Stratum corneum lipid abnormalities in surfactant-induced dry scaly skin.

Sodium dodecyl sulfate (SDS) has been used to induce a dry scaly skin condition in human subjects. Measurements of stratum (s.) corneum hydration, scaliness, and lipid composition reveal in vivo surfactant perturbations on desquamation. Subjects (n = 10) were briefly treated daily with a 4% aqueous solution of SDS on one lower leg over a period of 2 weeks. The other control leg received no treatments. At the end of the treatment period, both lower legs were evaluated for hydration using an electrical impedance technique and examined by an independent dermatologist using a visually based grading scale for surface roughness and scaliness. Shave biopsies were then excised from each lower leg for analysis of s. corneum lipids. Treatment resulted in decreased s. corneum hydration and increased surface scale/roughness. These physical changes were accompanied by significant changes in s. corneum lipid composition. While surfactant treatments did not alter the total quantity of lipids per gram s. corneum protein, significant changes in specific lipid classes were observed. The free cholesterol to cholesterol ester ratio increased while the quantity of total sterols remained constant. The distribution of certain ceramide species were altered while the quantity of total ceramides remained constant. Free fatty acids were resolved into 2 distinct bands, only one of which diminished upon treatment. These results are interpreted in terms of a model for surfactant-induced perturbation of keratinization which leads to abnormal s. corneum lipids and altered desquamation.

Higher order folding of two different classes of chromatin isolated from chicken erythrocyte nuclei. A light scattering study.

Bulk chromatin fragments were excised from chicken erythrocyte nuclei by digestion with micrococcal nuclease. Fractionation into S chromatin (soluble at physiological ionic strengths) and I chromatin (insoluble at physiological ionic strengths) was achieved by dialysis against buffers containing 0.15 M NaCl. The effects of NaCl concentration on the molecular dimensions of S and I chromatins were determined by dynamic and static light scattering. Series of fragment lengths were obtained by gel filtration of S and I chromatins under ionic conditions which lead to maximal intramolecular compaction. Hydrodynamic radii and radii of gyration were determined for fragment lengths ranging from 8 to 53 nucleosomes. These data are in excellent agreement with calculations for extended helical structures. Close-packed solenoidal or superbead structures are not compatible with these data. Comparisons of molecular dimensions derived from light scattering and electron microscopy indicate that considerable shrinkage of chromatin fragments can occur when common methods of sample preparation are used for microscopy.

Study of conformational states and reversibility of histone complexes.

The core histone complex (H3:H4:H2A:H2B)2 and products of dissociation, the H2A:H2B: dimer and the H3:H4 tetramer, were isolated from chicken erythrocyte chromatin by several literature methods as well as gel filtration on Bio-Gel A15m at various salt concentrations. The conformational and oligomeric characteristic of these histone complexes were compared to analogous histone complexes prepared by renaturation of individually acid-extracted histones by circular dichroism (CD) and analytical gel filtration chromatography. The salt-extracted core histone complex (independent of method of preparation), the purified dissociation products, the H2A:H2B dimer, and the H3:H4 tetramer in 2 M NaCl, 10 mM sodium phosphate, 0.25 mM EDTA and 0.1 mM DTT, pH 7.0, have conformation which are identical, by the criteria of similar CD spectra, with complexes prepared from acid-extracted histones, Likewise, the salt-extracted complexes may be cycled through solvents of low ionic strength (10 mM sodium phosphate, pH 7.0 or 50 mM NaOAc, pH 5.0) or 1 mM HCl and returned to 2.0 M NaCl, 10 mM sodium phosphate, 0.25 mM EDTA, and 0.1 mM DTT, pH 7.0, in a completely reversible manner. Thus it would appear that acid-denatured histones are capable of being fully renatured to yield native-like complexes.

Chicken erythrocyte nucleus contains two classes of chromatin that differ in micrococcal nuclease susceptibility and solubility at physiological ionic strength.

Inactive chromatin of the chicken erythrocyte nucleus is shown to consist of two distinct classes (I and S). I chromatin (approximately 60% of the total genome) is insoluble at greater than 0.1 M ionic strength whereas S chromatin (approximately 40% of the total genome) is soluble at all ionic strengths studied (0.01--0.3 M). These chromatins are released from nuclei upon digestion with micrococcal nuclease by two separate parallel processes that do not have a precursor--product relationship to each other. Isolated I-chromatin fragments show a progressive reduction in size from 250 to approximately 50 nucleosome equivalents with increasing digestion times at 0-2 degrees C. Prolonged digestion of nuclei at 37 degrees C results in conversion of I chromatin to mononucleosomes that are insoluble at greater than 30 mM NaCl. Isolated S-chromatin fragments show a constant size distribution, independent of digestion time, that peaks at approximately 35 nucleosome equivalents. Prolonged digestion of nuclei at 37 degrees C results in the conversion of S chromatin to mononucleosomes that are soluble at physiological ionic strength. Both I and S chromatins contain a full complement of histones with no nonhistone proteins.

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.

High resolution proton magnetic resonance spectroscopy of histones and histone--histone complexes in aqueous solution.

Low molecular weight histone complexes of H2A (congruent to dimer), H2B (congruent to tetramer), H3--H4 (congruent to tetramer), H2A--H2B (congruent to dimer), and H2B--H4 (congruent to dimer) have been prepared in 2 M NaCl and neutral pH at 4 degrees C. These materials are free of nonspecific aggregate and are suitable for study by high resolution proton magnetic resonance spectroscopy. Such spectra have been recorded in aqueous solutions under conditions allowing a study of the exchangeable proton resonances of histone complexes for the first time and indicate that the structured regions are rich in hydrophobic amino acids, as well as arginine and some acidic amino acids. Most of the lysine and probably alanine residues remain in a motile, random coil-like state after formation of the complexes. It is suggested that arginine residues may be important in inter- and/or intra-subunit interactions in histone complexes.

Interaction of histone f2al fragments with deoxyribonucleic acid. Circular dichroism and thermal denaturation studies.

The glycine-arginine-rich histone, f2al (IV) (102 amino acids), from calf thymus was cleaved at residue 84 with cyanogen bromide. Complexes containing homologous DNA and each f2al fragment were reconstituted by means of Gdn-HC1 gradient dialysis. The circular dichroic (CD) spectra of these complexes were all examined in 0.14 M NaC1. The CD spectra of the DNA-f2al fragment complexes did not differ appreciably from that of DNA alone in the wavelength region above 240 nm. However, intact f2al-DNA complexes yield CD spectra which differ significantly (enhanced, blue-shifted, 273-nm band) from that of native DNA (Shih and Fasman, 1971). The small C-terminal fragment (85-102) was bound weakly to DNA under the conditions used. However, the large basic N-terminal fragment (1-83) was bound as well to DNA as was whole f2al, but produced no CD distortion. The conformation of the N-terminal fragment, unlike intact f2al, was not changed upon increasing the ionic strength to 0.14 M NaF. These results complement previous studies on f2al and its N-terminal CNBr fragment (Ziccardi and Schumaker, 1973). Thermal denaturation of the complexes in 2.5 X 10(-4) M EDTA was monitored simultaneously by changes in the absorption and CD spectra. All complexes showed a thermal transition at 45 degrees (Tml), attributable to the melting of free, double-stranded DNA. In addition, f2al-DNA and N fragment-DNA complexes displayed melting phenomena at 88 and 78 degrees (Tm2), respectively, caused by the denaturation of the histone-bound DNA. This difference in Tm2 constitutes further evidence that loss of the 18-amino-acid carboxyl end segment of f2al prohibits the unique type of interaction which occurs between DNA and the intact histone.