Vincenzo Trefiletti

Viscoelastic properties of native DNA from intact nuclei of mammalian cells. Higher-order DNA packing and cell function.

Abstract Changes in reduced viscosity of nuclear lysates from rat liver cells have been studied, in conditions of very low shear stress by the use of an oscillating viscometer, as a function of incubation time in alkaline (pH 12.5) and neutral (pH 8.0) solutions. In non-denaturing conditions, nuclear DNA showed a stepwise, time-dependent increase of reduced viscosity, which suggests that it behaves as a single hydrodynamic unit that progressively changes its radius and viscoelastic properties because of a very slow unfolding, through discrete successive transitions, from a highly superpacked structure toward a linear relaxed B -form fiber. Experimental conditions shown to reduce chromatin-DNA superpacking without changing DNA length (e.g. G 1 cycling versus G 0 non-cycling liver cells, or young versus old rat liver cells) dramatically increased the initial value of reduced viscosity and its time-dependent increment. Conversely, in denaturing conditions, reduced viscosity increased in the initial phase (probably because DNA unfolding prevails on DNA unwinding), then exhibited a plateau level (when unfolding balances unwinding), and subsequently decreased progressively to the value of sheared DNA (when unwinding becomes more rapid due to the progressive breakage of phosphodiester bridges in alkali). Experimental conditions known to induce DNA single- or double-strand breaks (i.e. the use of liver cells from rats treated with dimethylnitrosamine or 2-acetylaminofluorene, or of liver cells exposed to X-rays) caused in both neutral and alkaline solution an increment in the initial reduced viscosity and in the slope of its time-dependent increase, which may be related to a reduction of chromatin-DNA superpacking. Moreover, it became evident in denaturing conditions that a decrease of the maximum viscosity and of the time taken to reach it both related to a reduced DNA length. These viscoelastic properties are constantly correlated with independent DNA structural measurements on the same nuclear lysates, to discriminate the effect due to mere aggregation and disaggregation.

Polyacetylenes Bearing Mesogenic Side Groups: Synthesis and Properties, 1. Mesogenic Substituents with a Short Flexible Spacer

We report on the synthesis and polymerization of novel acetylenic monomers derived from 3-butyn-1-ol and bearing different potentially mesogenic substitutuents. Polymerization is carried out in solution with typical metathesis catalysts based on Mo and W and yields polyacetylenes with fairly high molecular weight and soluble in common organic solvents. Polymers are fully characterized by the point of view of the molecular structure, by GPC, FT-IR, NMR and UV/VIS techniques, and of the thermal and morphological behavior, by TGA, DSC, POM and X-ray diffraction experiments. Polymers with different amounts of cis and trans units are obtained, depending upon polymerization conditions. Although the monomers do not show any liquid crystalline behavior, polymers P2 and P3, containing in the side-chain respectively biphenyl and benzoyloxy benzoate as mesogenic cores, exhibit an enantiotropic smectic phase.

Isolation and characterization of multiple forms of renin from bull kidney.

Different forms of renin have been purified from bull kidney by combined gel filtration, affinity chromatography and ion-exchange chromatography. The specific activity of the enzyme was determined by a biochemical method of synthetic substrate and by radioimmunoassay on both synthetic and natural substrates; molecular characterization was carried out by molecular weight determinations, polyacrylamide gel electrophoresis, isoelectric focusing, amino acid analysis and optical rotatory dispersion. Three forms (renin C, D, E) are distinct on the basis of amino acid composition and chromatographic behavior, while possessing the same molecular weight, and displaying only minor differences in specific activity, alpha-helix content and isoelectric point; the occurrence of a group of renin isoenzymes may be suggested. Another form (A) has a lower specific activity and a higher molecular weight (57 000) compared with C, D and E and further differs markedly in chromatographic behavior, amino acid composition, alpha-helix content and isoelectric point, as well as in substrate specificity; it may be regarded as a pseudorenin. The fifth form (B) possesses the highest specific activity and does not correspond to a single molecular form; the presence of two components of different molecular weight (27 000 and 46 000 respectively) has been established both by polyacrylamide gel electrophoresis and isoelectric focusing.