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Cardiolipin (CL) plays an essential role as a marker for cell apoptosis. Quantitative detection of phospholipids (PLs) by UV absorbance is problematic due to the presence of few double bonds in the structure. Although 10-N-nonyl acridine orange (NAO) has been utilized for fluorescent visualization of liposomes and mitochondria through its interaction with CL, in this work, we have developed a specific fluorescent method for CL in solution using NAO. The interaction of sodium n-dodecyl sulfate (SDS), used to treat cells prior to lipid extraction, and other PLs found in cell membranes such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidiylserine (PS), and sphingomyelin (SM) with NAO is investigated. The fluorescence intensity of the 0.5 microM NAO signal is strongly quenched by SDS below 25% methanol in water but with a methanol content above 50%, no quenching of NAO by SDS is observed. No fluorescence quenching of NAO with a 50% methanol/50% water solvent by the previously mentioned PLs or 4-20 microM cholesterol with the exception of PG at above 8 microM is noted. Using this 50% methanol/50% water solvent, the fluorescence signal due to the NAO-CL interaction is quite stable from 3 to at least 15 min. With excitation and emission wavelengths set at 518 and 530 nm, respectively, 20 microM NAO provides an inverse linear fluorescence response at 0.2-10 microM CL with a correlation coefficient of 0.9929. The detection limit is 0.2 microM and the limit of quantification is 0.6 microM. Structurally analogous acridine orange and phenosafranin dyes are less effective as fluorescent probes for CL. The CL in the whole cell and membrane samples is quantitatively determined by standard addition to range from 0.2 to 1.5 microM. The level of CL in cell membrane samples, previously subjected to staurosporine which initiates cell apoptosis, is increased but not significantly through use of the t-test.

A real-time fluorescence assay system using a series of 9- N -(alkylamino)acridine derivatives (methyl, ethyl, n -propyl, n -butyl, n -pentyl, and benzyl) that are N -dealkylated to 9-aminoacridine (9AA) is described. The product, 9AA, is approximately 27-fold more fluorescent than the substrates using excitation and emission wavelengths of 405 and 455 nm, respectively. Tests using expressed CYP1A1, 1A2, 3A4, 3A5, 1B1, 2C9, 2C19, and 2D6 indicated that N -dealkylase activity is specific for CYP1A1 and CYP2D6. CYP2D6 N -dealkylated methyl, ethyl, n -propyl, and n -butyl substrates, whereas CYP1A1 N -dealkylated these plus the n -pentyl derivative. Activities using 5 μM 9- N -(alkylamino)acridine substrates ranged from 0.1 to 0.9 pmol 9AA/min/pmol P450. Kinetic constants for CYP1A1 N -dealkylation of the 9- N -(methylamino)acridine (MAA) and 9- N -(ethylamino)acridine (EAA) were K m 1.09 ± 0.68 and 0.35 ± 0.21 μM and the V max 61.9 ± 48.5 and 113.8 ± 8.4 pmol 9AA/min/pmol CYP1A1, respectively. Kinetic constants for CYP2D6 N -dealkylation of MAA and EAA were K m 7.9 ± 5.4 and 3.2 ± 1.6 μM, and V max 501 ± 35.4 and 702.7 ± 257 pmol 9AA/min/pmol CYP2D6, respectively. The experimental binding energies (Δ G bind ) were calculated for MAA with CYP1A1 and CYP2D6 to be –8.266 and –7.074 kcal/mol, respectively. The Δ G bind values for EAA with CYP1A1 and CYP2D6 were –8.950 and –7.618 kcal/mol, respectively. The substrates were suitable for monitoring N -dealkylase activity in microsomal preparations (human, rat, and monkey hepatic preparations) and human hepatocellular carcinoma cell suspensions. Assays were conducted by monitoring reactions either in 96-well microtiter plates using a fluorescence plate reader or in cuvettes using a spectrofluorimeter.

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres withor =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.

The interaction of adenosine disodium triphosphate (ATP) with acridine yellow and its analytical application have been studied. In an alkalescent medium, adenosine disodium triphosphate react with acridine yellow to form an ion-association by virtue of electrostatic attraction and hydrophobic interaction, resulting in a remarkable enhancement of resonance light scattering (RLS) intensity of acridine yellow. The maximum scattering wavelength is at 325 nm. The spectral characteristics of the ion-associates, the effective factors and the optimum conditions have been investigated. The enhanced RLS intensity is directly proportional to the concentration of ATP in the range of 0.80-20.0 microg ml(-1) with the detection limit 0.086 microg ml(-1). The method has been successfully applied to the quick determination of ATP in tablet and injection samples. The results of the present method are in agreement with those obtained by the method in the Chinese Pharmacopoeia.

The conserved Glu540 in subunit I of Escherichia coli cytochrome bo (a H+ pump) is replaced by Asp544 in the Vitreoscilla enzyme (a Na+ pump). Site-directed mutagenesis of the Vitreoscilla cytochrome bo operon changed this Asp to Glu, and both wild type and mutant cyo’s were transformed into E. coli strain GV100, which lacks cytochrome bo. Compared to the wild type transformant the Asp544Glu transformant had decreased ability to pump Na+ as well as decreased stimulation in respiratory activity in the presence of Na+. Preliminary experiments indicated that this mutant also had increased ability to pump protons, suggesting that this single change may provide cation pumping specificity in this group of enzymes.

