Small angle scattering of cell nuclei

Neutron and X-ray small angle scattering techniques have been applied to study chromatin structure inside different types of cell nuclei. Scattering from genetically inactive chicken erythrocyte nuclei exhibits a maximum at Q = 0.1-0.15 nm-1 which cannot be observed by studying isolated chromatin derived from the same kind of cells. In highly active transcribing rat liver nuclei such a nuclear pattern is absent. The radius of gyration of isolated "superbeads" was determined. It is discussed whether the characteristic maximum of the nuclei originates from this superstructural organisation of chromatin. Rat liver nuclei were fractionated on sucrose gradients in order to determine whether the absence of the extra maximum in scattering profiles of these nuclei is due to overlapping effects of different chromatin organisation in the various cell types of the liver. As compared to unfractionated nuclei no strong deviations in the scattering profiles of the fractions could be observed. Erythrocyte nuclei were dialysed in buffers differing in the ionic strength of monovalent cations. The typical maximum from the nuclei is shifted from 60 nm (very low salt concentration) to about 35 nm (physiological ionic strength) and is linearly proportional to the decreasing radius of the nuclei. In conclusion, chromatin structure inside the nucleus has a scattering maximum due to an ordered packing of the fibres which is absent in nuclei with high genetic activity.     

Small angle X-ray scattering of intact and lysing cell nuclei

X-ray scattering at very low angles has been applied to the study of chromatin structure in intact and lying nuclei. From maxima and minima in the very low angle range, higher order or very large structures could be deduced, the existence of which has not been found in isolated chromatin; up to now, such structures could be demonstrated only be electron-micrographs of whole nuclei. A strong maximum at 0.12 nm^?1 or a shoulder at 0.08 nm^?1 is interpreted as a hollow or solid cylinder with diameter ～ 70 nm; however, another possible explanation, a side by side packing of 30 nm solenoids^1–4 with a distance of 52 nm, cannot be excluded. A shoulder at 0.033 nm^?1 leads to the conclusion that an even larger structure exist in interphase nuclei. A slight minimum at 0.2 nm^?1 is in the range where mildly isolated chromatin has its first order minimum. This accounts for a coil diameter ～ 30 nm. While in intact nuclei these characteristics of the scattering curve have only a low intensity (except the maximum at 0.12 nm^?1 lysing nuclei exhibit much more pronounced maxima.     

Low angle x-ray diffraction studies of chromatin structure in vivo and in isolated nuclei and metaphase chromosomes

Diffraction of x-rays from living cells, isolated nuclei, and metaphase chromosomes gives rise to several major low angle reflections characteristic of a highly conserved pattern of nucleosome packing within the chromatin fibers. We answer three questions about the x-ray data: Which reflections are characteristic of chromosomes in vivo? How can these reflections be preserved in vitro? What chromosome structures give rise to the reflections? Our consistent observation of diffraction peaks at 11.0, 6.0, 3.8, 2.7 and 2.1 nm from a variety of living cells, isolated nuclei, and metaphase chromosomes establishes these periodicities as characteristic of eukaryotic chromosomes in vivo. In addition, a 30-40- nm peak is observed from all somatic cells that have substantial amounts of condensed chromatin, and a weak 18-nm reflection is observed from nucleated erythrocytes. These observations provide a standard for judging the structural integrity of isolated nuclei, chromosomes, and chromatin, and thus resolve long standing controversy about the “tru” nature of chromosome diffraction. All of the reflection seen in vivo can be preserved in vitro provided that the proper ionic conditions are maintained. Our results show clearly that the 30-40-nm maximum is a packing reflection. The packing we observe in vivo is directly correlated to the side-by-side arrangement of 20- 30-nm fibers observed in thin sections of fixed and dehydrated cells and isolated chromosomes. This confirms that such packing is present in living cells and is not merely an artifact of electron microscopy. As expected, the packing reflection is shifted to longer spacings when the fibers are spread apart by reducing the concentration of divalent cations in vitro. Because the 18-, 11.0-, 6.0-, 3.8-, 2.7-, and 2.1-nm reflections are not affected by the decondensation caused by removal of divalent cations, these periodicities must reflect the internal structure of the chromaticn fibers.     

Genetic polymorphism of the CYP1A1, CYP2E1, GSTM1 and GSTT1 genes and lung cancer susceptibility in a north Indian population

The present study investigates in a experimental system in vitro the relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 degrees C. Approximately 50-55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH.     

