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At least 631 records · Page 35Linked to original sources
PubMed1987

The toxicity and mutagenicity of the anti-tumour drug 5-aziridino-2,4-dinitrobenzamide (CB1954) is greatly reduced in a nitroreductase-deficient strain of E. coli.

In the 1970s it was shown that the monofunctional alkylating agent CB1954 (5-aziridino-2,4-dinitrobenzamide) kills the Walker rat carcinoma 256 in vivo and in vitro, but is inactive against several other tumours. In studies with bacteria, the large differences in survival in DNA-repair-proficient and deficient strains of Escherichia coli B treated with CB1954 were characteristic of a difunctional cross-linking agent. It was concluded that DNA was the only target large enough to receive significantly more than one lethal hit per molecule and that mono-alkylation alone could not account for the lethal effects of CB1954. In 1986 it was shown that CB1954 induced DNA interstrand cross-links in CB1954-sensitive cultured Walker 256 cells, but not in resistant Chinese hamster V79 cells, suggesting that the sensitivity of Walker cells results mainly from their activation of the drug to a difunctional agent by nitroreduction. To test this, we assayed the toxicity and mutagenicity of CB1954 in nitroreductase-plus and -minus strains of E. coli WP2uvrA. Agar-overlay assays showed that CB1954 was mutagenic to several strains of E. coli WP2, in a dose range 1-100 micrograms per plate, with slopes (mutants/nmol) of 7.1, 1.05 and 0.16 for WP2uvrA pKM101, WP2uvrA and WP2. Assays with nitrofurazone showed that these strains possessed nitroreductase activity. However, E. coli NFR-343, a nitrofurazone-resistant mutant of WP2uvrA which lacks nitroreductase activity was markedly less sensitive to the mutagenicity of CB1954, giving a mean slope of 0.12 compared with 1.15 for WP2uvrA. Aroclor-induced rat-liver S9 did not change these responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1996

A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase.

In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2',3'-dideoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl) styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([gamma-32P]TDSddUTP) and poly(A).oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3'-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.

Affinity Labels↗
PubMed1997

Synthesis and evaluation of oligodeoxyribonucleotides containing an aryl(trifluoromethyl)diazirine moiety as the cross-linking probe: photoaffinity labeling of mammalian DNA polymerase beta.

Photolabile 2'-deoxy- E -5-[4-(3-trifluoromethyl-3 H-diazirin-3-yl)styryl]uridine and its protected phosphoramidite derivatives have been synthesized and introduced into DNA oligomers through solid-phase DNA synthesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In addition, this moiety was found to be stable at 60 degrees C in aqueous solution under the annealing conditions for duplex formation with complementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolabile residue was activated/decomposed by near-UV irradiation. In photoaffinity cross-linking experiments with recombinant rat DNA polymerasebeta, constituted from a 40 kDa polypeptide, using oligo(dT) 15mer analogs bearing the photolabile residue near the 3'-terminus, a covalently bound complex of 45 kDa was obtained in the presence of complementary templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DNA-related enzymes and DNA binding proteins.

Animals↗
PubMed2000

Assembly of archaeal signal recognition particle from recombinant components.

Signal recognition particle (SRP) takes part in protein targeting and secretion in all organisms. Searches for components of archaeal SRP in primary databases and completed genomes indicated that archaea possess only homologs of SRP RNA, and proteins SRP19 and SRP54. A recombinant SRP was assembled from cloned, expressed and purified components of the hyperthermophilic archaeon Archaeoglobus fulgidus. Recombinant Af-SRP54 associated with the signal peptide of bovine pre-prolactin translated in vitro. As in mammalian SRP, Af-SRP54 binding to Af-SRP RNA required protein Af-SRP19, although notable amounts bound in absence of Af-SRP19. Archaeoglobus fulgidus SRP proteins also bound to full-length SRP RNA of the archaeon Methanococcus jannaschii, to eukaryotic human SRP RNA, and to truncated versions which corresponded to the large domain of SRP. Dependence on SRP19 was most pronounced with components from the same species. Reconstitutions with heterologous components revealed a significant potential of human SRP proteins to bind to archaeal SRP RNAs. Surprisingly, M.jannaschii SRP RNA bound to human SRP54M quantitatively in the absence of SRP19. This is the first report of reconstitution of an archaeal SRP from recombinantly expressed purified components. The results highlight structural and functional conservation of SRP assembly between archaea and eucarya.

Animals↗
PubMed1989

Interaction of human apolipoprotein A-I with dipalmitoylphosphatidylcholine in vesicular and micellar complexes.

The interactions of human apolipoprotein A-I (apo A-I) with dipalmitoylphosphatidylcholine (DPPC) in vesicular complexes at low protein concentrations and in micellar complexes at high protein concentrations are compared. The C-terminal segment of this protein, with a relative molecular weight (Mr) of about 11,000, is protected on trypsin treatment of apo A-I-vesicle complexes. A segment within the sequence from Leu-189 to Arg-215 of apo A-I penetrates the hydrophobic interior of the membrane, as found in a hydrophobic labeling experiment involving 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID). No appreciable stretch of apo A-I in micellar complexes was found to be protected from the tryptic digestion. This indicates that the interactions of apo A-I with lipids in the vesicular and micellar complexes are different. The binding equilibrium of apo A-I as to DPPC vesicles at low protein concentrations, as studied by hydrophobic labeling of the bilayer-penetrating segment, is reached within about 1 h, while the formation of micellar complexes at high protein concentrations takes about 24 h at 42 degrees C. Time-dependent labeling studies involving photoreactive phosphatidylcholine (PC) with high apo A-I concentrations suggested an initial interaction with the head group region of the bilayer followed by interaction with the tail ends of the acyl chains of the lipid. A possible mechanism for the micellization process is discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Heterogeneity of dystrophin-associated proteins.

The proteins which compose the complex of dystrophin and its associated proteins were analyzed by two-dimensional PAGE, i.e., electrofocusing in the presence of 8 M urea followed by SDS PAGE. Silver-staining of the gel showed many more spots than were expected from the results of one-dimensional SDS PAGE. By examination of their reactivity with specific antibodies, various lectins and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine, most of these spots were identified as dystrophin and its associated proteins described previously. Several as yet unidentified minor proteins were also detected. Dystrophin-associated protein A1 was separated into two groups, alpha-A1 and beta-A1, both composed of numerous spots. These groups differed from each other in isoelectric point, molecular mass, and reactivity with antibodies. The beta-A1 group (64 kDa) was more basic than the alpha-A1 group (60 kDa). Beta-A1 but not alpha-A1 reacted with several lectins, indicating that beta-A1 is a glycoprotein. This is incompatible with the report that 59DAG, which corresponds to A1 (alpha-A1 + beta-A1), is not a glycoprotein [Ervasti et al. (1991) Cell 66, 1121-1131]. The charge heterogeneity observed in alpha-A1 and beta-A1 may be partially due to differential phosphorylation. The charge heterogeneity of A2 and A3a may, at least to some extent, be due to differential sialilation of their carbohydrates.

Alkaline Phosphatase↗
PubMed1980

Comparison of the kinetic properties of troponin-C and dansylaziridine-labeled troponin-C1.

Comparison of the kinetic properties of troponin-C and dansylaziridine-labeled troponin-C revealed that the calcium ion binding and removal reactions with the low affinity Ca2+-binding sites (sites I and II) and the resultant local conformational change are rapid processes, and that the calcium ion binding and removal reactions with the high affinity Ca2+-binding sites (sites III and IV) are also rapid, whereas the resultant local conformational change is a slow process. It is also shown in this work that the latter processes are independent of the former processes.

Animals↗