Biochemistry
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In this study, we have mapped the intracellular alkylation sites of adozelesin and bizelesin, two potent analogs of CC-1065, in individual genes at the single-nucleotide level. Human colon carcinoma cells were treated with adozelesin and bizelesin, and the position of adducts were mapped within the PGK-1 and p53 genes by means of ligation-mediated polymerase chain reaction. The monofunctional alkylating agent adozelesin was found to alkylate genomic DNA predominantly within 5'-(A/T)(A/T)A* sequences. ⋯ Both six- and seven-nucleotide regions were identified as putative sites of DNA interstrand cross-link formation with 5'-TTTTTTA*, 5'-TTTATCA* and 5'-GTACTAA* sequences being preferred. Non-adenine bases are not observed as potential intracellular sites of either DNA interstrand cross-linking formation or monofunctional alkylation. Thus, the patterns of alkylation induced by adozelesin and bizelesin in genomic DNA are similar but not identical to that observed in purified cell-free DNA.
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Comparative Study
Biologically active Arg-Gly-Asp oligopeptides assume a type II beta-turn in solution.
The sequence Arg-Gly-Asp (RGD) has been found to be the consensus sequence of matrix proteins for binding cell surface receptors (integrins). Studies with synthetic peptides containing the RGD sequence show that the biological activity of these oligopeptides is removed upon a conservative substitution of Glu for Asp in the RGD sequence. ⋯ In contrast, the sequence Tyr-Gly-Arg-Gly-Glu-Ser-Pro, which does not interfere with integrin-mediated cell attachment, is found to assume a type I or III beta-turn at both pH 4 and 7. This comparison confirms not only that oligopeptides can assume a secondary structure in aqueous solution, but also that these structures may be important to biological functions.
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The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. ⋯ The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.
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Covalent DNA adducts of the antitumor antibiotic CC-1065 and its analogues undergo a retrohomologous Michael reaction in aqueous/organic solvent mixtures to regenerate the initial cyclopropylpyrroloindole (CPI) structure and, presumably, intact DNA. This reaction, which at higher temperatures competes with depurination of the N3-alkylated adenine, also occurs to a significant extent at 37 degrees C in neutral aqueous solution. Tritium-labeled adozelesin, covalently bonded to a 3-kilobase DNA restriction fragment which was exhaustively extracted to remove unbonded drug, was efficiently transferred to a 1-kilobase fragment upon coincubation for 20 h at 37 degrees C in aqueous buffer. Covalent adducts of adozelesin, but not CC-1065, on calf thymus DNA were cytotoxic to L1210 cells after incubation for 3 days at 37 degrees C, indicating that reversal of DNA alkylation can mediate potent cellular effects for simplified CC-1065 analogues.
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CC-1065, a cyclopropylpyrroloindole (CPI), is a highly potent antitumor DNA-alkylating agent. We have devised a simple method to detect CPI bonding sites on double-stranded DNA (dsDNA). The technique utilizes a modified form of bacteriophage T7 polymerase, Sequenase, to synthesize a radiolabeled nascent strand from dsDNA that has been reacted in vitro with the CC-1065 analogue U-73975 (adozelesin). ⋯ Noncanonical sites were also found at poly(A) sites. This technique yielded a consensus sequence for U-73975 bonding that is similar to, but not identical with, the published consensus obtained for CC-1065 by a modified Maxam and Gilbert sequencing technique. We have also examined the bonding of [3H]U-73975 to the DNA of viable cultured mammalian cells, using gel electrophoresis and autoradiographic techniques.(ABSTRACT TRUNCATED AT 250 WORDS)