Ultrastructural features of chromatin nu bodies

Spread chromatin fibers and isolated chromatin fragments prepared from chicken erythrocyte nuclei were stained with dilute aqueous uranyl acetate. High-resolution electron micrographs reveal two new morphological features exhibited by many of the chromatin nu bodies: (a) lateral association of the nu body with the connecting strand, and (b) a centrally stained spot approximately 15 A wide, possibly corresponding to a hole or crevice within the nu body.     

Conformational states of annexin VI in solution induced by acidic pH

Acidic pH-induced folding of annexin (Anx)VI in solution was investigated in order to study the mechanism of formation of ion channels by the protein in membranes. Using 2-(p-toluidino)naphthalene-6-sulfonic acid as a hydrophobic probe, it was demonstrated that AnxVI exerts a large change in hydrophobicity at acidic pH. Moreover, circular dichroism spectra indicated that the native state of AnxVI changes at acidic pH towards a state characterized by a significant loss of alpha-helix content and appearance of new beta-structures. These changes are reversible upon an increase of pH. It is postulated that the structural folding of AnxVI could explain how a soluble protein may undergo transition into a molecule able to penetrate the membrane hydrophobic region. The physiological significance of these observations is discussed.     

Chromatin model calculations: Arrays of spherical nu bodies.

Chromatin fibers consists of globular nucleohistone particles (designated nu bodies) along the length of the chromatin DNA with approximately 6-to7-fold compaction of the DNA within the nu bodies. We have calculated theoretical small-angle x-ray scattering curves and have compared these with experimental data in the literature. Several models predict maxima at the correct angles. The first maximum (approximately 110 degrees A) results from interparticle interference, while both the spatial arrangement and the structure factor the nu bodies can contribute to the additional small-angle maxima. These calculations suggest models which can account for the electron microscopic observation that chromatin is seen as either approximately 100-or approximately 200-to 250 degrees A-diameter fibers, depending on the solvent conditions. They also account for the limited orientability of the x-ray pattern from pulled chromatin fibers.     

Conformational changes of actin induced by calponin.

Calponin, an actin-linked regulatory protein in smooth muscle, caused a remarkable change in the fluorescence intensity of pyrene-labeled actin in the filamentous form. Calponin, an equimolar ratio to actin, decreased the fluorescence intensity of pyrene-labeled F-actin by some 60% to the level near monomeric actin. This change was partially reversed by Ca2+, when calmodulin was present. Thus it appears that calponin causes conformational changes in actin molecules in an actin filament so as to inhibit their interactions with myosin.     

Conformational states of enkephalins in solution.

Chicken erythrocyte chromatin was partially digested with micrococcal nuclease and separated into multimeric subunit fractions by gel permeation chromatography. The fractions were characterized by their Svedberg constant, diffusion coefficient, circular dichroism, and electrophoresis pattern of the extracted DNA. The molecular weight dependence of the sedimentation coefficient was found to be S_20,w = .011 × M^.554. The molecular weight dependence of $\text{rmf}\text{f}{}_{\mathrm{o}}$ is best represented in the Kirkwood theory by either a helical superstructure $\text{or}$ a flexible coil $\text{with}\text{attractive}\text{interactions}$ between nucleosome units. The dimer calculations of $\text{f}\text{f}{}_{\mathrm{o}}$ suggest that the core particles are separated by spacer regions which contribute up to ～20% of the frictional properties of the molecule.     

Chromatin nu bodies: isolation, subfractionation and physical characterization.

Monomer chromatin subunit particles (nu1) have been isolated in gram quantities by large-scale zonal centrifugation of micrococcal nuclease digests of chicken erythrocyte nuclei. nu1 can be stored, apparently indefinitely, frozen in 0.2 mM EDTA (pH 7.0) at less than or equal to 25 degrees C. Aliquots of the stored monomers have been subfractionated by dialysis against 0.1 M KCl buffers into a soluble fraction containing equimolar amounts of H4, H3, H2A, H2B associated with a DNA fragment of approximately 130-140 nucleotide pairs, and a precipitated fraction containing all of the histones including H5 and H1 associated with DNA fragments. The total nu1 and the KCl-soluble fraction of nu1 have been examined by sedimentation, diffusion, sedimentation equilibrium ultracentrifugation, low-angle X-ray diffraction, and electron microscopy. Physical parameters from all of these techniques are presented and correlated in this study.     