Physical and conformational properties of staphylokinase in solution

The structure of staphylokinase has been analyzed by solution X-ray scattering, dynamic light scattering, ultracentrifugation and ultraviolet circular dichroism spectroscopy. Staphylokinase has a radius of gyration of 2.3 nm, a Stokes radius of 2.12 nm and a maximum dimension of 10 nm. The sedimentation coefficient is 1.71 S. These physical parameters indicate that the shape of staphylokinase is very elongated. The protein molecule consists of two folded domains of similar size. The mean distance of the centres of gravity of the domains is 3.7 nm. The mutual positions of the two domains are variable in solution. Thus, the molecule is shaped like a flexible dumbbell. About 18% of the amino acids of staphylokinase are organized in helical structures, 30% are incorporated in β-sheets and 20% form turns.     

Structure of myosin subfragment 1 from low-angle X-ray scattering

The X-ray scattering pattern produced by a solution of myosin subfragment 1 has been measured to a resolution (Bragg spacing) of 2 nm. We find that for subfragment 1 (S1) prepared by limited papain digestion in the presence of ethylenediaminetetraacetate the radius of gyration is 3.28 +/- 0.06 nm, the volume is 151 +/- 6 nm3, the surface area is 330 +/- 15 nm2, and the length of the maximum chord is 12.0 +/- 1.0 nm. The theoretical scattering patterns from several objects of uniform electron density have been calculated and compared with the observed scattering produced by S1. The recent three-dimensional electron micrograph reconstruction of S1-decorated actin by J. Seymour and E. O'Brien (private communication) generated the calculated pattern that best fit the observed scattering. This fit strongly suggests that this reconstruction resembles subfragment 1. The good correspondence between an S1 structure derived when S1 is attached to actin and a study of free S1 in solution strongly suggests that binding to actin does not grossly distort the shape of S1. This is consistent with the notion that S1 changes its orientation on actin, rather than its shape, in order to generate the contractile force in muscle.     

Investigation of Gold and Silver Nanoparticles on Absorption Heating and Scattering Imaging

Efficient conversion of absorbed light to heat energy and strong scattering by gold and silver nanoparticles suggest these nanoparticles as the agents of heating and imaging. Absorption efficiency and scattering efficiency of gold and silver nanoparticles were studied through numerical simulation using the discrete dipole approximation method. This study shows that the size of gold and silver nanoparticles can effect gold and silver nanoparticles’ absorption efficiency and scattering efficiency. The gold nanoparticle is found to possess the maximum absorption efficiency when the size of gold nanoparticle is 50 nm and the incident wavelength is 540 nm, and the increasing scattering efficiency with the increasing size of gold nanoparticle in the medium, and refractive index of the medium is around 1.33. However, the silver nanoparticle owns the maximum absorption efficiency when the size of silver nanoparticle is 20 nm and the incident wavelength is 396 nm, and the maximum scattering efficiency when the size of silver nanoparticle is 30 nm and the incident wavelength is 410 nm in the same medium. The conditions for achieving the maximum adsorption efficiency and scattering efficiency of gold and silver nanoparticle can be used for heating and imaging using visible and near-infrared light.     

Transcription in Isolated Wheat Nuclei: II. CHARACTERIZATION OF RNA SYNTHESIZED IN VITRO

Nuclei free of RNase activity were isolated from 3-hour-imbibed wheat (var. Yamhill) embryos by centrifugation through a discontinuous gradient of Percoll. The maximum rate of RNA synthesis observed in these nuclei was approximately 5 picomoles [(3)H]UTP per milligram DNA per minute. Two monovalent cation optima were found when measured in the presence of 15 millimolar MgCl(2) or 2 millimolar MnCl(2); 15 and 75 millimolar (NH(4))(2)SO(4). At the high monovalent cation optimum, the rate of RNA synthesis was linear for the first 10 to 15 minutes of incubation and still increasing after 3 hours. RNA synthesized in vitro (30-minute pulse followed by a 30-minute chase) showed distinct 18 and 26S RNA peaks comprising 13 and 17% of the total radioactivity, respectively. The over-all pattern of RNA synthesized in vitro was similar to the in vivo pattern. Approximately 40 to 50% of the RNA synthesized was inhibited by alpha-amanitin at 4 micrograms per milliliter. The newly synthesized 6 to 10S RNA appeared to be selectively inhibited by alpha-amanitin.     

