Frequent loss of chromosome 9, homozygous CDKN2A/p14(ARF)/CDKN2B deletion and low TSC1 mRNA expression in pleomorphic xanthoastrocytomas.

The molecular pathogenesis of pleomorphic xanthoastrocytoma (PXA), a rare astrocytic brain tumor with a relatively favorable prognosis, is still poorly understood. We characterized 50 PXAs by comparative genomic hybridization (CGH) and found the most common imbalance to be loss on chromosome 9 in 50% of tumors. Other recurrent losses affected chromosomes 17 (10%), 8, 18, 22 (4% each). Recurrent gains were identified on chromosomes X (16%), 7, 9q, 20 (8% each), 4, 5, 19 (4% each). Two tumors demonstrated amplifications mapping to 2p23-p25, 4p15, 12q13, 12q21, 21q21 and 21q22. Analysis of 10 PXAs with available high molecular weight DNA by high-resolution array-based CGH indicated homozygous 9p21.3 deletions involving the CDKN2A/p14(ARF)/CDKN2B loci in six tumors (60%). Interphase fluorescence in situ hybridization to tissue sections confirmed the presence of tumor cells with homozygous 9p21.3 deletions. Mutational analysis of candidate genes on 9q, PTCH and TSC1, revealed no mutations in PXAs with 9q loss and no evidence of TSC1 promoter methylation. However, PXAs consistently showed low TSC1 transcript levels. Taken together, our study identifies loss of chromosome 9 as the most common chromosomal imbalance in PXAs and suggests important roles for homozygous CDKN2A/p14(ARF)/CDKN2B deletion as well as low TSC1 mRNA expression in these tumors.

Comparative investigations of biopolymer hydration by physicochemical and modeling techniques.

The comparative investigation of biopolymer hydration by physicochemical techniques, particularly by small-angle X-ray scattering, has shown that the values obtained differ over a wide range, depending on the nature of the polymer and the environmental conditions. In the case of simple proteins, a large number of available data allow the derivation of a realistic average value for the hydration (0.35 g of water per gram of protein). As long as the average properties of proteins are considered, the use of such a default value is sufficient. Modeling approaches may be used advantageously, in order to differentiate between different assumptions and hydration contributions, and to correctly predict hydrodynamic properties of biopolymers on the basis of their three-dimensional structure. Problems of major concern are the positioning and the properties of the water molecules on the biopolymer surface. In this context, different approaches for calculating the molecular volume and surface of biopolymers have been applied, in addition to the development of appropriate hydration algorithms.

Calculation of hydrodynamic properties of macromolecular bead models with overlapping spheres.

For the calculation of hydrodynamic properties of rigid macromolecules using bead modelling, models with overlapping beads of different sizes are used in some applications. The hydrodynamic interaction tensor between unequal overlapping beads is unknown, and an oversimplified treatment with the Oseen tensor may introduce important errors. Here we discuss some aspects of the overlapping problem, and explore an ad hoc form of the interaction tensor, proposed by Zipper and Durchschlag. We carry out a systematic numerical study of the hydrodynamic properties of a two-spheres model, showing how the Zipper-Durchschlag correction removes efficiently the numerical instabilities, and predicts the correct limits.

Lumbricus terrestris hemoglobin: a comparison of small-angle x-ray scattering and cryoelectron microscopy data.

The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of clylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm-1 and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h > 1 nm-1) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex.

alpha-1,4-D-glucan phosphorylase of gram-positive Corynebacterium callunae: isolation, biochemical properties and molecular shape of the enzyme from solution X-ray scattering.

The alpha-1,4-D-glucan phosphorylase from gram-positive Corynebacterium callunae has been isolated and characterized. The enzyme is inducible approx. 2-fold by maltose, but remarkably not repressed by D-glucose. The phosphorylase is a homodimer with a stoichiometric content of the cofactor pyridoxal 5'-phosphate per 88-kDa protein subunit. The specificity constants (kcat/Km, glucan) in the directions of glucan synthesis and degradation are used for the classification of the enzyme as the first bacterial starch phosphorylase. A preference for large over small substrates is determined by variations in the apparent binding constants rather than catalytic-centre activities. The contribution of substrate chain length to binding energy is explained assuming two glucan binding sites in C. callunae phosphorylase: an oligosaccharide binding site composed of five subsites and a high-affinity polysaccharide site separated from the active site. A structural model of the molecular shape of the phosphorylase was obtained from small-angle solution X-ray scattering measurements. A flat, slightly elongated, ellipsoidal model with the three axes related to each other as 1:(0.87-0.95):0.43 showed scattering equivalence with the enzyme molecule. The model of C. callunae phosphorylase differs from the structurally well-characterized rabbit-muscle phosphorylase in size and axial dimensions.

