RESUMEN
Antibody inhibitor development in hemophilia A represents the most significant complication resulting from factor VIII (fVIII) replacement therapy. Recent studies have demonstrated that epitopes present in the C1 domain contribute to a pathogenic inhibitor response. In this study, we report the structure of a group A anti-C1 domain inhibitor, termed 2A9, in complex with a B domain-deleted, bioengineered fVIII construct (ET3i). The 2A9 epitope forms direct contacts to the C1 domain at 3 different surface loops consisting of Lys2065-Trp2070, Arg2150-Tyr2156, and Lys2110-Trp2112. Additional contacts are observed between 2A9 and the A3 domain, including the Phe1743-Tyr1748 loop and the N-linked glycosylation at Asn1810. Most of the C1 domain loops in the 2A9 epitope also represent a putative interface between fVIII and von Willebrand factor. Lastly, the C2 domain in the ET3i:2A9 complex adopts a large, novel conformational change, translocating outward from the structure of fVIII by 20 Å. This study reports the first structure of an anti-C1 domain antibody inhibitor and the first fVIII:inhibitor complex with a therapeutically active fVIII construct. Further structural understanding of fVIII immunogenicity may result in the development of more effective and safe fVIII replacement therapies.
Asunto(s)
Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Factor VIII/química , Proteínas Recombinantes de Fusión/química , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Factor VIII/genética , Factor VIII/inmunología , Factor VIII/metabolismo , Hemofilia A/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Dominios Proteicos/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , PorcinosRESUMEN
The expansion of humans into previously unoccupied parts of the globe is thought to have driven the decline and extinction of numerous vertebrate species. In New Zealand, human settlement in the late thirteenth century AD led to the rapid demise of a distinctive vertebrate fauna, and also a number of 'turnover' events where extinct lineages were subsequently replaced by closely related taxa. The recent genetic detection of an Australian little penguin (Eudyptula novaehollandiae) in southeastern New Zealand may potentially represent an additional 'cryptic' invasion. Here we use ancient-DNA (aDNA) analysis and radiocarbon dating of pre-human, archaeological and historical Eudyptula remains to reveal that the arrival of E. novaehollandiae in New Zealand probably occurred between AD 1500 and 1900, following the anthropogenic decline of its sister taxon, the endemic Eudyptula minor. This rapid turnover event, revealed by aDNA, suggests that native species decline can be masked by invasive taxa, and highlights the potential for human-mediated biodiversity shifts.
Asunto(s)
Distribución Animal , ADN Mitocondrial/genética , Filogenia , Spheniscidae/fisiología , Animales , Fósiles , Datos de Secuencia Molecular , Nueva Zelanda , Datación Radiométrica , Análisis de Secuencia de ADN , Spheniscidae/genéticaRESUMEN
While terrestrial megafaunal extinctions have been well characterized worldwide, our understanding of declines in marine megafauna remains limited. Here, we use ancient DNA analyses of prehistoric (<1450-1650 AD) sea lion specimens from New Zealand's isolated Chatham Islands to assess the demographic impacts of human settlement. These data suggest there was a large population of sea lions, unique to the Chatham Islands, at the time of Polynesian settlement. This distinct mitochondrial lineage became rapidly extinct within 200 years due to overhunting, paralleling the extirpation of a similarly large endemic mainland population. Whole mitogenomic analyses confirm substantial intraspecific diversity among prehistoric lineages. Demographic models suggest that even low harvest rates would likely have driven rapid extinction of these lineages. This study indicates that surviving Phocarctos populations are remnants of a once diverse and widespread sea lion assemblage, highlighting dramatic human impacts on endemic marine biodiversity. Our findings also suggest that Phocarctos bycatch in commercial fisheries may contribute to the ongoing population decline.