A new, fast, and sensitive DNA hybridization assay platform based on microwave-accelerated metal-enhanced fluorescence (MAMEF) is presented. Thiolated oligonucleotide anchors were immobilized onto silver nanoparticles on a glass substrate. The hybridization of the complementary fluorescein-labeled DNA target with the surface-bound oligonucleotides was completed within 20 s upon heating with low-power microwaves. In addition, the signal is optically amplified, a consequence of close proximity of the fluorophore to the silvered substrate. In this proof-of-principle methodology, as low as 50 nM of a target DNA was detected, although we envisage far-lower detection limits. Control experiments, where the surface-bound oligonucleotide was omitted, were also performed to determine the extent of non-specific binding. In these studies a significantly reduced non-specific adsorption was found when using microwave heating near to silvered structures as compared to room temperature incubation. These findings suggest that MAMEF could be a most useful alternative to the DNA hybridization assays used today, especially with regard to substantially increasing both the assay rapidity and sensitivity.

Urinary trypsin inhibitor (ulinastatin) is a characteristic protein pharmaceutical which contains both glycosaminoglycans and N-linked glycans in its molecule and has been used for treatment of acute pancreatitis. The comparability of ulinastatin preparations of different lots or from different companies was studied by using conventional analytical approaches such as SDS-PAGE, cellulose acetate membrane electrophoresis, and HP size-exclusion chromatography (SEC) and also by using newly developed techniques such as CE and MALDI-TOF MS. The methods using SEC and SDS-PAGE according to The Japanese Pharmacopoeia showed similar molecular masses for two different preparations, and the estimated molecular masses were significantly different from those observed with MALDI-TOF MS. We also showed that the electrophoretic methods using cellulose acetate membrane electrophoresis and CE can be used for comparability assessments of ulinastatin preparations. In addition, we analyzed the unsaturated disaccharides derived from glycosaminoglycan (chondroitin 4-sulfate chain) and N-linked oligosaccharides attached to ulinastatin by CE after releasing them by enzymatic digestion followed by fluorescent labeling with 2-aminoacridone and 2-aminobenzoic acid, respectively. The results indicated that carbohydrate chains are important as markers for comparability assessments of ulinastatin pharmaceutical preparations.

Aminoacridines have a long history in the drug and dye industries and display a wide range of biological and physical properties. Despite the historical relevance of 9-aminoacridines, there have been few studies investigating their stability. 9-Aminoacridines are known to hydrolyze at the C9-N15 bond, yielding acridones. In this study, the pH-dependent hydrolysis rates of a series of 9-substituted aminoacridines are investigated. In addition, ground-state physical properties of the compounds are determined using ab initio quantum mechanics calculations to gain insight into the forces that drive hydrolysis. An analysis of the bond orders, bond dissociation energies, and conformational energies show that the rate of hydrolysis depends on two main factors: delocalization across the C9-N15 bond and steric effects. The computational results are applied to explain the change in experimental rates of hydrolysis going from primary to secondary and to tertiary substituted 9-aminoacridines. In the case of tertiary substituted amines, the calculations indicate the C9-N15 bond is forced into a more gauche-like conformation, greatly diminishing delocalization (as shown by reductions in bond orders and bond energy), which leads to rapid hydrolysis. A model of intramolecular hydrogen bonding is also presented, which explains the increased rate of hydrolysis observed for highly substituted compounds under acidic conditions.

Mixed crystals of potassium hydrogen phthalate containing 3,6-diaminoacridine were photoexcited with visible light and the resulting triplet excited states were analyzed by time resolved EPR spectroscopy. Spectra from discrete growth sectors were compared with powders and polycrystalline glasses prepared at various pHs. The data yield the predominant protonation state and orientation of the triplets in each of a pair of growth sectors bounding the positive and negative ends of the polar crystal.

C1311 is the lead compound from the imidazoacridinones, a novel group of rationally designed anti-cancer agents. The compound is pharmaceutically formulated as a lyophilized product containing 100 mg C1311 (anhydrous free base) per dosage unit and requires reconstitution before intravenous administration. The aim of this study was to determine the stability of C1311 in the reconstituted solution and infusion solution and its compatibility with infusion devices. Moreover, the buffer capacity and haemolytic potential of C1311 infusion solutions, which exhibit a relatively low pH of 2-3, were evaluated in vitro. C1311 was shown to be stable in the reconstituted solution for at least 48 h and for at least 96 h after subsequent dilution in 0.9% sodium chloride and 5% dextrose. In vitro infusion simulation studies showed C1311 infusion solutions to be compatible with a low-density polyethylene administration set. Furthermore, the buffer capacity and haemolysis studies showed no indications for haemolysis or potential for vascular irritation upon continuous infusion of C1311. In conclusion, C1311 lyophilized product is adequately stable and compatible after reconstitution and in infusion fluids to be used in the clinic and is not expected to cause formulation-associated side effects in the intended administration schedule in the forthcoming Phase I clinical study.

C1311 (5-[[2-(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1-de]-acridin-6-one-dihydrochloride trihydrate) is the lead compound from the group of imidazoacridinones, a novel group of rationally designed anticancer agents. C1311 shows significant cytotoxic activity in vitro and in vivo toward a range of colon tumours. The aim of the present study is to develop a sterile and stable, injectable pharmaceutical product for C1311 to be used in phase I clinical trials. C1311 drug substance was structurally and analytically characterised by chromatographic, spectrometric, and diffraction techniques. C1311 was freely soluble in water, and its stability was investigated in several liquid and lyophilised formulations with or without the use of buffering, tonicity, and bulking agents. The final product, containing 100 mg/vial C1311 (as anhydrous free base), was stable for at least 3 months under accelerated storage conditions and at the designated long-term storage condition of 5 +/- 3 degrees C in the dark. The drug is currently used in phase I clinical trials.