Movement of histones in chromatin induced by shearing.

Methylation of accessible DNA within chromatin by restriction modification methylases from Haemophilus influenzae was used to detect movement of histones along the DNA strand during chromatin manipulation. Methylation at different stages of chromatin preparation was followed by titration of the nucleoprotein with ploy(D-lysine), digestion of chromosomal proteins with pronase and analysis of the DNA-poly(D-lysine) complex in steep cesium chloride gradients. Comparison of the specific radioactivities in the peak fractions of the free DNA and the DNA-poly(D-lysine) complex, respectively, reveals that lateral movement of histones, relative to specific sites in the DNA marked by restriction methylases, occurs during manipulation and fragmentation of chromatin.     

Conformational states of HRPA1 induced by thermal unfolding: effect of low molecular weight solutes

Fluorescence, CD, and activity measurements were used to characterize the different conformational states of horseradish peroxidase A1 induced by thermal unfolding. Picosecond time-resolved fluorescence studies showed a three-exponential decay dominated by a picosecond lifetime component resulting from energy transfer from tryptophan to heme. Upon thermal unfolding a decrease in the preexponential factor of the picosecond lifetime and an increase in the quantum yield were observed approaching the characteristics observed for apoHRPA1. The fraction of heme-quenched fluorophore decreased to 0.4 after unfolding as shown by acrylamide quenching. A new unfolding pathway for HRPA1 was proposed and the effect of the low molecular weight solutes trehalose, sorbitol, and melezitose on this pathway was analyzed. Native HRPA1 unfolds with an intermediate between the native and the unfolded conformation. The unfolded conformation can refold to the native state or to a native-like conformation with no calcium ions upon cooling or can give an irreversible denatured state. The refolded conformation with no calcium ions was clearly identified in a second thermal scan in the presence of EDTA and shows secondary and tertiary structures, heme reincorporation in the cavity, and at least 59% of activity. Melezitose stabilized the refolded Ca2+-depleted protein and induced a more complex mechanism for heme disruption. The effect of sorbitol and trehalose were mainly characterized by an increase in the temperature of unfolding.     

Conformational changes in rat liver chromatin after liver regeneration.

N-Pyrenemaleimide, a fluorescent probe that specifically labels histone H3 of rat liver chromatin in situ, was used to monitor the accessibility of histone H3 in chromatin isolated from rat liver at different times during degeneration. At times of maximum DNA synthesis (18--24 h after hepatectomy), the accessibility of the probe was found to be markedly (40--50%) increased. This increase is abolished, however, by treatment of the chromatin fibres with high salt (2 M-NaCl) or detergent. Tryptophan fluorescence was also enhanced at points of maximum DNA synthesis, suggesting that some non-histone tryptophan-containing protein was being synthesized. The polarization of the labelled histone H3 is not markedly altered, suggesting that fibre aggregation or dissociation does not occur. Mononucleosomes extracted from sham-operated and hepatectomized animals did not exhibit any difference in binding to the probe. Also, analysis of the chromatin protein by electrophoresis on detergent- and acid/urea/ Triton-X-100-containing polyacrylamide gels showed no detectable difference in histone H3 : 1, H3 : 2 or H3 : 3 subclasses.     

Conformational states of pancreatic lipase

Spin-label method was applied to the studies of conformation properties of pancreatic lipase. Spin-labelled derivatives of the enzyme in SH- and NH2-groups were obtained. ESR-spectra of both samples belong to the immobilized type, in the first case the ESR-spectrum corresponding to strong immobilization of the spin-label, and in the second--to the average one. In both cases the rotation correlation time of the enzyme molecule was measured. The time proved the same independent of the site of the label attachment; it corresponded to the rotation of macromolecule with molecular weight 50000. This fact points to the absence of both intramolecular flexibility of the enzyme molecule and of the association of lipase molecules in solution. It has been shown that introduction of substrates and inhibitors of the enzyme and the interface as well, induces no changes in the ESR spectra, which points to the absence of local conformation changes of protein near the spin-labels introduced.     

Carcinogenesis induced by foreign bodies

This review deals with the contemporary investigations of carcinogenesis induced by foreign bodies. The main attention is given to the interactions of macrophages with an implanted foreign body and their possible role in tumorigenesis.     

Making sense of chromatin states

Researchers find new pieces in the puzzle of genome regulation.