Multiconfigurational nature of 5f orbitals in uranium and plutonium intermetallics.

Uranium and plutonium's 5f electrons are tenuously poised between strongly bonding with ligand spd-states and residing close to the nucleus. The unusual properties of these elements and their compounds (e.g., the six different allotropes of elemental plutonium) are widely believed to depend on the related attributes of f-orbital occupancy and delocalization for which a quantitative measure is lacking. By employing resonant X-ray emission spectroscopy (RXES) and X-ray absorption near-edge structure (XANES) spectroscopy and making comparisons to specific heat measurements, we demonstrate the presence of multiconfigurational f-orbital states in the actinide elements U and Pu and in a wide range of uranium and plutonium intermetallic compounds. These results provide a robust experimental basis for a new framework toward understanding the strongly-correlated behavior of actinide materials.

Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is a common, non-specific renal lesion. Although it is often secondary to other disorders, including HIV infection, obesity, hypertension and diabetes, FSGS also appears as an isolated, idiopathic condition. FSGS is characterized by increased urinary protein excretion and decreasing kidney function. Often, renal insufficiency in affected patients progresses to end-stage renal failure, a highly morbid state requiring either dialysis therapy or kidney transplantation. Here we present evidence implicating mutations in the gene encoding alpha-actinin-4 (ACTN4; ref. 2), an actin-filament crosslinking protein, as the cause of disease in three families with an autosomal dominant form of FSGS. In vitro, mutant alpha-actinin-4 binds filamentous actin (F-actin) more strongly than does wild-type alpha-actinin-4. Regulation of the actin cytoskeleton of glomerular podocytes may be altered in this group of patients. Our results have implications for understanding the role of the cytoskeleton in the pathophysiology of kidney disease and may lead to a better understanding of the genetic basis of susceptibility to kidney damage.

Reduction in viscosity of cystic fibrosis sputum in vitro by gelsolin.

Obstruction of airways by viscous sputum causes lung damage in patients with cystic fibrosis (CF). Sputum samples from CF patients were shown to contain filamentous actin. Human plasma gelsolin, a protein that severs actin filaments, rapidly decreased the viscosity of CF sputum samples in vitro. Gc globulin and deoxyribonuclease I, proteins that sequester monomeric actin but do not sever actin filaments, were less efficient than gelsolin in diminishing sputum viscosity. These results suggest that gelsolin may have therapeutic potential as a mucolytic agent in CF patients.

Remodeling of neutrophil phospholipids with 15(S)-hydroxyeicosatetraenoic acid inhibits leukotriene B4-induced neutrophil migration across endothelium.

5-Lipoxygenase products, such as leukotrienes, are important stimuli for leukocyte-mediated tissue injury in acute inflammation. 15-Hydroxyeicosatetraenoic acid (15-HETE) is an eicosanoid generated by a variety of cell types via the actions of 15-lipoxygenases and, in addition, cyclooxygenases and epoxygenases. 15-HETE levels are frequently elevated at sites of inflammation, and extracellular 15(S)-HETE is esterified rapidly into neutrophil (PMN) phospholipids in vitro to levels that are comparable with arachidonic acid. We present evidence that remodeling of PMN phospholipids with 15(S)-HETE stereoselectively inhibits PMN migration across endothelium in response to leukotriene B4 (LTB4) and other chemoattractants. Esterified 15(S)-HETE causes a striking reduction in the affinity of LTB4 cell-surface receptors for their ligand and inhibition of LTB4-triggered stimulus-response coupling. As a result of these actions, esterified 15(S)-HETE attenuates the cytoskeletal rearrangements and CD11/CD18-mediated adhesive events that subserve directed locomotion of PMN across endothelium. These observations indicate that products of the 5-lipoxygenase and 15-lipoxygenase pathways can exert counterbalancing influences on PMN trafficking across endothelium. They suggest that 15(S)-HETE may be a potent endogenous inhibitor of PMN-endothelial interactions in vivo and serve to limit or reverse acute inflammation.

Neuroprotection mediated by changes in the endothelial actin cytoskeleton.

Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS(-/-)) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 microgram/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 +/- 3 hours to 24 +/- 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS(-/-) mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

Deconstructing gelsolin: identifying sites that mimic or alter binding to actin and phosphoinositides.

Gelsolin is involved in cytoskeletal remodeling as it can fragment and guide reassembly of actin networks. Recent advances in defining the structure of gelsolin identified functionally important sites. These structural insights could lead to the design of small molecule analogs to enhance, inhibit or mimic the functions of gelsolin.

Functional consequences of disulfide bond formation in gelsolin.

Gelsolin is an actin monomer binding and filament severing protein synthesized in plasma and cytoplasmic forms differing by an N-terminal amino acid extension and a disulfide bond between Cys-188 and Cys-201. To determine whether this bond altered gelsolin regulation or function, oxidized and reduced plasma gelsolins were assayed for severing, monomer binding and nucleation activity at a variety of rate-limiting calcium concentrations. The results indicate that the disulfide bond in domain 2 of gelsolin influences the transmission of information from C-terminal regulatory sites to functional sites in the N-terminus.