A parallel synthetic strategy to the 9-aminoacridine scaffold of the classical anti-malarial drug quinacrine (2) is presented. The method features a new route to 9-chloroacridines that utilizes triflates of salicylic acid derivatives, which are commercially available in a variety of substitution patterns. The route allows ready variation of the two diversity elements present in this class of molecules: the tricyclic aromatic heterocyclic core, and the disubstituted diamine sidechain. In this study, a library of 175 compounds was designed, although only 93 of the final products had purities acceptable for screening. Impurity was generally due to incomplete removal of 9-acridones (18), a degradation product of the 9-chloroacridine synthetic intermediates. The library was screened against two strains of Plasmodium falciparum, including a model of the drug-resistant parasite, and six novel compounds were found to have IC(50) values in the low nanomolar range.

We report the synthesis and characterization of photonucleases N,N'-bis[2-[bis(1H-imidazol-4-ylmethyl)amino]ethyl]-3,6-acridinediamine (7) and N-[2-[bis(1H-imidazol-4-ylmethyl)amino]ethyl]-3,6-acridinediamine (10), consisting of a central 3,6-acridinediamine chromophore attached to 4 and 2 metal-coordinating imidazole rings, respectively. In DNA reactions employing 16 metal salts, photocleavage of pUC19 plasmid is markedly enhanced when compound 7 is irradiated in the presence of either Hg(II), Fe(III), Cd(II), Zn(II), V(V), or Pb(II) (low-intensity visible light, pH 7.0, 22 degrees C, 8-50 microM 7). We also show that DNA photocleavage by 7 can be modulated by modifying buffer type and pH. Evidence of metal complex formation is provided by EDTA experiments and by NMR and electrospray ionization mass spectral data. Sodium azide, sodium benzoate, superoxide dismutase, and catalase indicate the involvement of type I and II photochemical processes in the metal-assisted DNA photocleavage reactions. Thermal melting studies show that compound 7 increases the Tm of calf thymus DNA by 10 +/- 1 degrees C at pH 7.0 and that the Tm is further increased upon the addition of either Hg(II), Cd(II), Zn(II), or Pb(II). In the case of Fe(III) and V(V), a colorimetric assay demonstrates that compound 7 sensitizes one electron photoreduction of these metals to Fe(II) and V(IV), likely accelerating the production of type I reactive oxygen species. Our data collectively indicate that buffer, pH, Hg(II), Fe(III), Cd(II), Zn(II), V(V), Pb(II), and light can be used to "tune" DNA cleavage by compound 7 under physiologically relevant conditions. The 3,6-acridinediamine acridine orange has demonstrated great promise for use as a photosensitizer in photodynamic therapy. In view of the distribution of iron in living cells, compound 7 and other metal-binding acridine-based photonucleases should be expected to demonstrate excellent photodynamic action in vivo.

Hyaluronan (HA) and chondroitin sulfate (CS) are glycosaminoglycans (GAGs) with great importance in biological events, since they participate in and regulate cell adhesion, migration and proliferation. Quantitation and analysis of the fine structure of GAGs are increasingly important for understanding many biological processes, among which are many critical aspects of pathology development and specific phenotype descriptions. Human umbilical cord and human synovial fluid are connective tissues containing high amounts of GAGs and change in the quantity and structure of these macromolecules is described in tissue development and is commonly associated with disease. Moreover, also in Xenopus laevis embryo development and chondrocyte cultures, the GAG content and structure play a critical role. A rapid analysis of hyaluronan and chondroitin sulfate Δ-disaccharides derived from the above human and animal samples, derivatized with 2-aminoacridone and analyzed by polyacrylamide gel electrophoresis, is described in this report. Qualitative and quantitative analysis were performed by comparing their migration and the pixel density with standard Δ-disaccharides, running in the same gel. Since this method allows the analysis of large numbers of samples simultaneously in one gel and has a relatively high sensitivity (less than 25 pmol), it is suggested as a cost-effective and useful tool for the fast screening of small amounts of hyaluronan and chondroitin sulfate disaccharides. Copyright © 2005 John Wiley & Sons, Ltd.

In near neutral to weak basic media, sodium carboxymethyl cellulose (NaCMC) will dissociate to become a macro polymeric anion, which can react with acridine yellow (AY) or acridine orange (AO) to form an ion-association complex resulting in fluorescence quenching of the acridine dyes. The maximum fluorescence quenching wavelength is 505 nm (lambda(ex)=440 nm) for AY system and 530 nm (lambda(ex)=493 nm) for AO system, respectively. The fluorescence quenching values (DeltaF) are directly proportional to the concentrations of NaCMC and the linear ranges are 20.0-4000 microg/L for AY system and 20.0-7000 microg/L for AO system, separately. This method has high sensitivity and the detection limits for NaCMC are 58.0 microg/L (AY system) and 157.2 microg/L (AO system). The effects of coexistent substance have been investigated, and the results show that this method has a relatively good selectivity. A fluorescence quenching method for the determination of NaCMC based on the ion-association reactions of CMC polymeric anion with a basic acridine dye was developed. The method is sensitive, simple and fast.