Lack of size and shape alteration of oxygenated and deoxygenated Lumbricus terrestris hemoglobin?

The giant extracellular hemoglobin of Lumbricus terrestris was investigated in the oxygenated, deoxygenated and reoxygenated state using small angle X-ray scattering. Scattering experiments of the oxygenated state of the protein yielded a radius of gyration of 10.71 +/- 0.02 nm, a maximum diameter of 29.37 +/- 0.21 nm and a volume of 6200 +/- 200 nm3. The values for the deoxygenated state of the hemoglobin are smaller than the values for the oxygenated state, but the differences hardly exceed the limits of error.

Molecular shape, dissociation, and oxygen binding of the dodecamer subunit of Lumbricus terrestris hemoglobin.

Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 +/- 0.01 nm, maximum diameter = 10.59 +/- 0.01 nm, and volume = 255 +/- 10 nm3, with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1 degrees C and 4 at 25 degrees C, providing DeltaH = -20 kcal/mol and DeltaS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39]-8. Equilibrium oxygen binding measurements at pH 6.5-8. 5, provided P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 5.2-9. 5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25 degrees C. P50 was decreased 3- and 2-fold in approximately 100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.

Small-angle X-ray scattering studies on cellobiose dehydrogenase from Phanerochaete chrysosporium.

Limited proteolysis of cellobiose dehydrogenase (CDH) from the white rot fungus Phanerochaete chrysosporium by papain cleaves the enzyme into two fragments containing flavin (FAD) and heme, respectively. Small-angle X-ray scattering (SAXS) was employed to investigate size and shape of intact CDH and of its fragments in solution. The largest dimension of CDH amounts to about 18 nm, whereas the corresponding quantity of each of the two fragments is only around 9 nm. CDH as well as its fragments appear to be of prolate shape, the cross-section of the FAD fragment (diameter 4.3 to 5.1 nm) being considerably larger than that of the heme fragment (diameter 3.3 nm). These findings suggest a collinear arrangement of the two domains in the CDH particle. Simulations based on the method of finite elements corroborate this structure model and furthermore suggest the existence of a possibly flexible linker between the two domains.

X-ray induced inactivation of the sulfhydryl enzyme malate synthase in the presence of various additives. Probing the extent of primary and post-irradiation inactivation and repair by rapid screening on the microlevel.

The sulfhydryl enzyme malate synthase was inactivated by X-irradiation in air-saturated aqueous solution, in the absence or presence of a variety of additives (thiols, antioxienzymes, typical radical scavengers, inorganic salts, buffer components, substrates, products, analogues). Radiation-induced changes of enzymic activity were registered immediately after stop of irradiation and in the post-irradiation period. Repair experiments were initiated by post-irradiation addition of dithiothreitol. Additionally, post-irradiation inactivation was modulated by some further additives. Probing the extent of primary and post-irradiation inactivation and repair was accomplished effectively by screening experiments on the microlevel, and by derivation of normalized efficiency parameters which allowed quick comparisons of the various additives with respect to their protective and repair-promotive efficiencies. Correlations between the efficiency parameters were studied by means of binary and ternary diagrams. Most of the substances added before irradiation were found to protect the enzyme against primary and post-irradiation inactivation and to increase the reparability of the enzyme by dithiothreitol, the extent of the effects depending on the nature (and concentration) of the additives used. Our results indicate that both specific protection (by substrates, products, analogues, and by sulfhydryl agents) and scavenging are responsible for the radioprotective efficiencies of the additives.

Primary and post-irradiation inactivation of the sulfhydryl enzyme malate synthase: correlation of protective effects of additives.

