L. Masotti

Lipid peroxidation in tumour cells.

Several studies point to the existence of a disturbance in the metabolism of the reactive species of oxygen in cancer cells. Based on this evidence, and in particular on a characteristic behaviour of tumour membrane lipids, namely their growth-related resistance to oxy-radical-induced peroxidation, a sequence of events is outlined that could hypothetically drive the transformed cell to an uncontrolled proliferation. The proposed scheme is also conceived as a framework for further in vivo investigations of the complex biological phenomena of tumour cell growth and invasion in more integrated and kinetically controlled cellular systems.

Lipid peroxidation and fluidity of plasma membranes from rat liver and Morris hepatoma 3924A

Plasma membranes isolated from the fast‐growing, maximal‐deviation, Morris hepatoma 3924A exhibit remarkable changes in lipid composition, lipid peroxidation and to some extent in the physical state with respect to rat liver plasmalemmas. A correlation appears to exit between the lower phospholipid: protein ratio, higher cholesterol: phospholipid ratio, lower rate of lipid peroxidation and decrease in fluidity in tumor plasma membranes.

Conformational studies on the gramicidin A transmembrane channel in lipid micelles and liposomes

The interaction of gramicidin A with lysolecithin micelles and with lecithin liposomes is demonstrated by circular dichroism to result in several metastable conformational states. A stable state can be obtained after extensive heating when the gramicidin A was added dry or in ethanol solution to the phospholipid dispersion but the stable state is readily obtained when gramicidin A is added in a trifluoroethanol solution. The circular dichroism of the stable conformational state is characterized by negative ellipticity below 205 nm and principally by a positive 220 nm band on which is superposed a weak 230 nm band (the latter likely arising from tryptophan side chains). The stable conformational state is considered to be that of the functional transmembrane channel primarily on the basis of extensive studies on its interaction with sodium ions.

A time-resolved fluorescence study of 4′,6′-diamidine-2-phenylindole dihydrochloride binding to polynucleotides

At phosphate/dye (P/D) ratios greater than 30 the quantum yield of 4,6-diamidine-2-phenylindole dihydrochloride (DAPI)-DNA and DAPI-poly(d(A-T)) complexes was found to be 0.62 and 0.66, respectively. Contrary to earlier reports a fluorescence enhancement of DAPI-poly(d(G-C)) complexes was observed with a quantum yield of 0.22. Time-resolved fluorescence measurements of complexes with a P/D ratio of 150:1 indicate that there were three fluorescent components in DAPI-DNA complexes with lifetimes of 3.86, 1.79 and 0.13 ns. In DAPI-poly(d(A-T)) complexes the lifetimes were 3.91, 1.20 and 0.11 ns. Also, three components with lifetimes of 3.98, 0.87 and 0.12 ns were found in DAPI-poly(d(G-C)) complexes. At low P/D ratios (< 5) another binding form of DAPI was observed which was assigned to the interaction of one or more molecules of DAPI with one previously bound to DNA. It is concluded that DAPI does not exhibit A-T binding specificity and that at high P/D ratios there are two types of binding having similar binding constants.

Supramolecular organization of lysophosphatidylcholine-packaged Gramicidin A.

Heat derived gramicidin A/L-alpha-lysophosphatidylcholine complexes were separated on a sucrose gradient to form two fractions: Fraction A which had an approximately constant Gramicidin A to phospholipid ratio of 8 to 10 lipid molecules per Gramicidin A molecule and Fraction B which had a larger but variable ratio. Fluorescence and circular dichroism studies confirmed Fraction A to be a lipid-incorporated channel state. Electron microscopic studies, using uranyl acetate negative staining, showed fraction A to be a membranous state with the formation of bilayer vesicles, that is, the interaction of peptide and phospholipid micelles causes the lipid to reorganize into a bilayer structure. Freeze-fracture replicas of the channel incorporated state demonstrated the presence of a supramolecular organization of particles exhibiting a tendency to form rows with a 50-60 A periodicity along the row and with 70-80 A distance between rows. An idealized working model for the incorporated state is presented.