In T-cell Acute Lymphocytic Leukemia (T-ALL), the inhibitors of cyclin-dependent kinases (CDK) 4 and 6, p16 and p15, are inactivated almost universally at the DNA, RNA and protein levels. This suggests that CDK-targeting may be an effective therapeutic approach for T-ALL and other cancers. In this study, we tested 3 inhibitors of CDK4, 3-aminothioacridone (3-ATA), thioacridone (TA), and oxindole, for their effects on DNA synthesis and viability in primary T-ALL. Each compound was an effective inhibitor, with overall IC(50)s in similar ranges. In colony formation assay, leukemic cells were approximately 10-fold more sensitive to 3-ATA than normal bone marrow cells. When sorted by G1 protein status of T-ALL, p16(+), p15(+) or pRb(-) samples were significantly less sensitive to 3-ATA and TA, but not to oxindole, than p16(-), p15(-) or pRb(+) samples. There was no relationship of sensitivity with ARF expression. Despite their in vitro function as inhibitors of CDK4, 3-ATA did not inhibit pRb phosphorylation or cause G1 arrest, but did cause DNA damage and result in the induction and phosphorylation of p53. We conclude that 3-ATA efficacy can be predicted by p16 status in T-ALL, but the mechanism of action may be distinct from their in vitro ability to regulate CDK4 kinase activity

This study was designed to provide more detailed information on the subcellular sites of binding of the porphycene, termed 9-capronyloxytetrakis (methoxyethyl) porphycene (CPO), with a fluorescence resonance energy transfer (FRET) technique. The proximity of CPO to two fluorescent probes was determined: nonyl acridine orange (NAO), a dye with specific affinity for the mitochondrial lipid cardiolipin, and dihexa-oxacarbocyanine iodide (DiOC6), an agent that labels the endoplasmic reticulum (ER). FRET spectra indicated energy transfer between DiOC6 and CPO but no significant transfer between NAO and CPO. These results confirm data obtained by fluorescence microscopy, suggesting a similar pattern of subcellular localization by CPO and DiOC6 but not by CPO and NAO. However, when cells containing CPO were irradiated and then loaded with NAO, FRET between the two fluorophores was observed. Hence, a relocalization of CPO can occur during irradiation. These data provide an explanation for recent studies on CPO-catalyzed photodamage to both ER and mitochondrial Bcl-2.

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.

The anticancer effect of 1-nitro-9-hydroxyethylamino acridine (C-857), a compound belonging to the 1-nitroacridine class, has been well documented. Despite its therapeutic efficacy, the clinical development of C-857 has been impeded partly due to its high systemic toxicity. In an effort to enhance antitumor efficacy and lower toxicity, derivatives of C-857 have been synthesized with substitutions made at position C-4 and/or an esterified hydroxyl group in side chain at the C-9 position. The introduction of a methyl group at C-4 resulted in C-1748, which has a significantly higher therapeutic efficacy and is being clinically developed as an anticancer agent for solid tumors. The present study was undertaken to correlate the mutagenicity of C-857, C-1748, C-1790, C-1872 and C-1873 with their cytotoxicity and their anti-tumor efficacy. The mutagenicity of these drugs was determined using three Ames Salmonella typhimurium strains TA1537, TA98 and TA102. The bacteria were treated with different molar concentrations, ranging from 10(-3) to 10(-12) M, of the drugs and drug-induced histidine revertants were then counted after a 48 h incubation. C-1748 did not induce any revertants in both TA1537 and TA98 at a dose of 10(-6) M, whereas, C-857 at the same dose induced approximately 842 and approximately 1034 revertants respectively. In TA102, mutagenicity was lower than observed with TA98 and TA1537 with highest revertants observed at 10(-5) M with C-857 (approximately 606) and C-1748 (approximately 108). Higher mutagenicity was observed in the derivatives C-1790, C-1872 and C-1873 compared to C-1748, but lower than C-857. These studies demonstrate that C-1748 has the least mutagenic potential, with a much higher antitumor effect in prostate cancer and is a promising chemotherapeutic agent for clinical development.

RNA tetraloops are common secondary structural motifs in many RNAs, especially ribosomal RNAs. There are few studies of small molecule recognition of RNA tetraloops although tetraloops are known to interact with RNA receptors and proteins, and to form nucleation sites for RNA folding. In this paper, we investigate the binding of neomycin, kanamycin, 2,4-diaminoquinazoline, quinacrine, and an aminoacridine derivative (AD1) to a GAAA tetraloop using fluorescence spectroscopy. We have found that AD1 and quinacrine bind to the GAAA tetraloop with the highest affinity of the molecules examined. The equilibrium dissociation constant of the AD1–GAAA tetraloop complex was determined to be 1.6 μM. RNase I and lead acetate footprinting experiments suggested that AD1 binds to the junction between the loop and stem of the GAAA tetraloop.

International audience; The proton-pumping and the ATP hydrolysis activities of the ATP synthase of Rhodobacter capsulatus have been compared as a function of the ADP and P(i) concentrations. The proton pumping was measured either with the transmembrane pH difference probe, 9-amino-6-chloro-2-methoxyacridine, or with the transmembrane electric potential difference probe, bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, obtaining consistent results. The comparison indicates that an intrinsic uncoupling of ATP synthase is induced when the concentration of either ligand is decreased. The half-maximal effect was found in the submicromolar range for ADP and at about 70 microM for P(i). It is proposed that a switch from a partially uncoupled state of ATP synthase to the coupled state is induced by the simultaneous binding of ADP and P(i).

C1311 (5-[[2-(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1-de]-acridin-6-one-dihydrochloride trihydrate) is the lead compound from the group of imidazoacridinones, a novel group of rationally designed anticancer agents. The pharmaceutical development of C1311 necessitated the availability of an assay for the quantification and purity determination of C1311 active pharmaceutical ingredient (API) and its pharmaceutical dosage form. A reversed-phase liquid chromatographic method (RP-LC) with ultraviolet (UV) detection was developed, consisting of separation on a C18 column with phosphate buffer (60 mM; pH 3 with 1 M citric acid)–acetonitrile–triethylamine (83:17:0.05, v/v/v) as the mobile phase and UV-detection at 280 nm. The method was found to be linear over a concentration range of 2.50–100 μg/mL, precise and accurate. Accelerated stress testing showed degradation products, which were well separated from the parent compound, confirming its stability-indicating capacity. Moreover, the use of LC–MS and on-line photo diode array detection enabled us to propose structures for four degradation products. Two of these products were also found as impurities in the API and more abundantly in an impure lot of API.