Semi-Rigid Solution Structures of Heparin by Constrained X-ray Scattering Modelling: New Insight into Heparin-Protein Complexes

The anionic polysaccharides heparin and heparan sulphate play essential roles in the regulation of many physiological processes. Heparin is often used as an analogue for heparan sulphate. Despite knowledge of an NMR solution structure and 19 crystal structures of heparin-protein complexes for short heparin fragments, no structures for larger heparin fragments have been reported up to now. Here, we show that solution structures for six purified heparin fragments dp6-dp36 (where dp stands for degree of polymerisation) can be determined by a combination of analytical ultracentrifugation, synchrotron X-ray scattering, and constrained modelling. Analytical ultracentrifugation velocity data for dp6-dp36 showed sedimentation coefficients that increased linearly from 1.09 S to 1.84 S with size. X-ray scattering of dp6-dp36 gave radii of gyration R_G that ranged from 1.33 nm to 3.12 nm and maximum lengths that ranged from 3.0 nm to 12.3 nm. The higher resolution of X-ray scattering revealed an increased bending of heparin with increased size. Constrained molecular modelling of 5000 randomised heparin conformers resulted in 9-15 best-fit structures for each of dp18, dp24, dp30, and dp36 that indicated flexibility and the presence of short linear segments in mildly bent structures. Comparisons of these solution structures with crystal structures of heparin-protein complexes revealed similar ranges of phi (φ) and psi (ψ) angles between iduronate and glucosamine rings. We conclude that heparin in solution has a semi-rigid and extended conformation that is preformed for its optimal binding to protein targets without major conformational changes.     

The superstructure of chromatin and its condensation mechanism

Solutions of rat liver and chicken erythrocyte chromatin at different ionic strngths were characterized by synchrotron X-ray solution scattering, ultracentrifugation, density and viscosity measurements. Previous observations on nuclei were extended to rat liver, calf thymus and yeast nuclei.It is shown that with monovalent cations condensation is independent of the nature of the cation whereas with divalent cations there are significant differences related to the preference of base binding over phosphate binding. The consistency of hydrodynamic and scattering results confirm the view that chromatin in solution at low ionic strength has a helix-like superstructure. A survey of X-ray and neutron scattering results in the literature shows that previous interpretations, e.g. in terms of a 10 nm filament, are incompatible with the experimental data at low resolution.     

Characterization of a quasicrystalline phase in codispersions of phosphatidylethanolamine and glucocerebroside

Synchrotron x-ray diffraction, differential scanning calorimetry, and electron spin resonance spectroscopy have been employed to characterize a quasicrystalline phase formed in aqueous dispersions of binary mixtures of glucocerebroside and palmitoyloleoylphosphatidylethanolamine. Small- and wide-angle x-ray scattering intensity patterns were recorded during temperature scans between 20 degrees and 90 degrees C from mixtures of composition 2, 5, 10, 20, 30, and 40 mol glucocerebroside per 100 mol phospholipid. The quasicrystalline phase was characterized by a broad lamellar d-spacing of 6.06 nm at 40 degrees C and a broad wide-angle x-ray scattering band centered at approximately 0.438 nm, close to the gel phase centered at approximately 0.425 nm and distinct from a broad peak centered at 0.457 nm observed for a liquid-crystal phase at 80 degrees C. The quasicrystalline phase coexisted with gel and fluid phase of the pure phospholipid. An analysis of the small-angle x-ray scattering intensity profiles indicated a stoichiometry of one glucosphingolipid per two phospholipid molecules in the complex. Structural transitions monitored in cooling scans by synchrotron x-ray diffraction indicated that a cubic phase transforms initially into a lamellar gel. Thermal studies showed that the gel phase subsequently relaxes into the quasicrystalline phase in an exothermic transition. Electron spin resonance spectroscopy using spin labels located at positions 7, 12, and 16 carbons of phospholipid hydrocarbon chains indicated that order and motional constraints at the 7 and 12 positions were indistinguishable between gel and quasicrystalline phases but there was a significant decrease in order and increase in rate of motion at the 16 position on transformation to the quasicrystalline phase. The results are interpreted as an arrangement of polar groups of the complex in a crystalline array and a quasicrystalline packing of the hydrocarbon chains predicated by packing problems in the bilayer core requiring disordering of the highly asymmetric chains. The possible involvement of quasicrystalline phases in formation of membrane rafts is considered.     

DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved.

Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5-15 mM) to hydrolyze the DNA substrate while the 40 kDa-activity is inhibited by added divalent cations (5-15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.     

