Planar wire-array Z-pinch implosion dynamics and X-ray scaling at multiple-MA drive currents for a compact multisource hohlraum configuration.

An indirect drive configuration is proposed wherein multiple compact Z-pinch x-ray sources surround a secondary hohlraum. Planar compact wire arrays allow reduced primary hohlraum surface area compared to cylindrical loads. Implosions of planar arrays are studied at up to 15 TW x-ray power on Saturn with radiated yields exceeding the calculated kinetic energy, suggesting other heating paths. X-ray power and yield scaling studied from 1-6 MA motivates viewfactor modeling of four 6-MA planar arrays producing 90 eV radiation temperature in a secondary hohlraum.

A consistent picture of the actin filament related to the orientation of the actin molecule.

We show that freeze-dried actin filaments which have been rotary shadowed with a light coat of platinum appear very similar in morphology and width to negatively-stained filaments. The addition of a thicker coat of platinum to such preparations gives the actin filaments a different morphology and width, which are similar to those of the rotary-shadowed, quick-frozen filaments described by Heuser and Kirschner (J. Cell Biol. 1980, 86:212-234). The consistent view of the actin filament presented here, particularly its 7-8-nm width, can be interpreted in terms of the overall orientation of the actin subunit in the actin filament.

Polymorphism of actin paracrystals induced by polylysine.

We describe a method for the induction of different polymorphic forms of actin filament paracrystals. This polymorphism is probably based on differences in the stagger and/or polarity of adjacent filaments in single-layered paracrystals and by superposition of different layers in multilayered paracrystals. The helical parameters defining the filament geometry are indistinguishable for the different polymorphic forms observed and for the four different actins used. Analysis of these paracrystals, some of which are ordered to better than 2.5 nm, should provide a reference structure suitable for alignment and orientation within the actin filament of high resolution models of the actin monomer obtained from crystal data.

The fibrillar substructure of keratin filaments unraveled.

We show that intermediate-sized filaments reconstituted from human epidermal keratins appear unraveled in the presence of phosphate ions. In such unraveling filaments, up to four "4.5-nm protofibrils" can be distinguished, which are helically twisted around each other in a right-handed sense. Lowering the pH of phosphate-containing preparations causes the unraveling filaments to further dissociate into "2-nm protofilaments." In addition, we find that reconstitution of keratin extracts in the presence of small amounts of trypsin yields paracrystalline arrays of 4.5-nm protofibrils with a prominent 5.4-nm axial repeat. Limited proteolysis of intact filaments immobilized on an electron microscope grid also unveils the presence of 4.5-nm protofibrils within the filament with the same 5.4-nm axial repeat. These results, together with other published data, are consistent with a 10-nm filament model based on three distinct levels of helical organization: (a) the 2-nm protofilament, consisting of multi-chain extended alpha-helical segments coiled around each other; (b) the 4.5-nm protofibril, being a multi-stranded helix of protofilaments; and (c) the 10-nm filament, being a four-stranded helix of protofibrils.

Crystalline actin sheets: their structure and polymorphism.

Crystalline sheets of Acanthamoeba actin induced by the trivalent lanthanide gadolinium exist in three different polymorphic forms, which show different striation patterns and surface topographies. We have called these different forms "rectangular" and "square" sheets, and "cylinders" and have shown that each of the three forms is constructed from common "basic" lattices associated in different ways. We have used image processing of electron micrographs to obtain a model for the actin molecule in projection to a resolution of 1.5 nm. The overall dimensions observed in these images are 5.6 x 3.3 x 4.5 nm, and the molecule itself appears distinctly bilobed with the two lobes separated by a cleft. actin monomers in the sheets are arranged with P2 symmetry and are therefore packed in a manner different from that of the molecules in actin filaments. Because approximately 35% of the surface area of the actin molecule is exposed on the surface of these sheets, the sheets should be useful to study the stoichiometric binding of actin-binding proteins to the actin molecule.

Electron microsocpy of plasmic fragments of human fibrinogen as related to trinodular structure of the intact molecule.