In a rotary motor F1F0-ATP synthase, F0 works as a proton motor; the oligomer ring of F0c-subunits (c-ring) rotates relative to the F0ab2 domain as protons pass through F0 down the gradient. F0ab2 must exert dual functions during rotation, that is, sliding the c-ring (motor drive) while keeping the association with the c-ring (anchor rail). Here we have isolated thermophilic F1F0(-a) which lacks F0a. F1F0(-a) has no proton transport activity, and F0(-a) does not work as a proton channel. Interestingly, ATPase activity of F1F0(-a) is greatly suppressed, even though its F1 sector is intact. Most likely, F0b2 associates with the c-ring as an anchor rail in the intact F1F0; without F0a, this association prevents rotation of the c-ring (and hence the gamma-subunit), which disables ATP hydrolysis at F1. Functional F1F0 is easily reconstituted from purified F0a and F1F0(-a), and thus F0a can bind to its proper location on F1F0(-a) without a large rearrangement of other-subunits.

We studied fluorescence of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride (tacrine) in the presence of serum cholinesterase. Quenching of tacrine fluorescence dependent on cholinesterase activity has been revealed. A quenching mechanism is proposed; it includes formation of a complex and charge transfer mediated by excited tacrine molecule as well as indole of tryptophan residue from the periphery of cholinesterase active site.

Microchip electrophoresis is a powerful tool for fast analysis of nucleic acids and has expanded its applicability to the analysis of various biological materials including proteins and carbohydrates. Glycosaminoglycans have intrinsic negative charges, and are good targets for electrophoretic analysis. In the present paper, we developed a method to analyze oligosaccharides and unsaturated disaccharides derived from some glycosaminoglycans after digestion with specific enzymes followed by derivatization with 2-aminoacrydone (AMAC) by reductive amination. The method described here allowed rapid analysis of oligosaccharides derived from glycosaminoglycans within 150 s with high sensitivity. We show an application of the present technique to the glycosaminoglycan analysis in cultured HeLa cells.

The fluorescent dye 10-N-nonyl acridine orange (NAO) is extensively used for location and quantitative assays of cardiolipin in living cells on the assumption of its high specificity for cardiolipin; however, the limits and the mechanism of this specificity are not clear. Moreover, whether factors such as the membrane potential in mitochondria may limit the consistency of the results obtained by this method is open to discussion. The aim of this research was to investigate the effects of some experimental factors on the selective fluorescence of NAO in the presence of cardiolipin in artificial and natural membranes (mitochondria). The results show that the fluorescence of NAO, due to interaction with cardiolipin, is significantly modified by factors that control the spatial arrangement of cardiolipin molecules within the space of the membrane under investigation. Moreover, the present observations suggest that the specific effect of cardiolipin is to facilitate the dimerization of this fluorescent dye, thus confirming that reliable measurements of cardiolipin concentration can be obtained only when the NAO/cardiolipin molar ratio is equal to 2. The finding is also reported that in isolated respiring mitochondria the interaction of NAO with cardiolipin is somewhat related to the respiratory state of mitochondria.

The imidazoacridinone derivative, C-1311, is an antitumor agent that has been under phase I of clinical trial. The work presented here aims to elucidate the molecular mechanism of the enzymatic oxidative activation of this drug in such a model metabolic system, where the covalent binding to DNA was previously demonstrated. The oxidative activation of C-1311 was performed with HRP/H(2)O(2) and MPO/H(2)O(2) systems. The obtained final products of such transformations were separated and analysed by HPLC. The structures of the products were identified by means of ESI-MS and NMR. It was demonstrated that C-1311 was oxidised with HRP and MPO in the manner dependent on the drug:H(2)O(2) ratio and the drug was more susceptible to HRP oxidation than to MPO. Structural studies showed compounds C0 and C1 to be the result of dealkylation, which occurred in the amino groups of the side chain. The structures of C3 and C4 products were identified as dimers, whose monomers held the imidazoacridinone core. The activation of the imidazoacridinone ring system in position ortho to 8-hydroxyl group was necessary to form such dimers. We suggest that similar mechanism of C-1311 activation should occur in the presence of DNA when, instead of the dimer formation, the covalent binding to DNA, showed earlier for this drug, was formed. Since peroxidase-type enzymes are present in the cell nucleus of tumour cells the activation mechanisms of the C-1311 proposed here may be expected to take place in the cellular environment in vivo.

The title compound exhibits a selective enhancement of its fluorescence intensity in the presence of AT-rich DNA.

An energy transfers technique between 4-(2-Pyridylazo) resorcinol(PAR) and acridine yellow is studied, and optimum experimental conditions of energy transfer is done as well. It is found that in the aqueous solution of sodium lauryl sulfate, NH3.H2O-NH4Cl buffer solution at pH = 9.2, the energy transfers from acridine yellow to iron(II)-PAR complexes. So a new method of fluorometric energy transfers determination of trace iron(II) by acridine yellow--PAR was described. The range of determination of iron is 0-10 micrograms.L-1. The detection limit for iron is 0.06 microgram.L-1. The method has been applied to the determination of iron in water sample and hair with satisfactory results.