Sensory specificity of apparent motion.

Previous reports of intermodal apparent motion claim that the various senses contribute to or are under the guidance of a single synthesizing or organizing system, a common sense, and that under proper circumstances the separate heteromodal stimuli may be combined into a novel perceptual object. Our first experiment suggests that the term intermodal apparent motion has been misapplied in the past to one of the many idiosyncratic interpretations to which relatively meaningless patterns of heteromodal stimulation lend themselves. In our second experiment, we found that a properly timed visual stimulus can facilitate the perception of auditory apparent motion without its peculiarly auditory qualities of the moving percept. We find no evidence for a common or suprasensory organizing principle that integrates the separate visual and auditory stimuli.

Investigation of Aquo and Chloro Complexes of UO(2)(2+), NpO(2)(+), Np(4+), and Pu(3+) by X-ray Absorption Fine Structure Spectroscopy.

U, Np, and Pu L(II,III)-edge X-ray absorption fine structure (XAFS) spectra were collected for the UO(2)(2+), NpO(2)(+), Np(4+), and Pu(3+) ions as a function of chloride concentration in aqueous solution. At low chloride concentration, the hydration numbers and corresponding bond lengths for the different ions are as follows: UO(2)(2+), N= 5.3, R = 2.41 Å; NpO(2)(+), N = 5.0, R = 2.50 Å; Np(4+), N = 11.2, R = 2.40 Å; Pu(3+), N = 10.2, R = 2.51 Å. As the Cl(-) concentration increases, inner-sphere Cl(-) complexation occurs, resulting in a decrease in the hydration numbers and an expansion of the actinide-oxygen (water) bond lengths. The Pu(3+) ion shows only a decrease in hydration number (40%) and no inner-sphere Cl(-) complexation for [Cl(-)] < 14 M. For concentrations up to 10-14 M Cl(-), the average Cl(-) coordination numbers and bond lengths are as follows: UO(2)(2+), N = 2.6, R = 2.73 Å; NpO(2)(+), N = 1.0, R = 2.84 Å; Np(4+), N = 2.0, R = 2.61 Å. Structural changes are observed in the near-edge spectral region as shown by significant changes in the white line intensities upon Cl(-) complexation. For ions with similar structures, i.e. Pu(3+) and Np(4+) or the actinyl ions NpO(2)(+) and UO(2)(2+), positive energy shifts are observed with increasing oxidation state. The ability to use XAFS speciation results to calculate equilibrium constants and the relationship of these results to previous studies are discussed.

The natural decline of an introduced species following its initial increase in abundance; an explanation for Ommatoiulus moreletii in Australia.

The black Portuguese millipede, Ommatoiulus moreletii, an exotic species first reported in Australia in 1953, shows a pattern of initial eruption and subsequent decline in abundance following its introduction to sites in South Australia. Comparative sampling of new, erupted populations and older, declined populations was done in an attempt to find testable hypotheses to account for the decline. We report on laboratory and field experiments which show that a native rhabditid nematode appears to be the causal agent for the decline of populations of O. moreletii in South Australia. Implications for the biological control of introduced species are discussed in terms of this work.

237Np: oxidation state in vivo and chelation by multidentate catecholate and hydroxypyridinonate ligands.

Chemically, 237Np(V) is as toxic as U(VI), and radiologically, about as toxic as 239Pu. Depending on redox conditions in vivo, 237Np exists as weakly complexing Np(V) (NpO2+) or as Np(IV), which forms complexes as stable as those of Pu(IV). Ten multidentate catecholate (CAM) and hydroxypyridinonate (HOPO) ligands with great affinity for Pu(IV) were compared with CaNa3-DTPA for in vivo chelation of 237Np. Mice were injected intravenously with 237NpO2Cl: those in a kinetic study were killed 1 to 2880 min; in ligand studies, fed mice were injected intraperitoneally with a ligand 5, 60, or 1440 min after 237Np(V) (molar ratio 5.6 to 73), mice fasted for 16 h were gastrically intubated with a ligand 3 min after 237Np(V) (molar ratio 5.6 to 274), and all were killed 24 h after ligand administration; tissues and excreta were radioanalyzed. Rapid plasma clearance and urinary excretion of 237Np(V) resemble U(VI); deposition and early retention in skeleton and liver resemble Pu(IV). The x-ray absorption near edge structure spectroscopy (XANES) spectra of femora of 237Np(V)-injected mice, compared with spectra of Np(V) and Np(IV) from reference solids, showed predominantly Np(IV). Significant in vivo 237Np chelation was obtained with all of the HOPO and CAM ligands injected at molar ratio 22; the HOPO ligands reduced 237Np in skeleton, liver, and other soft tissue, on average, to 72, 25, and 25% of control, respectively, while CaNa3-DTPA was ineffective. Two HOPO ligands injected 60 min after 237Np (molar ratio 5.6) significantly reduced body and liver 237Np, and three HOPO ligands given orally (molar ratio > or = 73) significantly reduced body and liver 237Np, compared with controls. Combined with earlier work, these results indicate that: the dominant neptunium species circulating and excreted in urine is Np(V), while that in bone and liver deposits is Np(IV); Np(V) must be reduced to Np(IV) before it can be stably chelated; efficient decorporation of neptunium requires multidentate ligands that form exceptionally stable actinide(IV) chelates and facilitate Np(V) reduction.