We have examined rotary shadowed, purified plasmic fragments of human fibrinogen with the electron microscope and have determined the relation of these fragments to the intact fibrinogen molecule. Both intact fibrinogen and its earliest cleavage product, fragment X, are trinodular. The next largest product, fragment Y, consists of two linked nodules. The two terminal products, fragments D and E, are single nodules. From measurements of simultaneously shadowed specimens of these different species, we conclude that the outer nodules of the trinodular fibrinogen molecule are the fragment D-containing regions and the central nodule is the fragment E-containing region.

Substructure of human von Willebrand factor.

Using electron microscopy, we have visualized the substructure of human von Willebrand factor (vWf) purified by two different approaches. vWf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. Examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 X 6.5 nm) connected to a small central node (6.4 X 3.4 nm) by two flexible rod domains each approximately 34 nm long and approximately 2 nm in diameter. The protomer is 120 nm in length when fully extended. These same structural features are seen both in vWf molecules that were rapidly purified from fresh plasma by a new two-step procedure and in those purified from lyophilized intermediate-purity Factor VIII/vWf concentrates. The 240,000-mol wt subunit observed by gel electrophoresis upon complete reduction of vWf apparently contains both a rod domain and a globular domain and corresponds to one half of the protomer. Two subunits are disulfide-linked, probably near their carboxyl termini, to form the protomer; disulfide bonds in the amino-terminal globular ends link promoters to form vWf multimers. The vWf multimer strands have at least two morphologically distinct types of ends, which may result from proteolytic cleavage in the globular domains after formation of large linear polymers. In addition to releasing fragments that were similar in size and shape to the repeating protomeric unit, plasmic degradation of either preparation of vWf reduced the size of multimers, but had no detectable effect on the substructure of internal protomers.

Structure of the actin molecule determined from electron micrographs of crystalline actin sheets with a tentative alignment of the molecule in the actin filament.

Electron microscopy and image processing of negatively stained crystalline sheets induced from Acanthamoeba actin have been used to yield a three-dimensional reconstruction of the actin molecule, including data to a maximum resolution of 15 A. This model shows actin to be an asymmetric, wedge-shaped molecule. A three-dimensional reconstruction of an averaged, polar actin filament from negatively stained polylysine-induced actin filament paracrystals has also been computed. We show two possible ways in which the wedge-shaped actin molecule from the sheets can be placed into such a filament reconstruction. In both, the major intermolecular contacts are formed on complementary surfaces of the actin subunit and follow the left-handed genetic helix of the filament, a feature also found in the filament reconstruction.

Characteristics and scaling of tungsten-wire-array z -pinch implosion dynamics at 20 MA.

We present observations for 20-MA wire-array z pinches of an extended wire ablation period of 57%+/-3% of the stagnation time of the array and non-thin-shell implosion trajectories. These experiments were performed with 20-mm-diam wire arrays used for the double- z -pinch inertial confinement fusion experiments [M. E. Cuneo, Phys. Rev. Lett. 88, 215004 (2002)] on the Z accelerator [R. B. Spielman, Phys. Plasmas 5, 2105 (1998)]. This array has the smallest wire-wire gaps typically used at 20 MA (209 microm ). The extended ablation period for this array indicates that two-dimensional (r-z) thin-shell implosion models that implicitly assume wire ablation and wire-to-wire merger into a shell on a rapid time scale compared to wire acceleration are fundamentally incorrect or incomplete for high-wire-number, massive (>2 mg/cm) , single, tungsten wire arrays. In contrast to earlier work where the wire array accelerated from its initial position at approximately 80% of the stagnation time, our results show that very late acceleration is not a universal aspect of wire array implosions. We also varied the ablation period between 46%+/-2% and 71%+/-3% of the stagnation time, for the first time, by scaling the array diameter between 40 mm (at a wire-wire gap of 524 mum ) and 12 mm (at a wire-wire gap of 209 microm ), at a constant stagnation time of 100+/-6 ns . The deviation of the wire-array trajectory from that of a thin shell scales inversely with the ablation rate per unit mass: f(m) proportional[dm(ablate)/dt]/m(array). The convergence ratio of the effective position of the current at peak x-ray power is approximately 3.6+/-0.6:1 , much less than the > or = 10:1 typically inferred from x-ray pinhole camera measurements of the brightest emitting regions on axis, at peak x-ray power. The trailing mass at the array edge early in the implosion appears to produce wings on the pinch mass profile at stagnation that reduces the rate of compression of the pinch. The observation of precursor pinch formation, trailing mass, and trailing current indicates that all the mass and current do not assemble simultaneously on axis. Precursor and trailing implosions appear to impact the efficiency of the conversion of current (driver energy) to x rays. An instability with the character of an m = 0 sausage grows rapidly on axis at stagnation, during the rise time of pinch power. Just after peak power, a mild m = 1 kink instability of the pinch occurs which is correlated with the higher compression ratio of the pinch after peak power and the decrease of the power pulse. Understanding these three-dimensional, discrete-wire implosion characteristics is critical in order to efficiently scale wire arrays to higher currents and powers for fusion applications.

