RESUMO
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.
RESUMO
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.
RESUMO
Extended x-ray absorption fine structure (EXAFS), using a laser-imploded target as a source, can yield the properties of laser-shocked metals on a nanosecond time scale. EXAFS measurements of vanadium shocked to approximately 0.4 Mbar yield the compression and temperature in good agreement with hydrodynamic simulations and shock-speed measurements. In laser-shocked titanium at the same pressure, the EXAPS modulation damping is much higher than is warranted by the predicted temperature increase. This is shown to be due to the alpha-Ti to omega-Ti crystal phase transformation, known to occur below approximately 0.1 Mbar for slower shock waves.
RESUMO
The local structures of Am, Nd and Er-Benzimidazole (Biz) in solution were determined by EXAFS. The BIZ molecule coordinated to Am and Nd through two nitrogen atoms in a bidentate fashion. Two nitrogen atoms of BIZ ligated to Am and Nd with the bond distances R(Am-n) N=2.63A and R(Nd-N) = 2.65 A, respectively. The total coordination number of the Am BIZ complexes (at a molar ratio of metal ion to ligand of 1:20) was approximately 10 but that of Nd BIZ complex was approximately 9.
RESUMO
We have determined the structure of uranyl, UO(2)(2+), and Th(4+) complexes formed in aqueous solution with 4,5-dihydroxy-3,5-benzenedisulfonate (Tiron) as function of pH and concentration. At equimolar concentrations of 0.05 M UO(2)(2+) and Tiron, the predominant species was found to be aqueous uranyl at pH = 2.0. At pH = 6.0, the formation of a 3:3 UO(2)(2+):Tiron trimer (proposed in earlier studies) was observed. In this structure, bidentate catecholate complexation to Tiron as well as oxygen bridging between uranyl units is detected. Th(4+) structural changes were observed both as a function of pH and Th:L (L = Tiron) ratio. At Th:L = 1:1 and pH = 1.4, a monomeric complex is observed with each Th center complexing monodentate to approximately 2 sulfonate functional groups. At pH 4.0 similar sulfonate ligation is observed along with oligomer formation. At pH 6.0 thorium hydrolysis products are detected, with little evidence for inner-sphere Tiron coordination. When the Th:L is changed to 1:2 at pH = 6.0, a stable oligomeric complex is formed that dominates the speciation for Th:L ratios up to 1:5. This complex is characterized by bidentate catechol and monodentate sulfonate ligation to Tiron along with oxygen bridging between Th(4+) atoms and is consistent with the formation of the 2:3 Th:L polymeric species proposed from earlier work. At a Th:L ratio of 1:10, Th(4+) complexation is dominated by bidentate catechol ligation and the formation of a monomeric Th(Tiron)(x) species, where x > or = 2.
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An initial step in platelet shape change is disassembly of actin filaments, which are then reorganized into new actin structures, including filopodia and lamellipodia. This disassembly is thought to be mediated primarily by gelsolin, an abundant actin filament-severing protein in platelets. Shape change is inhibited by VASP, another abundant actin-binding protein. Paradoxically, in vitro VASP enhances formation of actin filaments and bundles them, activities that would be expected to increase shape change, not inhibit it. We hypothesized that VASP might inhibit shape change by stabilizing filaments and preventing their disassembly by gelsolin. Such activity would explain VASP's known physiological role. Here, we test this hypothesis in vitro using either purified recombinant or endogenous platelet VASP by fluorescence microscopy and biochemical assays. VASP inhibited gelsolin's ability to disassemble actin filaments in a dose-dependent fashion. Inhibition was detectable at the low VASP:actin ratio found inside the platelet (1:40 VASP:actin). Gelsolin bound to VASP-actin filaments at least as well as to actin alone. VASP inhibited gelsolin-induced nucleation at higher concentrations (1:5 VASP:actin ratios). VASP's affinity for actin (K(d) approximately 0.07 microM) and its ability to promote polymerization (1:20 VASP actin ratio) were greater with Ca(++)-actin than with Mg(++)-actin (K(d) approximately 1 microM and 1:1 VASP), regardless of the presence of gelsolin. By immunofluorescence, VASP and gelsolin co-localized in the filopodia and lamellipodia of platelets spreading on glass, suggesting that these in vitro interactions could take place within the cell as well. We conclude that VASP stabilizes actin filaments to the severing effects of gelsolin but does not inhibit gelsolin from binding to the filaments. These results suggest a new concept for actin dynamics inside cells: that bundling proteins protect the actin superstructure from disassembly by severing, thereby preserving the integrity of the cytoskeleton.