The presence of additives during X-irradiation of malate synthase led to radioprotective effects against primary and post-irradiation inactivation. Pronounced effects were provided by typical scavengers, sulfhydryl reagents and specific ligands (substrates, products, analogues). The results show that scavenging and specific protection are responsible for the protective efficiency of additives. Scavengers delete noxious species formed during irradiation or post-radiationem. Sulfhydryl reagents may act as repair substances. Specific ligands protect the active site of the enzyme and the essential sulfhydryls; specific protection is more pronounced post-radiationem. Ligands and sulfhydryl reagents may additionally act as scavengers. A cumulative index for the protective power of additives against both sorts of inactivation was established.

A small-angle X-ray scattering study on pre-irradiated malate synthase. The influence of formate, superoxide dismutase, and catalase on the X-ray induced aggregation of the enzyme.

The sulfhydryl enzyme malate synthase from baker's yeast was X-irradiated with 6 kGy in air-saturated aqueous solution (enzyme concentration: congruent to 10 mg/ml; volume: 120 microliters), in the absence or presence of the specific scavengers formate, superoxide dismutase, and catalase. After X-irradiation, a small aliquot of the irradiated solutions was tested for enzymic activity while the main portion was investigated by means of small-angle X-ray scattering. Additionally, an unirradiated sample without additives was investigated as a reference. Experiments yielded the following results: X-irradiation in the absence of the mentioned scavengers caused considerable aggregation, fragmentation, and inactivation of the enzyme. The dose Dt37 for total (= repairable + non-repairable) inactivation resulted as 4.4 kGy. The mean radius of gyration was found to be about 13 nm. The mean degree of aggregation was obtained as 5.7, without correction for fragmentation. An estimation based on the thickness factor revealed that about 19% of material might be strongly fragmented. When this amount of fragments was accordingly taken into account, a value of 7.1 was obtained as an upper limit for the mean degree of aggregation. The observed retention of the thickness factor and the finding of two different cross-section factors are in full accord with the two-dimensional aggregation model established previously (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99-121 (1980)). The presence of catalytic amounts of superoxide dismutase and/or catalase, in the absence of formate, during X-irradiation reduced both aggregation and inactivation significantly. The presence of formate (10 or 100 mM) during X-irradiation led to a strong decrease of aggregation and inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Small-angle X-ray and light scattering studies on the influence of Mg2+ ions on the structure of the RNA from bacteriophage MS2.

The influence of Mg2+ ions on the secondary and tertiary structure of the RNA from bacteriophage MS2 was investigated by small-angle X-ray scattering and light scattering and by sedimentation experiments. The analysis of the outer part of the X-ray scattering curve obtained at low temperature in the absence of Mg2+ yielded a cross-section radius of gyration of 0.88 nm and a mass per unit length of 1720 g mol-1 nm-1. Very similar values for these parameters, which refer to the secondary structure of the RNA molecule, were also derived from the X-ray scattering curves obtained in the presence of different amounts of Mg2+ (0.07 to 1 ions per nucleotide). On the contrary, the inner part of the X-ray scattering curves turned out to be highly dependent on the Mg2+ concentration: the cross-section radius of gyration and the mass per unit length, which were determined from the scattering curves at small angles as parameters related to the tertiary structure of the RNA, amounted to 3.11 nm and 4000 g mol-1 nm-1, respectively, in the absence of Mg2+ and increased significantly upon raising the concentration of Mg2+. The increase of these structural parameters was found to be accompanied by a decrease of the overall radius of gyration (as revealed indirectly by X-ray scattering and directly by light scattering measurements) and by an increase of the sedimentation coefficient. The results from the investigations of the RNA at low temperature clearly establish the existence of double-stranded structures down to very low Mg2+ concentrations as well as the occurrence of Mg2+ induced changes of the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase. Effects of formate, superoxide dismutase, catalase, and dithiothreitol.

Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0, 30, 60 h after irradiation. To elucidate the role of OH., O-.2, and H2O2 in the X-ray inactivation of the enzyme, experiments were performed in the absence or presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Ar = 3% (corresponding to a D37 = 0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t = 0 led to Ar = 21%; repairs started later on resulted in somewhat lower activities. The decay of repairability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Ar = 72% and D37 = 6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate. Both the presence of specific additives during irradiation and the addition of additives after irradiation may alter the post-irradiation inactivation. Catalase turned out to be the most potent inhibitor of post-irradiation inactivation; superoxide dismutase showed an ambivalent behaviour, it accelerated or impeded post-irradiation inactivation; formate, when added after irradiation, exhibited a moderate protective effect. The presence of specific additives, added before and/or after irradiation, influenced the repair behaviour to some extent. The highest activity achieved by repair amounted to about 90% of the activity of the corresponding unirradiated sample. The percentual gain of activity was found to be the greater the lower the residual activity of the enzyme was before initiation of repair.(ABSTRACT TRUNCATED AT 400 WORDS)

Post-irradiation inactivation of the sulfhydryl enzyme malate synthase.

The sulfhydryl enzyme malate synthase was inactivated in air-saturated aqueous solution by X-irradiation (2 kGy). Changes of activity were registered up to 60 h after irradiation. Effects of specific additives (formate, superoxide dismutase, catalase), added before and/or after irradiation, revealed the role of the deleterious radical and non-radical species responsible for the radiation damage: inactivation during irradiation is mainly due to the action of OH, to a minor extent to O(2) and H2O2; post-irradiation inactivation is mainly caused by H2O2. A partial restoration of enzyme activity by dithiothreitol, added after irradiation, was found for all systems investigated; repairs were significant even when they were initiated 60 h after irradiation.

The interaction of calf thymus DNA with mercuric acetate and 3,6-bis-(acetatomercurimethyl)-dioxane. Small-angle X-ray scattering and viscosity studies.

The binding of Hg2+ and 3,6-bis-(acetatomercurimethyl)-dioxane (BAMD) to sonicated calf thymus DNA was studied by small-angle X-ray scattering and viscosity measurements. The scattering experiments with DNA complexed by different amounts of mercurials (for Hg2+ rb=0-0.79, for BAMD rb=0-0.86 mol of mercurial bound per mol of base pairs) established that the rod-like character of the DNA molecules is maintained up to high binding ratios. They revealed further a steady decrease of the cross-section radius of gyration Rc for the DNA X Hg2+ complex and a similar decrease of Rc for the DNA X BAMD complex up to Rb=0.35. This behaviour is certainly caused by the incorporation of both mercurials near the axis of the DNA helix. Binding of BAMD at rb greater than 0.35 led to an increase of Rc which behaviour obviously reflects the location of mercury atoms at large distances from the axis, possibly on the surface of the helix. The increase of the mass per unit length Mc upon binding of the mercurials was found to be much higher than expected. This finding established that the length of the DNA helix decreases by 0.10 +/- 0.01 nm per bound mercurial at low binding ratios (i.e. up to rb=1/3 for BAMD, up to possibly rb=0.5 for Hg2+). A similar conclusion was also drawn from the observed decrease of intrinsic viscosity [n] with increasing rb. The analysis of Mc at high binding ratios suggests that every BAMD molecule bound beyond rb=1/3 decreases the length of the DNA by 0.21 +/- 0.05 nm whereas Hg2+ when bound beyond rb=0.5 causes no change of the length.

Electrophoretic and chemical studies on the X-ray damage of malate synthase.