Three-dimensional structure of proteins determined by electron microscopy.

Recent developments in specimen preparation and image processing techniques have made it possible to determine the three-dimensional structure of proteins by electron microscopy. Periodic supramolecular aggregates of the protein under investigation are requiring to minimize radiation damage and to maximize the signal-to-noise ratio of structural detail. Useful information about the fine structure of the protein (e.g. binding sites for interacting molecules, antigenic determinants) can often be obtained by stoichiometric labeling of the ordered arrays with interacting molecules or antibody fragments, and computing difference maps from the reconstructions of the labeled and native structures. The use of this approach to molecular structure determination of proteins will be discussed in light of our work with bacteriophage and actin.

Towards an alignment of the actin molecule within the actin filament.

Electron micrographs of negatively stained actin filament paracrystals and single-layered filament rafts showing different interfilament spacings have been studied and three-dimensional reconstructions have been computed from them. Lateral ordering of the filaments in rafts was lost when interfilament spacings exceeded 8.5 nm, suggesting this distance as an upper limit for the filament diameter. Further, all reconstructions showed the same structural features at the 3 nm resolution level, except that the filaments from ordered single-layered rafts appeared 10-20% wider than those from multi-layered paracrystals. A comparison between electron microscopical and X-ray filament data, and synthetic filaments generated using different tentative molecular models and/or orientations for actin did not allow a single best model to be selected.

Movement disorders after status epilepticus and other brain injuries.

A retrospective medical record review was conducted of 173 consecutive children hospitalized for acquired brain injuries on a specialized pediatric rehabilitation service. The chart review identified children who developed movement disorders with acquired brain injuries: 8 with status epilepticus, 2 with trauma, and 1 with anoxia. Movement disorders were observed more frequently following status epilepticus (8 of 12) than following other causes of acquired brain injury (3 of 161; P = .0001). Four additional children had severe neurologic deficits following status epilepticus but did not develop movement disorders. The 11 patients who developed movement disorders had choreiform movements predominantly. Even though status epilepticus is a clinical phenomenon resulting from a variety of etiologies, the features of movement disorders in these children were strikingly similar. The pathophysiology of this complication is unknown.

Preparation of single molecules and supramolecular complexes for high-resolution metal shadowing.

We have compared the appearance and preservation of molecular and supramolecular structures in preparations that were dried in vacuo at room temperature or freeze-dried. Fibrinogen and brain spectrin molecules appear similar in both types of preparation provided that drying at room temperature is performed in the presence of glycerol, which results in an even and reproducible distribution of such molecules (Shotton et al., 1979, J. Mol. Biol. 131, 303-329; Fowler and Erickson, 1979, J. Mol. Biol. 134, 241-249). In the case of crystalline actin sheets, actin filaments, and keratin filaments, freeze-drying preserves structural details that are often completely lost during drying at room temperature, whether or not glycerol is used. On the other hand, keratin filaments prepared by drying in the presence of glycerol display a beaded axial repeat that is probably due to "glycerol decoration." We conclude that although freeze-drying has no clear advantage over glycerol spraying/vacuum-drying in the case of single extended molecules, it may provide insight into the multiple effects of glycerol in specimen preparation. In the case of supramolecular assemblies such as filaments or crystalline sheets, freeze-drying preserves significantly more substructure and surface detail. The loss of such detail during drying at room temperature, probably through collapse phenomena such as distortion and flattening, cannot be prevented by glycerol.