Assuntos
Actinas/metabolismo , Plaquetas/metabolismo , Moléculas de Adesão Celular/fisiologia , Gelsolina/metabolismo , Fosfoproteínas/fisiologia , Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Gelsolina/isolamento & purificação , Humanos , Cinética , Listeria/metabolismo , Magnésio/metabolismo , Proteínas dos Microfilamentos , Microscopia de Fluorescência , Modelos Biológicos , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Pseudópodes/metabolismo , Fatores de TempoRESUMO
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.
Assuntos
Actinas/fisiologia , Citoesqueleto/fisiologia , Endotélio Vascular/fisiologia , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/fisiologia , Actinas/antagonistas & inibidores , Animais , Circulação Cerebrovascular/efeitos dos fármacos , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , RNA Mensageiro/análise , Sinvastatina/farmacologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
The gelsolin family of actin filament binding proteins have highly homologous structures. Gelsolin and adseverin, also known as scinderin, are the most similar members of this family, with adseverin lacking a C-terminal helix found in gelsolin. This helix has been postulated to serve as a calcium-sensitive latch, keeping gelsolin inactive. To test this hypothesis, we have analyzed the kinetics of severing by gelsolin, adseverin, and a gelsolin truncate which lacks the C-terminal latch. We find that the relationship between severing rate and calcium ion concentration differs between gelsolin and adseverin, and suggest that calcium controls one rate-limiting step in the activation of adseverin and two in the activation of gelsolin. In contrast, both proteins are activated equally by protons, and have identical severing kinetics at pHs below 6.3. The temperature sensitivity of severing by adseverin and gelsolin is remarkably different, with gelsolin increasing its severing rate 8-fold per 10 degrees C increase in temperature and adseverin increasing its rate only 2-fold per 10 degrees C increase in temperature. Analysis of the gelsolin construct lacking the C-terminal helix demonstrates that this helix is responsible for the regulatory differences between gelsolin and adseverin. These results support the C-terminal latch hypothesis for the calcium ion activation of gelsolin.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Gelsolina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática , Gelsolina/química , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteínas dos Microfilamentos/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes , TemperaturaRESUMO
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.
Assuntos
Actinina/fisiologia , Cromossomos Humanos Par 19/genética , Glomerulosclerose Segmentar e Focal/genética , Proteínas dos Microfilamentos , Actinina/deficiência , Actinina/genética , Actinas/metabolismo , Sequência de Aminoácidos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Análise Mutacional de DNA , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Predisposição Genética para Doença , Humanos , Falência Renal Crônica/etiologia , Falência Renal Crônica/genética , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Mammalian cells express several isoforms of beta-thymosin, a major actin monomer sequestering factor, including thymosins beta4, beta10, and beta15. Differences in actin-binding properties of different beta-thymosin family members have not been investigated. We find that thymosin beta15 binds actin with a 2.4-fold higher affinity than does thymosin beta4. Mutational analysis was performed to determine the amino acid differences in thymosin beta15 that specify its increased actin-affinity. Previous work with thymosin beta4 identified an alpha-helical domain, as well as a conserved central motif, as crucial for actin binding. Mutational analysis confirms that these domains are also vital for actin binding in thymosin beta15, but that differences in these domains are not responsible for the variation in actin-binding properties between thymosins beta4 and beta15. Truncation of the unique C-terminal residues in thymosin beta15 inhibits actin binding, suggesting that this domain also has an important role in mediating actin-binding affinity. Replacement of the 10 C-terminal amino acids of thymosin beta15 with those of thymosin beta4 did, however, reduce the actin-binding affinity of the hybrid relative to thymosin beta15. Similarly, replacement of the thymosin beta4 C-terminal amino acids with those of thymosin beta15 led to increased actin binding. We conclude that functional differences between closely related beta-thymosin family members are, in part, specified by the C-terminal variability between these isoforms. Such differences may have consequences for situations where beta-thymosins are differentially expressed as in embryonic development and in cancer.