The frameshift mutagenicity of 9-aminoacridine (9AA) was compared with that of quinacrine, the acridine mustards ICR-191 and quinacrine mustard (QM), and the nitroacridine Entozon in the lacZ reversion assay in Escherichia coli. As intercalating agents, 9AA and quinacrine cause mutations through noncovalent associations with DNA. Mustards and nitroacridines form covalent adducts in DNA and give rise to different spectra of mutations. Quinacrine and 9AA most effectively induced -1 frameshifts in a run of guanine residues, with 9AA being the more potent mutagen. They also induced +G frameshifts. The acridine mustard ICR-191 was a stronger mutagen than 9AA, owing largely to its potent induction of +G frameshifts. QM induced +G frameshifts more strongly than did its nonreactive counterpart quinacrine. The nitroacridine Entozon differed from the other acridines in being a potent inducer of -2 frameshifts, but it was less effective in inducing +/-1 frameshifts. Quinacrine, although a simple intercalator, induced all five kinds of frameshift mutations detected in the assay, as did the acridine mustards. Although +A and -A frameshifts were induced, adenine runs were less susceptible to acridine mutagenesis than guanine runs. The patterns of frameshift mutagenicity in the lacZ assay are similar to those in an assay based on the reversion of mutations in the tetracycline-resistance gene of the plasmid pBR322. The similarity suggests that the responses reflect the inherent bacterial mutagenicity of the compounds in the local sequence context and are not highly dependent on the broader sequence context. The results are interpreted with respect to slipped mispairing models of frameshift mutagenesis.

Ningguo Gao and Mark A. Lehrman Dept. Pharmacology UT-Southwestern Medical Center 5323 Harry Hines Boulevard Dallas, TX 75390-9041 # Contact information: (214)-648-2323; mark.lehrman@utsouthwestern.edu Fluorophore-assisted carbohydrate electrophoresis (FACE) is a simple, sensitive, and versatile method for analysis of both monosaccharides and oligosaccharides, and has become an important method in Glycobiology. Recently, the major provider of reagents and supplies for FACE has ceased production and shipping. To assist those who have relied upon the major provider for FACE reagents and supplies, the Editors have asked us to summarize alternative commercial sources and procedures that we have used (Gao and Lehrman, 2002a; Gao and Lehrman, 2002b). Our procedures are modified from those published previously (Jackson, P., 1994; Starr, C. M. et al., 1996).

3-Amino- and 3-alkylamino-4-hydroxymethylacridines bearing various substituents on the C ring have been prepared by regioselective electrophilic aromatic substitution of the corresponding 3-aminoacridines and ring opening of the dihydrooxazinoacridine key intermediates. Most of the new compounds show potent cytotoxic activities against murine L1210 (leukemia), human A549 (lung), and HT29 (colon) cancer cell lines. The most cytotoxic molecules, 1 and 13, are active at nanomolar concentrations. As predicted for acridine derivatives, the new compounds intercalate in DNA, but interestingly they do not interfere with topoisomerase I and II activities. The mode of action remains uncertain because intracellular distribution indicated very different behaviors for 1 and 13. Compound 13 is uniformly distributed in the cell both in the cytoplasm and in the nucleus, whereas compound 1 is essentially localized in cytoplasmic granules.

Here we show for the first time that the novel designed drug, Antoksyd S and its polyphenolic flavones (baicalin and baicalein), act as cell proliferation modifiers of mouse leukemia cells (L1210). The cytotoxicity of Antoksyd S and baicalein in vitro was expressed as ED50 and compared with those of the cytostatics doxorubicin, cisplatin and DACA, under the same experimental conditions. Cell viability was determined by modified tetrazolium dye assay, using as a model cells with neoblastic phenotype (L1210). The antiradical activity of Antoksyd S and baicalin were investigated using the DPPH test in order to obtain their antioxidant characteristics. Structure- and concentration-dependent one electron bioactivations (peroxidative oxidation) of Antoksyd S and baicalin were performed in the absence or in the presence of GSH or SOD. It appears that Antoksyd S is a low toxic novel drug which could be effective in providing concentration-dependent antioxidative activity, acting as a cell proliferation modifier and, probably, as an apoptosis inducer in vitro, though this remains to be explored. These findings are discussed from a mechanistic standpoint as well as in terms of potential pharmacological applications under acute oxidative stress and apoptotic events. This work provides the basis for further investigations of Antoksyd S action in vitro and in vivo, since the presented results are indicative of its intracellular metabolic activation, which can only be partially associated with its observed action towards model cancer cells (mouse leukemia L1210).

Fluorophore-assisted carbohydrate-electrophoresis (FACE) was developed as a bioassay for environmental stressors in larval Chironomus riparius. This quantitative technique involved acid hydrolysis and 2-aminoacridone labeling of monosaccharides followed by carbohydrate gel electrophoresis. Methods for carbohydrate isolation from whole tissue homogenates as well as migration distances for 23 different monosaccharides and 4 disaccharides were established. Sensitivity of the technique (5 microg/ml saccharide) exceeded those of other detection methods. Results showed seven distinct bands in larvae. Four migrated distances similar to those of ribose, glucose, galactose, and fructose. Three proved to be alternative reaction products (ARP). Experiments determined that two of the ARPs were primarily from glucose and one was from glycogen. FACE allowed different saccharides from multiple larval samples to be analyzed in parallel. Effects of toxicants and diet on bioenergetics could be studied using this technique.

9-Aminoacridines and (terpyridine)platinum(II) complexes are competitive and irreversible inhibitors, respectively, of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. Four chimeric compounds in which 2-methoxy-6-chloro-9-aminoacridine was covalently linked to the (2-hydroxyethanethiolate)(2,2':6',2' '-terpyridine)platinum(II) complex were synthesized and studied as inhibitors of the parasite enzyme. The derivatives differed by the nature and/or the length of the spacer connecting the two aromatic systems. All four compounds were effective mixed type inhibitors of trypanothione reductase with K(i) and K(i)' values of 0.3-4 and 2-11 microM, respectively. The most potent inhibitor had an ethylthioether linkage between the two aromatic ring systems, and the other compounds contained an alkyl ether group with 4-6 methylene groups. In contrast to the parasite enzyme, human glutathione reductase, the closest related host enzyme was not inhibited by these compounds. The finding that the conjugation of a competitive and an irreversible inhibitor can give rise to reversible mixed type inhibitors underlines the difficulties associated with inhibitor design based on the three-dimensional structure of trypanothione reductase.