The sulfhydryl enzyme malate synthase was shown to undergo an X-ray induced aggregation and inactivation in solution (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99-121 (1980). Further evidence for the occurrence of aggregation and inactivation and also of fragmentation and partial unfolding of the enzyme upon X-irradiation was obtained by chemical and electrophoretic studies. Irradiation was carried out in a specially designed microcell, experiments were performed on the microlevel. Under conditions of the experiments the formation of H2O2 upon X-irradiation could be proven; therefore the influence of H2O2 on the enzyme was investigated too. Though the quantitative results of the damaged enzyme particles are influenced by many disturbing factors, the findings allow clear statements on the nature of the effects under investigation. 1) Both X-irradiation and treatment with H2O2 caused a decrease of total and an increase of available sulfhydryl groups of the enzyme and led to a loss of enzymic activity. The presence of dithiothreitol turned out to be able to protect the enzyme against X-ray or H2O2 induced inactivation. Moreover, addition of dithiothreitol after X-irradiation or H2O2 treatment allowed a considerable repair of enzymic activity. 2) Polyacrylamide gel disc electrophoreses of X-irradiated enzyme solutions, performed in the presence of sodium dodecyl sulfate, showed the occurrence of covalently cross-linked subunits (preferably dimers and trimers) and of various definite fragments. Electrophoreses in the absence of the denaturant indicated the occurrence of enzyme aggregation. The effects were more pronounced with increasing X-ray doses. The electrophoreses also clearly reflected a radioprotection by dithiothreitol against cross-linking, but not against fragmentation. Addition of excess of 2-mercaptoethanol or of dithiothreitol to the X-irradiated enzyme clearly demonstrated that part of the covalent cross-links were disulfide bridges; the aggregates themselves, however, were held together primarily by non-covalent bonds. Blocking of exposed enzyme sulfhydryls by means of Ellman's reagent prevented both covalent cross-linking and enzyme aggregation. 3) Similar electrophoretic patterns as found for the X-irradiated enzyme were obtained for the unirradiated enzyme after treatment with H2O2. The similarity of the sulfhydryls in the presence of H2O2, suggest an involvement of H2O2 in the radiation damage of the enzyme. It seems plausible that oxidation reactions are responsible for the effects caused by X-irradiation or H2O2 treatment.

Structure of two subfractions of normal porcine (Sus domesticus) serum low-density lipoproteins. X-ray small-angle scattering studies.

Two subfractions of low-density lipoproteins (LDL) were isolated from normal pig (Sus domesticus) serum by a combined method including precipitation, ultracentrifugation, and gel chromatography. The fractions recovered from the buoyant density ranges 1.020-1.050 and 1.050-1.090 g/mL, denoted as LDL1 and LDL2, respectively, were studied with regard to structure and thermotropic behavior by X-ray small-angle scattering and were compared to human serum low-density lipoprotein of density 1.020-1.063 g/mL. The average molecular weights determined from the scattering intensities on absolute scale were 2.6 X 10(6) and 2.0 X 10(6) for LDL1 and LDL2, respectively. The maximum particle diameters were found to be 24 and 21 nm, respectively. Both species were found to have quasi-spherical symmetry and to display the thermotropic transition of the apolar lipids within the particle core similar to human LDL. The width of the transition was approximately 9 degrees C in both cases, but the midpoint transition temperature was higher by 8 degrees C for LDL1 (33 degrees C) than for LDL2 (25 degrees C). Despite their different sizes and thermotropic behavior, the two porcine LDL subfractions appear to be built according to the same structural principle as human LDL in the molecular organization of the apolar lipids within the particle core.

Small-angle X-ray scattering studies on the X-ray aggregation of malate synthase. Computer simulations and models.

Malate synthase undergoes an X-ray induced aggregation which can be monitored in situ by small-angle X-ray scattering; the analysis of scattering curves, taken at subsequent stages of aggregation, has led to the establishment of a tentative model for an aggregation in two dimensions (Zipper and Durchschlag (1980) Rad. and Environm. Biophys., in press). This model was checked by comparison of appropriate theoretical curves with the experimental curves. The theoretical scattering curves for this comparison were obtained by weighted averaging over the scattering curves calculated for various species of hypothetical aggregates. Based on the approximation of the unaggregated enzyme particle by an oblate cylinder, the aggregates were assumed to be composed of 2, 3, 4 or 6 of such cylinders, associated side-by-side in one and later on in two linear rows. THe weight fractions of the species were chosen so, that an optimum fit of the experimental mean radii of gyration and mean degrees of aggregation was achieved. The distance distribution functions calculated for the model are very similar to the functions derived from the scattering experiment. Cross-section Guinier plots of the scattering curves of the model reveal the occurrence of one and later on of two pseudo cross-section factors similar to those observed in the experimental scattering curves. The pseudo thickness factor of the model of the unaggregated particle is found to be retained in the model curves for all stages of aggregation. From these results it can be concluded that the model for the aggregation process is essentially consistent with the scattering behaviour of the aggregating enzyme. Small differences between the theoretical and experimental curves may be explained by the idealizations of the model. The comparison of theoretical curves for alternative models, assuming aggregation in three dimensions, suggests that these models are unlikely, though small amounts of three-dimensional aggregates cannot be ruled out completely.