Assuntos
Actinas/metabolismo , Variação Genética , Timosina/genética , Timosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Movimento Celular/fisiologia , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
We have used EXAFS spectroscopy to investigate the inner sphere coordination of trivalent lanthanide (Ln) and actinide (An) ions in aqueous solutions as a function of increasing chloride concentration. At low chloride concentration, the hydration numbers and corresponding Ln,An-O bond lengths are as follows: La3+, N = 9.2, R = 2.54 A; Ce3+, N = 9.3, R = 2.52 A; Nd3+, N = 9.5, R = 2.49 A; Eu3+, N = 9.3, R = 2.43 A; Yb3+, N = 8.7, R = 2.32 A; Y3+, N = 9.7, R = 2.36 A; Am3+, N = 10.3, R = 2.48 A; Cm3+, N = 10.2, R = 2.45 A. In ca. 14 M LiCl, the early Ln3+ ions (La, Ce, Nd, and Eu) show inner sphere Cl- complexation along with a loss of H2O. The average chloride coordination numbers and Ln-Cl bond lengths are as follows: La3+, N = 2.1, R = 2.92 A; Ce3+, N = 1.8, R = 2.89 A; Nd3+, N = 1.9, R = 2.85 A; Eu3+, N = 1.1, R = 2.81 A. The extent of Cl- ion complexation decreases going across the Ln3+ series to the point where Yb3+ shows no Cl- complexation and no loss of coordinated water molecules. The actinide ions, Am3+ and Cm3+, show the same structural effects as the early Ln3+ ions, i.e., Cl- ion replacement of the H2O at high chloride thermodynamic activities. The Clion coordination numbers and An-Cl bond lengths are: Am3+, N = 1.8, R = 2.81 A; Cm3+, N = 2.4, R = 2.76 A. When combined with results reported previously for Pu3+ which showed no significant chloride complexation in 12 M LiCl, these results suggest that the extent of chloride complexation is increasing across the An3+ series. The origin of the differences in chloride complex formation between the Ln3+ and An3+ ions and the relevance to earlier work is discussed.
RESUMO
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.
Assuntos
Encéfalo/metabolismo , Citoesqueleto/fisiologia , Proteínas de Filamentos Intermediários/metabolismo , Actinas/metabolismo , Animais , Humanos , Proteínas de Filamentos Intermediários/genética , Camundongos , Fenótipo , Fosfatidilinositol 4,5-Difosfato/metabolismo , PlectinaRESUMO
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.
Assuntos
Quelantes/farmacologia , Netúnio/farmacocinética , Administração Oral , Animais , Radiação de Fundo , Osso e Ossos/metabolismo , Catecóis/metabolismo , Catecóis/farmacologia , Quelantes/metabolismo , Relação Dose-Resposta a Droga , Feminino , Ligantes , Camundongos , Netúnio/administração & dosagem , Oxirredução , Piridinas/metabolismo , Piridinas/farmacologiaRESUMO
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.
Assuntos
Actinas/metabolismo , Gelsolina/química , Fosfatidilinositóis/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Citoesqueleto/química , Gelsolina/metabolismo , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de ProteínaAssuntos
Citoesqueleto/fisiologia , Citoesqueleto de Actina , Actinas , Cátions , Polímeros , Estresse MecânicoRESUMO
HIV-1 RNA was quantitated directly by capillary electrophoresis with laser-induced fluorescence (CE-LIF). CE-LIF was used to analyze cellular RNA and various nucleotide complexes. A fluorescently labeled DNA probe (DNA/RNA complex) in conjunction with thiazole orange intercalator was determined to have optimal stability and sensitivity for RNA analysis. Based on this observation, a hybridization method using a HIV-specific fluorescently labeled probe with analysis by CE-LIF was developed. Plasma samples from a HIV-seropositive patient were lysed to obtain RNA, hybridized with the HIV-specific probe and analyzed by CE-LIF. As little as 19 fg (1710 copies per 1 ml of starting plasma) of HIV RNA can be reliably and quantitatively detected. CE-LIF appears to be an efficient and sensitive method to quantitatively analyze RNA from a variety of sources.
Assuntos
Genes Virais , HIV-1/genética , RNA Viral/sangue , Animais , Benzotiazóis , Sondas de DNA , Eletroforese Capilar , Fluoresceínas , Corantes Fluorescentes , Humanos , Substâncias Intercalantes , Lasers , Nucleotídeos/análise , Quinolinas , RNA/análise , RNA/genética , RNA/isolamento & purificação , RNA Viral/genética , Sensibilidade e Especificidade , Espectrometria de Fluorescência , Spodoptera/genética , TiazóisRESUMO
Gelsolin is a calcium-activated actin-binding protein with six subdomains. The N-terminal (G1) domain is essential for actin-filament-severing activity while other domains within G2-3 position the protein on the filament side allowing G1 to sever. In order to generate reagents capable of competitively inhibiting endogenous gelsolin and, potentially, other actin filament regulatory protein, we expressed several truncates of gelsolin in Escherichia coli, and analyzed how they affected the in vitro activity of two different actin-binding proteins, gelsolin and cofilin. A Ca2+-sensitive truncate containing G2-6 inhibited the F-actin-depolymerizing activities of both gelsolin and cofilin, while a G2-3 truncate was less effective. Using two independent assays, our results support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kinetics. Cosedimentation assays in the presence of calcium demonstrate that the G2-6 truncate binds to F-actin more strongly than the G2-3 truncate consistent with a protection mechanism by conformational change of F-actin and/or competitive binding to actin filaments which depends upon the presence of actin filament binding domains.