPT0 partition to the ISA grid and convergence parameter, orbital localization metric, and induction coupling treatment, and recommend a set of practical choices which closes the definition of the A-SAPT0 partition. We demonstrate the utility and computational tractability of the A-SAPT0 partition in the context of side-on cation-π interactions and the intercalation of DNA by proflavine. A-SAPT0 clearly shows the key processes in these complicated noncovalent interactions, in systems with up to 220 atoms and 2845 basis functions.« less

Depth-sensitive optical spectroscopy for noninvasive diagnosis of oral neoplasia

NASA Astrophysics Data System (ADS)

Schwarz, Richard Alan

Oral cancer is the 11th most common cancer in the world. Cancers of the oral cavity and oropharynx account for more than 7,500 deaths each year in the United States alone. Major advances have been made in the management of oral cancer through the combined use of surgery, radiotherapy and chemotherapy, improving the quality of life for many patients; however, these advances have not led to a significant increase in survival rates, primarily because diagnosis often occurs at a late stage when treatment is more difficult and less successful. Accurate, objective, noninvasive methods for early diagnosis of oral neoplasia are needed. Here a method is presented to noninvasively evaluate oral lesions using depth-sensitive optical spectroscopy (DSOS). A ball lens coupled fiber-optic probe was developed to enable preferential targeting of different depth regions in the oral mucosa. Clinical studies of the diagnostic performance of DSOS in 157 subjects were carried out in collaboration with the University of Texas M. D. Anderson Cancer Center. An overall sensitivity of 90% and specificity of 89% were obtained for nonkeratinized oral tissue relative to histopathology. Based on these results a compact, portable version of the clinical DSOS device with real-time automated diagnostic capability was developed. The portable device was tested in 47 subjects and a sensitivity of 82% and specificity of 83% were obtained for nonkeratinized oral tissue. The diagnostic potential of multimodal platforms incorporating DSOS was explored through two pilot studies. A pilot study of DSOS in combination with widefield imaging was carried out in 29 oral cancer patients, resulting in a combined sensitivity of 94% and specificity of 69%. Widefield imaging and spectroscopy performed slightly better in combination than each method performed independently. A pilot study of DSOS in combination with the optical contrast agents 2-NBDG, EGF-Alexa 647, and proflavine was carried out in resected tissue specimens from 15 oral cancer patients. Improved contrast between neoplastic and healthy tissue was observed using 2-NBDG and EGF-Alexa 647.

Energy component analysis of π interactions.

PubMed

Sherrill, C David

2013-04-16

Fundamental features of biomolecules, such as their structure, solvation, and crystal packing and even the docking of drugs, rely on noncovalent interactions. Theory can help elucidate the nature of these interactions, and energy component analysis reveals the contributions from the various intermolecular forces: electrostatics, London dispersion terms, induction (polarization), and short-range exchange-repulsion. Symmetry-adapted perturbation theory (SAPT) provides one method for this type of analysis. In this Account, we show several examples of how SAPT provides insight into the nature of noncovalent π-interactions. In cation-π interactions, the cation strongly polarizes electrons in π-orbitals, leading to substantially attractive induction terms. This polarization is so important that a cation and a benzene attract each other when placed in the same plane, even though a consideration of the electrostatic interactions alone would suggest otherwise. SAPT analysis can also support an understanding of substituent effects in π-π interactions. Trends in face-to-face sandwich benzene dimers cannot be understood solely in terms of electrostatic effects, especially for multiply substituted dimers, but SAPT analysis demonstrates the importance of London dispersion forces. Moreover, detailed SAPT studies also reveal the critical importance of charge penetration effects in π-stacking interactions. These effects arise in cases with substantial orbital overlap, such as in π-stacking in DNA or in crystal structures of π-conjugated materials. These charge penetration effects lead to attractive electrostatic terms where a simpler analysis based on atom-centered charges, electrostatic potential plots, or even distributed multipole analysis would incorrectly predict repulsive electrostatics. SAPT analysis of sandwich benzene, benzene-pyridine, and pyridine dimers indicates that dipole/induced-dipole terms present in benzene-pyridine but not in benzene dimer are relatively unimportant. In general, a nitrogen heteroatom contracts the electron density, reducing the magnitude of both the London dispersion and the exchange-repulsion terms, but with an overall net increase in attraction. Finally, using recent advances in SAPT algorithms, researchers can now perform SAPT computations on systems with 200 atoms or more. We discuss a recent study of the intercalation complex of proflavine with a trinucleotide duplex of DNA. Here, London dispersion forces are the strongest contributors to binding, as is typical for π-π interactions. However, the electrostatic terms are larger than usual on a fractional basis, which likely results from the positive charge on the intercalator and its location between two electron-rich base pairs. These cation-π interactions also increase the induction term beyond those of typical noncovalent π-interactions.