We report the application of capillary electrophoresis (CE) with postcolumn laser-induced fluorescence (LIF) detection to measure the cardiolipin content of individual mitochondria from cultured NS1 cells. Mitochondria were isolated by differential centrifugation and stained with the fluorescent dye 10-N-nonyl acridine orange which stoichiometrically binds to cardiolipin in a 1:1 or 2:1 ratio depending on the dye concentration. The green fluorescence resulting from the 1:1 complex was chosen for analysis because it is substantially more intense than the red fluorescence resulting from the 2:1 complex. Two dye concentrations that resulted in maximal and submaximal formation of the 1:1 10-N-nonyl acridine orange-cardiolipin complex were identified by spectrofluorometry. Individual mitochondria stained with both dye concentrations were separated and detected by CE with LIF detection. The data from mitochondria dosed with the lower dye concentration, where it is assumed that all the dye added to the mitochondrial sample was bound to cardiolipin, were used to derive a sensitivity factor relating fluorescence intensity of a mitochondrial event to its cardiolipin content. Using this factor, the cardiolipin contents of individual mitochondria stained with the higher dye concentration were determined, and ranged from 1.2 to 920 amol, with a median value of 4 amol. These results suggest a new strategy for estimating the organellar content of compounds that can be fluorescently tagged.

In quest for high sensitivities, we developed an ultrahigh capillary electrophoresis (CE) method for the structural analysis of heparin and heparan sulfate (HS) in biologic samples. Heparin and HS were digested with an equi-unit mixture of heparin lyases I, II and III and the obtained Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone. All known twelve non-, mono-, di- and trisulfated Delta-disaccharides were completely resolved in a single run, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV. Relative standard deviation in migration times and peak areas as well as day-to-day variance ranged from 0.9 to 2.4%, suggesting a reproducible and precise method. Detection of 2-aminoacridone (AMAC)-derivatives of Delta-disaccharides by UV at 255 nm showed 2.8 and 10 times higher sensitivity than that of derivatized and non-derivatized ones at 232 nm. Laser-induced fluorescence detection with an Ar-ion laser source showed an approximately 100 times higher sensitivity than that obtained at 232 nm of the non-derivatized species. Application of this method to quantitative analysis of Delta-disaccharides derived from porcine intestinal mucosa heparin and bovine kidney HS showed excellent agreement with previously published methods, suggesting an accurate method. The developed method can be easily applied for the disaccharide analysis of heparin/HS at the attomole level with high accuracy, for distinguishing between heparin and HS and may be of value for studying their interactions with matrix effective molecules.

A flow-injection configuration for the determination of formaldehyde is proposed. The method is based on the enhancing effect of formaldehyde on the oxidation of acridine yellow by bromate in acidic medium. The proposed procedure is simple, inexpensive, sensitive and suitable for concentrations of formaldehyde between 1 and 56 microg mL(-1). A sampling-rate of 60 samples h(-1) was achieved. The effect of several organic and inorganic species was studied. The method was applied to the determination of formaldehyde in pharmaceuticals, milk and air in work environments. The accuracy of the method was confirmed by comparing the results with those obtained using the standard acetylacetone method.

The aim of this study was to investigate the influence of reactive oxygen species (ROS) on the activity of complex I and on the cardiolipin content in bovine heart submitochondrial particles (SMP). ROS were generated through the use of xanthine/xanthine oxidase (X/XO) system. Treatment of SMP with X/XO resulted in a large production of superoxide anion, detected by acetylated cytochrome c method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to ROS generation resulted in a marked loss of complex I activity and to parallel loss of mitochondrial cardiolipin content. Both these effects were completely abolished by SOD+catalase. Exogenous added cardiolipin was able to almost completely restore the ROS-induced loss of complex I activity. No restoration was obtained with other major phospholipid components of the mitochondrial membrane such as phosphatidylcholine and phosphatidylethanolamine, nor with peroxidized cardiolipin. These results demonstrate that ROS affect the mitochondrial complex I activity via oxidative damage of cardiolipin which is required for the functioning of this multisubunit enzyme complex. These results may prove useful in probing molecular mechanisms of ROS-induced peroxidative damage to mitochondria, which have been proposed to contribute to those pathophysiological conditions characterized by an increase in the basal production of reactive oxygen species such as aging, ischemia/reperfusion and chronic degenerative diseases.

Background Bioreduction of water-soluble tetrazolium salts (e.g., MTS, XTT, and MTT) to their respective formazans is generally regarded as an indicator of cell “redox activity.” The reaction is attributed mainly to mitochondrial enzymes and electron carriers. However, MTT reduction may also be catalyzed by a number of other nonmitochondrial enzymes. The goal of this work was to establish the sites of MTT reduction in intact HepG2 human hepatoma cells in culture. Methods In order to establish the subcellular localization of the sites of reduction of MTT, we imaged the formation of MTT-formazan deposits using backscattered light confocal microscopy. Mitochondria were visualized in viable cells using fluorescent dyes that bind in a manner dependent (JC-1 and TMRE) or independent (NAO) of mitochondrial electric potential. Results Only 25–45% of MTT-formazan was associated with mitochondria after 25 min of incubation. No more than 25% of the mitochondrial area on images was occupied by MTT-formazan. Mitochondrial fluorescence of TMRE, NAO, and the monomeric form of JC-1 decreased rapidly in cells incubated with MTT. However, the intensity of fluorescence of JC-1 aggregates dropped by less than 30% at the onset of incubation and remained constant as reduction of MTT proceeded further. Conclusions (1) Most of MTT-formazan deposits are not coincident with mitochondria. (2) Monomeric JC-1, as well as TMRE and NAO, accumulating in mitochondria may be displaced by MTT. Thus, the presence of positively charged organic compounds (like MTT) may distort measurements of mitochondrial transmembrane electric potential, which are based on accumulation of fluorescent dyes. Cytometry 47:236–242, 2002. © 2002 Wiley-Liss, Inc.