Asunto(s)
Biodiversidad , Conservación de los Recursos Naturales , Extinción Biológica , Leones Marinos/genética , Animales , ADN Antiguo/análisis , ADN Mitocondrial/genética , Explotaciones Pesqueras , Actividades Humanas , Humanos , Islas , Nueva ZelandaRESUMEN
The biological status of the so-called 'Upland seal' has remained contentious ever since historical records described a distinct seal from the uplands of New Zealand's (NZ) remote sub-Antarctic islands. Subsequent genetic surveys of the NZ fur seal (Arctocephalus forsteri) detected two highly-divergent mtDNA clades, hypothesized to represent a post-sealing hybrid swarm between 'mainland' (Australia-NZ; A. forsteri) and sub-Antarctic (putative 'Upland'; A. snaresensis) lineages. We present ancient-DNA analyses of prehistoric mainland NZ and sub-Antarctic fur seals, revealing that both of these genetic lineages were already widely distributed across the region at the time of human arrival. These findings indicate that anthropogenic factors did not contribute to the admixture of these lineages, and cast doubt on the validity of the Upland seal. Human-mediated impacts on Arctocephalus genetic diversity are instead highlighted by a dramatic temporal haplotype frequency-shift due to genetic drift in heavily bottlenecked populations following the cessation of industrial-scale harvesting. These extinction-recolonisation dynamics add to a growing picture of human-mediated change in NZ's coastal and marine ecosystems.
Asunto(s)
ADN Mitocondrial/genética , Lobos Marinos/clasificación , Lobos Marinos/genética , Animales , Regiones Antárticas , Australia , Ecosistema , Flujo Genético , Variación Genética , Haplotipos , Actividades Humanas , Criaturas Legendarias , Nueva Zelanda , FilogeniaRESUMEN
Unravelling prehistoric anthropogenic impacts on biodiversity represents a key challenge for biologists and archaeologists. New Zealand's endemic Stewart Island Shag (Leucocarbo chalconotus) comprises two distinct phylogeographic lineages, currently restricted to the country's south and southeast. However, fossil and archaeological remains suggest a far more widespread distribution at the time of Polynesian settlement ca. 1280 AD, encompassing much of coastal South Island. We used modern and ancient DNA, radiocarbon dating, and Bayesian modelling, to assess the impacts of human arrival on this taxon. Our analyses show that the southeast South Island (Otago) lineage was formerly widespread across coastal South Island, but experienced dramatic population extinctions, range retraction and lineage loss soon after human arrival. By comparison, the southernmost (Foveaux Strait) lineage has experienced a relatively stable demographic and biogeographic history since human arrival, retaining much of its mitochondrial diversity. Archaeological data suggest that these contrasting demographic histories (retraction vs. stability) reflect differential human impacts in mainland South Island vs. Foveaux Strait, highlighting the importance of testing for temporal and spatial variation in human-driven faunal declines.
Asunto(s)
Aves/genética , Variación Genética , Filogenia , Animales , Teorema de Bayes , ADN Mitocondrial/genética , Extinción Biológica , Fósiles , Humanos , Funciones de Verosimilitud , Modelos Genéticos , Datos de Secuencia Molecular , Nueva Zelanda , Filogeografía , Dinámica Poblacional , Datación Radiométrica , Análisis de Secuencia de ADN , Análisis Espacio-TemporalRESUMEN
The competitive processes that result in elimination/pruning of developing synapses are incompletely understood. Serial electron microscopy was used to image postnatal mouse neuromuscular junctions where elimination is well studied and events at every synaptic contact can be examined. Glial or Schwann cells (SCs) are shown to have two activities during elimination: their processes separate nerve terminals from each other and from the muscle fiber; they contact the plaque of acetylcholine receptors, apposing this surface as closely as the nerve, limiting the area where synaptic transmission occurs. SCs phagocytose nerve terminals contacting the muscle fiber. This phagocytosis involves all axons; SCs are not selecting the winner but rather driving turnover. Previous modeling of stochastic turnover and reoccupation of nerve contacts shows that single innervation of synaptic sites can result. Thus, our study shows roles of SCs in neuromuscular development beyond the previous demonstration of consumption of synaptic inputs after their elimination.