EXCITED STATE pKa BEHAVIOUR OF DAPI. A RATIONALIZATION OF THE FLUORESCENCE ENHANCEMENT OF DAPI IN DAPI‐NUCLEIC ACID COMPLEXES

Abstract— The steady state and time resolved fluorescence of the drug and chromosomal staining agent, 4′,6‐diamidino‐2‐phenylindole dihydrochloride, DAPI, was examined under different solvent conditions. In solutions between pH 3 and pH 9 the fluorescence spectral maximum of DAPI was found at 460 nm. The fluorescence decayed with double exponential kinetics, with decay times of 2.86 and 0.144 ns, at all wavelengths below 550 nm. At 550 nm single exponential decay kinetics with a lifetime of 0.153 ns was observed. The fluorescence spectrum could be resolved into two components, the 2.86 ns component having a spectral maximum near 450 nm and the 0.144 ns component having a spectral maximum near 490 nm. The results are rationalized in terms of there being two different configurations of DAPI, one of which undergoes a rapid protonation of the indole ring by proton transfer from the 6‐amidinium group in the excited singlet state. The 0.144 ns component is assigned as the fluorescence from the excited state of the protonated indole ring. The results provide an explanation of the fluorescence enhancement in DAPI‐nucleic acid complexes.

Interactions between ubiquinones and phospholipid bilayers. A spin-label study.

Abstract The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.

Gramicidin a induces lysolecithin to form bilayers

Heat-induced association of Gramicidin A with lyso-lecithin micelles results in the formation of lipid bilayer structures. The capacity of the Gramicidin A peptide to transform the lysolecithin lipid structure from micelle to bilayer is considered in terms of molecular packing mechanisms and relevance to membrane processes in general. The resulting lipid-bilayer-packaged channel system has particular usefulness in c h a r a c t e r i z i n g channel structure and mechanism.

DNA-4′-6-diamidine-2-phenylindole interactions: A comparative study employing fluorescence and ultraviolet spectroscopy

DAPI is a drug that interacts with double-stranded nucleic acids, binding preferentially to A + T base pairs. The interaction is not intercalative, therefore providing a useful model for mimicking the effect of functional molecules in modifying specific sites, namely, A + T segments, of significance in gene expression. Knowledge of the nature of such interaction has been enriched by additional information obtained from comparative analysis of the data acquired by uv spectroscopy and fluorescence. Two classes of binding sites, defined by different apparent affinity constants and numbers of binding sites, are evident. All types of interaction are dependent on the nucleic acid/dye ratio and on the ionic strength of the medium.

Membrane Alterations in Cancer Cells

Radiation, chemicals, and some viruses are known to be environmental causes of cancer. The origin of tumors of different kinds has been associated for a long time to exposure to radiations of different energy.. Chemicals also can cause cancer either by a direct action on the cells or by enzymatic conversion to an active carcinogenic form. This process often involves the NADPH-cytochrome P-450 electron transport system, located mainly in the endoplasmic reticulum of the hepatocyte but also of several other tissues.3v4 Free radicals are among the products of this cytochrome P-450-mediated reactions. Substances that give free radical intermediates are halogenoalkanes, nitro-compounds, aromatic amines, quinones, nitrosoamines, polycyclic hydrocarbons, and polyunsaturated fatty acids. These intermediates have been found to play a significant role in chemical carcin~genesis.~*~ Free radicals are indeed responsible for a series of damaging reactions within the cell. Among their targets are DNA, nucleotidecoenzymes, thiol-dependent enzymes and membranes, whose damage is known to be induced by lipid peroxidation, responsible for structural and functional modifications as well as production of toxic intermediates. It has of course to be considered that highly reactive radicals are very short lived and cannot diffuse far from the site of generation. This consideration is particularly relevant for radicals that may have DNA as a target and are produced far from the nucleus. On the other hand, slow reacting radicals should not have too great a damaging effect even if they can diffuse more freely within cell. Among the damaging effects of free radicals, lipoperoxidation has been studied with particular attention because of a number of important biological consequences. It consists of a chain reaction resulting in the oxidative degradation of unsaturated lipids, particularly of polyunsaturated fatty acid residues of phospholipids that constitute the bilayer of the rnembrane~..~ Several are the products of lipoperoxidation: alkanals,