The recent investigation of the apparently anomalous attenuation factor (beta1.5 A(-1)) for photoinduced hole injection into DNA duplexes modified by protonated 9-amino-6-chloro-2-methoxyacridine (X+) led to the conclusion that in addition to the electronic couplings, the activation energy must also be distance-dependent. In this communication we report the verification of this postulate by direct measurements of the activation energies for a series of (X+)-modified DNA duplexes which sample an appreciable range of donor-acceptor distances (approximately 4-11 A). The resulting changes in thermal activation energy can be explained within the framework of a distance-dependent reorganization energy.

To evaluate the antitumor efficacy against metastatic breast cancer of fluoropyrimidines alone and combined with other chemotherapeutic agents, we developed a murine model of breast cancer metastatic to the lung by orthotopically implanting MDA-MB-435S breast tumors into mice. MDA tumor cells greatly metastasized to lung tissue only after the orthotopically implanted tumors were surgically removed. Measurement of the expression of enzymes involved in 5-FU metabolism showed significantly higher activity of dihydropyrimidine dehydrogenase (DPD) and lower activity of thymidylate synthase (TS) in the MDA metastases than in the orthotopically implanted tumors. Based on the enzymatic properties of metastatic tumors, the minimum toxic doses of UFT (17.5 mg/kg/day) as a DPD-inhibitory fluoropyrimidine (DIF), and of 5'-DFUR (120 mg/kg/day) as a non-DIF, were orally administered to mice with pulmonary metastasis of the breast tumor. The results showed that UFT significantly inhibited the growth of pulmonary metastases of the breast tumors, but 5'-DFUR did not. UFT seemed to inhibit the growth of the pulmonary metastases of the breast tumors in combination with paclitaxel (50 mg/kg) more than in combination with 5'-DFUR, although the antitumor efficacy of neither combination was significantly different from that of paclitaxel alone. These results suggest that combination of DIF with other chemotherapeutic drugs, such as taxanes, is required to attain high antimetastatic and antitumor efficacy against breast tumor metastases, based on the molecular characteristics of the metastatic tumors.

N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (AC) is an experimental antitumour agent that is being considered for phase I trials. After i.p. administration of 150 mg/kg [3H]-AC to tumour-bearing mice, AC was absorbed rapidly into the plasma and tissues such as the heart, liver, kidney and brain but more slowly into the s.c. tumour. The maximal AC concentration (86±36 μmol/kg) in the tumour occurred at 35–60 min and was 3-fold the maximal plasma concentration, which occurred at 15 min. Although higher maximal concentrations were observed in other tissues, these concentrations fell rapidly in parallel with plasma concentrations. In contrast, AC concentrations in the tumour remained elevated, thet1/2 value (16.3 h) and mean residence time (MRT, 9.5 h) being prolonged in comparison with those in the plasma and other tissues (t1/2 range, 1.0–2.9 h; MRT, 1.2–1.4 h). AC concentrations were not detectable by our high-performance liquid chromatographic (HPLC) method (limit of detection, 0.02 μmol/l) in the plasma or other tissues at 24 or 48 h after administration but were measurable in the tumour (1.6±0.8 and 0.6±0.3 μmol/kg, respectively). Radioactivity concentrations in the plasma, tissues and tumour were very variable but were greater than the corresponding levels of unchanged parent AC. By 24 h, radioactivity concentrations in the plasma, tissues and tumour had fallen to similar levels with prolonged elimination profiles. Thus, the exposure of the s.c. implanted tumour to a threshold AC concentration for a prolonged time (>24 h) may partly explain the greater efficacy of AC against this tumour, whereas the shorter period of exposure of blood and other tissues may explain its low haematological toxicity.

The pharmacokinetics of N-[2-(dimethyl-amino)ethyl]acridine-4- carboxamide (AC) were investigated in rats after i.v. administration of 18, 55 and 81 mumol/kg [3H]-AC. The plasma concentration-time profiles of AC (as measured by high-performance liquid chromatography) typically exhibited biphasic elimination kinetics over the 8-h post-administration period. Over this dose range, AC's kinetics were first-order. The mean (+/- SD) model-independent pharmacokinetic parameters were: clearance (Cl), 5.3 +/- 1.1 1 h-1 kg-1; steady-state volume of distribution (Vss), 7.8 +/- 3.0 l/kg; mean residence time (MRT), 1.5 +/- 0.4 h; and terminal elimination half-life (t1/2Z), 2.1 +/- 0.7 h (n = 10). The radioactivity levels (expressed as AC equivalents) in plasma were 1.3 times the AC concentrations recorded at 2 min (the first time point) and remained relatively constant for 1-8 h after AC administration. By 6 h, plasma radioactivity concentrations were 20 times greater than AC levels. Taking into account the species differences in the unbound AC fraction in plasma (mouse, 16.3%; rat, 14.8%; human, 3.4%), allometric equations were developed from rat and mouse pharmacokinetic data that predicted a Cl value of 0.075 (range, 0.05-0.10; 95% confidence limits) 1 h-1 kg-1 and a Vss value of 0.63 (range, 0.2-1.1) l/kg for total drug concentrations in humans.