Asunto(s)
Axones/fisiología , Unión Neuromuscular/fisiología , Células de Schwann/fisiología , Animales , Axones/ultraestructura , Ratones , Unión Neuromuscular/ultraestructura , Receptores Colinérgicos/fisiología , Células de Schwann/ultraestructuraRESUMEN
A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and ß-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.
Asunto(s)
Phytophthora/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Plantas/microbiología , Australia , Frutas/microbiología , Datos de Secuencia Molecular , Filogenia , Phytophthora/clasificación , Phytophthora/genética , Phytophthora/crecimiento & desarrollo , Esporas/crecimiento & desarrollo , Estados UnidosRESUMEN
The reaction between CH radicals and NH(3) molecules is known to be rapid down to at least 23 K {at which temperature k = (2.21 ± 0.17) × 10(-10) cm(3) molecule(-1) s(-1): Bocherel ; et al. J. Phys. Chem. 1996, 100, 3063}. However, there have been only limited theoretical investigations of this reaction and its products are not known. This paper reports (i) ab initio quantum chemical calculations on the energy paths that lead to various reaction products, (ii) calculations of the overall rate constant and branching ratios to different products using transition state and master equation methods, and (iii) an experimental determination of the H atom yield from the reaction. The ab initio calculations show that reaction occurs predominantly via the initial formation of a datively bound HC-NH(3) complex and reveal low energy pathways to three sets of reaction products: H(2)CNH + H, HCNH(2) + H, and CH(3) + NH. The transition state calculations indicate the roles of "outer" and "inner" transition states and yield rate constants between 20 and 320 K that are in moderate agreement with the experimental values. These calculations and those using the master equation approach show that the branching ratio for the most exothermic reaction, to H(2)CNH + H, is ca. 96% throughout the temperature range covered by the calculations, with those to HCNH(2) + H and CH(3) + NH being (4 ± 3)% and <0.3%, respectively. In the experiments, multiple photon dissociation of CHBr(3) was used to generate CH radicals and laser-induced fluorescence at 121.56 nm (VUV-LIF) was employed to observe H atoms. By comparing signals from CH + NH(3) with those from CH + CH(4), where the yield of H atoms is known to be unity, it is possible to estimate that the yield of H atoms from CH + NH(3) is equal to 0.89 ± 0.07 (2σ), in satisfactory agreement with the theoretical estimate.
RESUMEN
We report the first measurements of rate constants for the reaction in which OH radicals associate with O(2) to form HO(3). Our recent measurements (Science, 2010, 328, 1258) have shown that the HO-O(2) bond dissociation energy is only (12.3 ± 0.3) kJ mol(-1). Consequently, above ca. 90 K under attainable experimental conditions, the rate of the reverse dissociation of HO(3) becomes comparable to, and then greater than, the rate of the forward association reaction. We have used the CRESU (Cinétique de Réaction en Ecoulement Supersonique Uniforme) method to access low temperatures and have explored the kinetics of OH + O(2) + M â HO(3) + M in two series of experiments. At temperatures between 55.9 and 79.2 K, the OH radicals, created by pulsed laser photolysis of H(2)O(2) and observed by laser-induced fluorescence, decayed by pseudo-first-order kinetics to effectively zero concentration at longer times. The third-order rate constants derived from these experiments fit the expression: k(3rd)(o) (T) = (4.2 ± 1.9) × 10(-34) (T/298 K)(-(3.5 ± 0.3)) cm(6) molecule(-2) s(-1). At temperatures between 87.4 and 99.8 K, rate constants for the association reaction were determined allowing for the significant occurrence of the reverse dissociation reaction. The values of the derived rate constants are consistent with those obtained in the lower temperature range, though the errors are larger. The experimental values of k(3rd)(o) (T) are compared with (a) those for other association reactions involving species of similar complexity, and (b) values of k(3rd)(o) (T) estimated according to both the energy transfer (ET) and the radical-complex (RC) mechanisms. We conclude that the RC mechanism probably makes the major contribution to the association of OH + O(2) at the low temperatures of our experiments.