EXAFS measurement of iron bcc-to-hcp phase transformation in nanosecond-laser shocks.

Extended x-ray absorption fine structure (EXAFS) measurements have demonstrated the phase transformation from body-centered-cubic (bcc) to hexagonal-close-packed (hcp) iron due to nanosecond, laser-generated shocks. The EXAFS spectra are also used to determine the compression and temperature in the shocked iron, which are consistent with hydrodynamic simulations and with the compression inferred from velocity interferometry. This is a direct, atomic-level, and in situ proof of shock-induced transformation in iron, as opposed to the previous indirect proof based on shock-wave splitting.

Gelsolin displaces phalloidin from actin filaments. A new fluorescence method shows that both Ca2+ and Mg2+ affect the rate at which gelsolin severs F-actin.

We describe an assay for measuring both the extent and kinetics of the severing of F-actin, based on the enhanced fluorescence emission of tetramethylrhodamine-phalloidin bound to F-actin. The enhanced fluorescence is lost after exposure to active gelsolin by displacement of the phalloidin from actin during severing. This assay requires small amounts of actin and gelsolin, can be used to measure reaction times ranging from 1 to 10(3) s, and does not require covalent modification of either protein. The rate of fluorescence loss is linearly related to the concentrations of both actin and gelsolin. However, the apparent rate constant of the reaction is highly dependent on the divalent cation concentration, varying between 10(4) and 10(6) M-1 s-1 when the [Ca2+] varies between 20 and 200 microM. Addition of Mg2+ increases the apparent rate constant at equivalent Ca2+ concentration. These results suggest that in vitro the rate-limiting step in the severing process is the activation of gelsolin by the binding of Ca2+ and Mg2+ to several low affinity (Kd approximately 100 microM) sites on gelsolin. While activation of gelsolin by Ca2+ is a slow process, the binding and severing of actin occurs at a rate approaching the diffusion limit.

Brains and brawn: plectin as regulator and reinforcer of the cytoskeleton.

Plectin is a 580 kDa intracellular protein, previously shown to link intermediate filaments with microtubules, actin filaments, and membrane components. Disruption of the plectin gene in humans and in mice results in severe skin blistering and muscular degeneration, consistent with plectin's structural role in stabilizing cells against mechanical force. However, recent work by Andra et al. characterizing cells from plectin-deficient mice demonstrates that in addition to this structural role, plectin also modulates the dynamics of the actin cytoskeleton. This makes plectin unusual in that it serves both to reinforce and crosslink intermediate filament attachments to membranes and other cytoskeletal polymers and to regulate actin dynamics in cells.

Phagocytosis in Acanthamoeba: II. Soluble and insoluble mannose-rich ligands stimulate phosphoinositide metabolism.

The generation of second messengers during phagocytosis of yeast by Acanthamoeba castellanii was examined. The kinetics of binding and internalization of yeast by Acanthamoeba were measured and this was compared with the generation of known second messengers. We observed stimulated degradation of PI-4, 5-P2 to 1,4,5 IP3 with kinetics similar to that observed for the binding of yeast to amoeba. Similar production of IP3 could be induced upon treatment with a soluble mannosylated glycoprotein. We propose that the Acanthamoeba mannose receptor stimulates the degradation of PI-4, 5-P2 to 1,4,5 IP3 as an initial event in phagocytosis.

Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast.

We have examined the initial events in phagocytosis by Acanthamoeba castellanii in order to understand this process at the molecular level and have determined that phagocytosis in this organism is mediated by a receptor which recognizes mannose-rich elements in the particle to be phagocytosed. We demonstrate that the binding and internalization of yeast particles can be inhibited by the sugars (D(+)-mannose and D(-)-fructose in a stereospecific, concentration-dependent manner. This inhibition is specific; these sugars did not inhibit the uptake of latex beads by this organism. Using mannosylated neoglycoproteins, which are much more potent inhibitors of particle binding as compared with the free sugar, we demonstrate the presence of a receptor on the amoeba cell surface which is necessary for the binding of yeast as the initial event of phagocytosis. The Acanthamoeba mannose receptor also appears to be able to mediate the delivery of soluble mannose-rich molecules to a degradative compartment such as the lysosome. Knowledge of this receptor will allow a better understanding of the molecular events of phagocytosis.