Brain spectrin, a membrane-associated protein related in structure and function to erythrocyte spectrin.

An immunoreactive analogue of erythrocyte spectrin has been purified from brain membranes. This protein co-sediments with and cross-links actin filaments, associates with spectrin-binding sites on erythrocyte membranes, and has been visualized by rotary shadowing as an extended, flexible rod. The brain spectrin comprises 3% of the total membrane protein, and may have a major role in mediating linkage of actin to membranes.

Tubular arrays of the actin-DNase I complex induced by gadolinium.

We describe the preparation and structural analysis of ordered tubular arrays of the actin-DNase I complex. These structures consist of helically stacked rings; each ring is 73 A thick, has a 240 A outer and a 120 A inner diameter, and has 7-fold rotational symmetry. The actin-DNase I complex forms tubes under conditions in which actin alone aggregates into crystalline sheets-i.e., in the presence of the trivalent cation gadolinium. Moreover, upon addition of an equimolar amount of DNase I, crystalline actin sheets are slowly converted to tubes. The rings making the tubes contain a radial dyad axis that may be identical to the dyad axis of the unit cell of the crystalline actin sheet. Evidence is presented for this identification, which in turn allows tentative assignment of actin- and DNase I-containing regions in three-dimensional reconstructions of the rings. The structural analysis presented here may be useful in aligning available three-dimensional molecular models of actin determined from crystals of the actin-DNase I complex and from crystalline actin sheets with each other and ultimately within the biologically important actin filament.

Electron microscopy of human factor V and factor VIII: correlation of morphology with domain structure and localization of factor V activation fragments.

Clotting factor V and factor VIII are each represented by the domain structure A1-A2-B-A3-C1-C2 and share 40% sequence homology in the A and C domains. Rotary-shadowed samples of human factor V and factor VIII were examined in the electron microscope. Single-chain factor V molecules exhibited a globular "head" domain 12-14 nm in diameter. In addition, up to 25% of these molecules showed a rod-like "tail" of up to 50 nm. Glycerol-gradient centrifugation of factor V treated with thrombin partially resolved the factor Va heterodimer from a larger activation peptide of 150 kDa, as determined by gel electrophoresis. Electron microscopy of factor Va revealed globular molecules with several smaller appendicular structures but lacking the tails seen in factor V. Images of the 150-kDa activation peptide showed rod-like structures, similar in width to the tail of intact factor V and approximately 34 nm long. Rotary shadowing was also used to visualize factor VIII that had been fractionated into heterodimers containing heavy chains of distinct sizes. Each factor VIII preparation showed a globular structure approximately 14 nm in diameter, but the associated tails were observed much more frequently with factor VIII heterodimers containing the higher-molecular-weight heavy chains. These results, in conjunction with results of studies using other biophysical techniques, suggest a model in which the A and C domains of each cofactor constitute a globular head and the connecting B domain is contained in a two-stranded tail that is released by thrombin cleavage.

Structure of the fibrin protofibril.

We identified the two-stranded fibrin protofibril and studied its structure in electron micrographs of negatively stained specimens. Based on these images and on considerations of symmetry, we constructed a model of the protofibril in which the two strands of trinodular fibrin molecules are related by a two-fold screw axis between the strands and two-fold axes perpendicular to them. The two strands are held together by staggered lateral contacts between the central nodules of one strand and outer nodules of the other. The molecules within a strand are joined by longitudinal contacts between outer nodules. This interpretation of the structure of protofibrils is supported by images of trimer complexes whose preparation and structure are described here, in which the central nodule of a fibrin monomer is attached to the crosslinked outer nodules of two other molecules. We conclude that the association of protofibrils to form thicker fibers must involve a second type of lateral contact, probably between outer nodules of adjacent, in-register strands. In total, we identify three intermolecular contacts involved in the polymerization of fibrin.