Uptake of acridinecarboxamide derivatives by L1210 cells. (1/161)

The uptake of six 9-aminoacridinecarboxamide derivatives by L1210 cells in relation to their lipophilicity and cytotoxic activity was studied. The amount of acridines taken up by cells was estimated by fluorimetric measurements. It was found that the uptake efficiency of this class of compounds by cells depends on the size of carboxamide residue as well as on position of the substituent. The increase of size of carboxamide chain resulted in the loss of capability of acridines to penetrate cell membrane. Cytotoxic effects of acridines were well correlated with the level of drugs accumulated by cells, whereas no clear correlation between uptake and lipophilicity was observed. It is concluded that uptake of 9-aminoacridinecarboxamides is the most important factor determining their antiproliferative activity.  (+info)

F0 complex of the Escherichia coli ATP synthase. Not all monomers of the subunit c oligomer are involved in F1 interaction. (2/161)

The antigenic determinants of mAbs against subunit c of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic heptapeptides. All epitopes recognized are located in the hydrophilic loop region and are as follows: 31-LGGKFLE-37, 35-FLEGAAR-41, 36-LEGAAR-41 and 36-LEGAARQ-42. Binding studies with membrane vesicles of different orientation revealed that all mAbs bind to everted membrane vesicles independent of the presence or absence of the F1 part. Although the hydrophilic region of subunit c and particularly the highly conserved residues A40, R41, Q42 and P43 are known to interact with subunits gamma and epsilon of the F1 part, the mAb molecules have no effect on the function of F0. Furthermore, it could be demonstrated that the F1 part and the mAb molecule(s) are bound simultaneously to the F0 complex suggesting that not all c subunits are involved in F1 interaction. From the results obtained, it can be concluded that this interaction is fixed, which means that subunits gamma and epsilon do not switch between the c subunits during catalysis and furthermore, a complete rotation of the subunit c oligomer modified with mAb(s) along the stator of the F1F0 complex, proposed to be composed of at least subunits b and delta, seems to be unlikely.  (+info)

Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311. (3/161)

C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy.  (+info)

Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a. (4/161)

We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of succinate-sustained ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the succinate-sustained ATP synthesis rate of the particles was found, but both the ATP hydrolysis rate and ATP-driven proton translocation (when the protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human F(1)F(0)-ATPase that causes a severe impairment of ATP synthesis. This is possibly due to a defect in either the vectorial proton transport from the cytosol to the mitochondrial matrix or the coupling of proton flow through F(0) to ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired ATP synthesis. On the other hand, ATP hydrolysis that involves proton flow from the matrix to the cytosol is essentially unaffected.  (+info)

Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors. (5/161)

We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4-tetrahydro-N-(phenylmethyl)-9-acridinamine and 1,2,3,4-tetrahydro-N-(3-iodophenyl-methyl)-9-acridinamine--all three at 2.7 A resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active-site gorge, but some of the surface loops deviate by up to 8 A from their position in the vertebrate structures, and the C-terminal helix is shifted substantially. The active-site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is approximately 50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active-site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three-dimensional structures.  (+info)

A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)(2). (6/161)

The structures of the complexes formed between 9-amino-[N:-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG(5Br)UACG)(2) and d(CGTACG)(2) have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 A. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).  (+info)

Acridine-a neglected antibacterial chromophore. (7/161)

The use of acridines as antimicrobial agents was first proposed by Ehrlich and Benda in 1912, and the first clinical use of these agents occurred in 1917. Many compounds containing the acridine chromophore were synthesized and tested, and the aminoacridines found wide use, both as antibacterial agents and as antimalarials, during World War II. The emergence of the penicillins eclipsed the acridines in antisepsis due to the greater therapeutic efficacies of the former. However, with the current massive increases in drug-resistant bacterial infection, new acridine derivatives may be of use. In addition, the topical utilization of aminoacridines in conjunction with directed low-power light offers bactericidal action at much lower doses.  (+info)

Protein-free parallel triple-stranded DNA complex formation. (8/161)

A 14 nt DNA sequence 5'-AGAATGTGGCAAAG-3' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5'-end of the probe strand as donor and BODIPY-Texas Red on the 3'-amino group of either strand of the target duplex as acceptor. There was full protection from OsO(4)-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe-intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.  (+info)

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