Intercalation of water-soluble bis-porphyrins into poly(dA)-poly(dT) double helix.

The association constants (K) of nucleic acid monomers with a series of water-soluble bis-porphyrins (bisMC1, bisMC3, bisMC5, bisMC7, and bisMC11) in which two porphyrin units were linked by a methylene chain of various lengths were estimated spectrophotometrically. Among the bis-porphyrins, the K values are similar for each nucleic acid monomer, indicating that the bridging chain length does not affect the association of the bis-porphyrins with the nucleic acid monomers. The melting curves of poly(dA)-poly(dT) in the presence of bisMC3 or bisMC5 were found to be biphasic, suggesting that bisMC3 and bisMC5 are bound to poly(dA)-poly(dT) with a binding mode different from the groove binding exhibited by the corresponding porphyrin monomers. A negative-induced CD peak in the Soret region of bisMC3 and bisMC5 with poly(dA)-poly(dT) is observed and the visible spectral changes of bisMC3 and bisMC5 upon addition of poly(dA)-poly(dT) are accompanied by a large red shift of the Soret band (bisMC3: 21 nm, bisMC5: 23 nm) with substantial hypochromicity (bisMC3: 49%, bisMC5: 40%). Therefore, it is reasonable to conclude that both of the porphyrin units of bisMC3 and bisMC5 intercalate into poly(dA)-poly(dT). In contrast to poly(dA)-poly(dT), the melting curves of poly(dA.dT)(2) in the presence of the bis-porphyrins did not show such biphasic behavior. Together with the CD and visible absorption data, it is certain that these bis-porphyrins do not intercalate into poly(dA.dT)(2).

Synthesis and nucleic acid-binding properties of water-soluble porphyrins appending platinum(II) complexes.

We synthesized two water-soluble porphyrins appending platinum(II) complexes [alpha,beta-(4a) and alpha,alpha-(4b) 5,15-bis(2-trans-[PtCl(NH3)2]N-2-aminoethylaminocarbonylphenyl) 2,3,7,8,12,13,17,18-octamethylporphyrin] and studied their reactions with a variety of nucleic acids [disodium adenosine-5'-monophosphate (AMP), disodium guanosine-5'-monophosphate (GMP), disodium thymidine-5'-monophosphate (TMP), disodium cytidine-5'-monophosphate (CMP), synthetic polymer poly(dG)-poly(dC), poly(dA)-poly(dT)] by 1H-NMR, UV-vis and FAB-MS spectroscopies. Based on the denaturation experiments of synthetic nucleic acid polymers, we conclude that the presence of the porphyrins (5.6 microM) does not cause significant changes in the melting temperature of poly(dA)-poly(dT) (28 microM) (deltaT=1 degrees C) and shows reannealing. On the other hand, gradual melting of poly(dG)-poly(dC) (28 microM) occurs at a low temperature (deltaT= -27 degrees C) in the presence of the porphyrins (5.6 microM), and the solutions do not show reannealing phenomena. The results of UV-vis and 1H-NMR experiments revealed that the porphyrins bind to guanine bases and that the porphyrins bind to GMP more strongly than to the other nucleotides. The binding modes between the porphyrins and synthetic nucleic acids are affected more by the coordination of the nucleobase [poly(dG)-poly(dC)] to the Pt(II) in the porphyrins than by Coulomb and hydrophobic interactions.

A marked stimulation of Fe2+-initiated lipid peroxidation in phospholipid liposomes by a lipophilic aluminum complex, aluminum acetylacetonate.

In the present study, the efficacy of a lipophilic Al complex, aluminum acetylacetonate, as a stimulator of Fe2+-initiated lipid peroxidation in phospholipid liposomes was examined, and results were compared with those from the liposomes treated with AlCl3. The extent of lipid peroxidation was assessed by the formation of thiobarbituric acid-reactive substances (TBARS). The results indicated that the stimulatory effect of Al complex on Fe2+-initiated lipid peroxidation in phosphatidylcholine liposomes was more effective than that of AlCl3 under the same conditions. The concentration dependence of Al complex on TBARS production showed that the concentration of the complex required to induce half-maximal stimulation of TBARS production was 43 microM. In contrast, the stimulatory effect of AlCl3 was not observed until the AlCl3 concentration is increased above 300 microM. In addition, it was found that there is a linear relationship between the TBARS values and the residual amounts of Fe2+ at an earlier stage (within 2 min after the addition of Fe2+) of the lipid peroxidation in PC liposomes with different concentrations of Al complex, suggesting that Fe2+ oxidation process is closely related to the stimulatory effect of Al complex. The stimulatory effect of Al complex upon the lipid peroxidation completely disappeared by treatment of Al complex-treated liposomes with Triton X-100. The results of fluorescence anisotropy measurements using 12-(9-anthroyloxy)stearic acid-labeled liposomes suggested that treatment of the liposomes with Al complex caused a decrease in their lipid fluidity. Furthermore, it was found that there is a correlation between the extents of the fluorescence anisotropy and the Fe2+ oxidation parameters in the liposomes with different concentrations of Al complex. From these results, it is suggested that the Al effect on Fe2+-initiated lipid peroxidation in the phospholipid liposomes is markedly enhanced by incorporation of Al complex into the liposomal membranes and that an acceleration of Fe2+ oxidation due to a strengthened packing between the acyl chains in the lipid layer may be one possible mechanism for the occurrence of a marked stimulatory effect of Al complex on Fe2+ initiated lipid peroxidation.

A 31-kDa recombinant fibronectin cell-binding domain fragment: its binding to receptor, cell adhesive activity, and fusion proteins.

The binding of fibronectin to fibronectin receptor was studied using a recombinant 31-kDa cell-binding domain fragment of fibronectin (C279), which consisted of three type III repeats (III8-III9-III10). Fibronectin receptor in several cell lysates was bound to a column of C279-immobilized Sepharose HP and obtained in a highly purified form by elution with a synthetic peptide, GRGDSP. alpha 5 beta 1-Integrin was detected in the GRGDSP-eluted fraction by immunoblotting. The cell-adhesive activity of C279 was inhibited by GRGDSP peptide, an anti-integrin a5 subunit antibody, and an anti-integrin beta 1 subunit antibody. The cell adhesion of fusion proteins of the 31-kDa fragment with biologically interesting polypeptides (heparin-binding domain of fibronectin, and basic fibroblast growth factor) was also studied. In the presence of an anti-integrin a5 subunit antibody, human fibrosarcoma HT-1080 cells attached to the fusion protein containing fibroblast growth factor, giving rise to changes the morphology of the attached cells. The cell adhesion of C279 was inhibited by GRGDSP peptide but that of the fusion protein with the heparin-binding domain of fibronectin was not completely inhibited by the peptide. These results suggest that these biologically interesting polypeptides contribute to the cell adhesion of the fusion proteins.