Effect of triterpene glycosides on plasma membrane permeability for UV-absorbing substances in Saccharomyces carlsbergensis yeast cells]

The effect of triterpene glycosides of cauloside C from Caulophyllum robustum M, stichoposide A from Stichopus japonicus S. and theasaponine from Thea sinensis Z. on permeability of plasmic membranes of Saccharomyces carlsbergensis for UV-absorbing substances was studied. It was found that incorporation of 14C-uridine from the endocellular pool into the yeast acid-insoluble fraction decreased under the effect of the triterpene glycosides as a result of the precursor leakage from the cell into the medium. It was found that the triterpene glycosides stimulated the leakage of the UV-absorbing substances with an absorption maximum at 260 nm from the cells. The maximum membranotropic effect was observed at 30--40 degrees C and in the presence of monovalent potassium, sodium, ammonium and lithium ions in the medium. Cauloside C and theasaponine, pentacyclic glycosides had the highest effect on the permeability at pH 4.8--5.6, while stichoposide A, a tetracyclic glycoside, had the highest effect at pH 7.0.     

Small-angle X-ray study on the quaternary structure of erythrocruorin from Caenestheria inopinata.

The erythrocruorin of the clam shrimp Caenestheria inopinata was studied in sodium phosphate buffer at pH 6.8 by small-angle X-ray scattering. The following molecular parameters were determined: radius of gyration 4.77 +/- 0.05 nm, maximum dimension 14.0 +/- 0.5 nm and a volume of 640 +/- 40 nm3. A model which fits the experimental data well is presented. The model is composed of 10 subunits arranged symmetrically in two layers, whereby five subunits are always forming a ring.     

Investigation of cellobiohydrolase from trichoderma reesei by small angle X-ray scattering

Summary Trichoderma reesei was grown on sulfite pulp and the major cellobiohydrolase of the culture filtrate was purified to homogeneity. The distance distribution function p(r) measured by the small angle X-ray scattering technique indicates that the enzyme molecule has a rather unusual tadpole like shape with an isotropic head and a long tail. The maximum length is 18 nm and the largest diameter is 4.4 nm.     

Noncovalent interaction of MNSFbeta, a ubiquitin-like protein, with histone 2A

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSFbeta, an isoform of the MNSF, has been isolated and characterized. MNSFbeta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Most recently, we observed that Ubi-L covalently conjugates to Bcl-G, a novel pro-apoptotic protein. In this study, we observed that Ubi-L noncovalently and specifically binds to histone 2A. The maximum binding was observed at a molar ratio equal to 1 for GST-Ubi-L and 2 for histone 2A. Ubi-L formed complex with histone 2A in the presence of 1% Triton X-100. Free Ubi-L was detected in nuclei from unstimulated murine helper T cell line, D10. The increased amounts of free Ubi-L and some Ubi-L adducts were observed in nuclei from mitogen-activated D10 cells. Interestingly, two Ubi-L adducts were unique to the chromatin fraction of nuclei from the activated D10 cells.     

水痘-带状疱疹病毒地方分离株在兔脑神经细胞中的形态与形态发生特征

为阐明水痘-带状疱疹病毒济南分离株（VZVJ1）在兔脑神经细胞（RNC）中的形态与形态发生特征,我们利用超薄切片电子显微镜技术对感染VZVJ1的RNC进行了观察研究。结果表明RNC在感染VZVJI6h后核内可见散在的核衣壳,12h后细胞核和细胞质内核衣壳明显增多,24h达高峰,而细胞核和细胞质内的成熟病毒颗粒较少见。病毒大小、形态基本一致,呈圆形或椭圆形,核心直径30～50nm,核衣壳74～96nm,成熟病毒110～180nm。核衣壳内有3种类型的核心,即电子致密核心、部分致密核心和电子透明核心。细胞核和细胞质内均可见核心样电子致密体和布纹样结构。在细胞质内还可见少量“繁殖复合体”,由膜性结构包绕多个囊泡构成。提示VZVJ1在RNC中的形态发生不同于其它性质的细胞。     

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.     

Synthesis and chemical transformations of N-hydroxy- and N-hydroxyalkylcycloimides of chlorin p6

Two series of chlorin p6 13,15-cycloimides that differ in their substituents at the nitrogen atom of the additional six-membered ring were synthesized. The compounds of the first series have a hydroxyl, alkoxyl, or acyloxy group at the 13,15-cycloimide nitrogen and those of the second series, residues of aliphatic alcohols. The cycloimides synthesized are satisfactorily stable and display an intensive light absorption maximum at 710-718 nm. Treatment of the cycloimides with sodium periodate in the presence of osmium tetroxide and with the Vilsmeier reagent resulted in the formation of 3-formyl- and 3-(2-formylvinyl)derivatives, respectively. The conversion of vinyl into formyl group or 2-formylvinyl group leads to an additional bathochromic shift of the long-wave maximum by 30 nm on an average. An extra hydroxy group was introduced at position 18 of the macrocycle to increase the cycloimide hydrophilicity. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.