RESUMEN
Anti-drug antibodies to coagulation factor VIII (fVIII), often termed inhibitors, present the greatest economical and treatment related obstacle in the management of hemophilia A. Although several genetic and environmental risk factors associated with inhibitor development have been identified, the precise mechanisms responsible for the immune response to exogenous fVIII therapies remain undefined. Clinical trials suggest there is an increased immunogenic potential of recombinant fVIII compared to plasma-derived products. Additional biochemical and immunological studies have demonstrated that changes in recombinant fVIII production and formulation can alter fVIII structure and immunogenicity. Recently, one study demonstrated increased immunogenicity of the recombinant fVIII product Helixate in hemophilia A mice following oxidation with hypochlorite (ClO-). It is widely reported that protein aggregates within drug products can induce adverse immune reactions in patients. Several studies have therefore investigated the prevalence of molecular aggregates in commercial recombinant products with and without use-relevant stress and agitation. To investigate the potential link between oxidation-induced immunogenicity and molecular aggregation, we analyzed the recombinant fVIII product, Helixate, via sedimentation velocity analytical ultracentrifugation following oxidation with ClO-. At 80 µM ClO-, a concentration that reduced the specific-activity by 67%, no detectable increase in large molecular aggregates (s > 12 S) was observed when compared to non-oxidized fVIII. This lack of aggregates was demonstrated both in commercial excipient as well as a HEPES buffered saline formulation. These data suggest that oxidation induced immunogenicity is independent of aggregate-mediated immune response. Therefore, our data support multiple, independent mechanisms underlying fVIII immunogenicity.
Asunto(s)
Factor VIII/química , Factor VIII/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Hemofilia A/tratamiento farmacológico , Hemofilia A/inmunología , Humanos , Oxidación-Reducción , UltracentrifugaciónRESUMEN
BACKGROUND: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. OBJECTIVES: To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. METHODS: The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. RESULTS: Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. CONCLUSIONS: These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.
Asunto(s)
Factor VIII/química , Hemofilia A , Animales , Dominios C2 , Cromatografía Liquida , Hemofilia A/tratamiento farmacológico , Humanos , Ingeniería de Proteínas , Proteínas Recombinantes/química , Porcinos , Espectrometría de Masas en TándemRESUMEN
The reaction between CN radicals and NH3 molecules has been studied experimentally over an unusually wide range of temperature (25-716 K). Below 295 K, the rate constant exhibits a strong negative dependence on temperature; that is, it increases sharply as the temperature is lowered. The present work analyses the kinetics of this reaction theoretically, both to explain this unusual temperature-dependence and to identify the major products of the reaction--which have not been well established by experiment. Quantum chemical calculations at the CCSD(T) theoretical level show that the minimum energy path for reaction proceeds: (a) first, via a potential well, which is 39.3 kJ mol(-1) below the energy of the separated reactants, when allowance is made for zero-point energies, corresponding to a quite strongly bound NC-NH3 complex, and (ii) then over a 'submerged' barrier with a crest 10.9 kJ mol(-1) below the energy of the reactants to the products HCN + NH2. These ab initio calculations also demonstrate that there is no low energy path to the products NCNH2 + H. The dynamics of the main reaction have been further investigated using the two transition state model of Klippenstein and co-workers, in which transition state theory is applied at the selected E, J microcanonical level. The rate constants calculated for temperatures between 25 and 200 K are in excellent agreement with the experimental values.
RESUMEN
By comparing H-atom yields from the reactions of CN with C(2)H(2) and NH(3), it has been confirmed that the latter reaction produces insignificant amounts of H-atoms, implying that it proceeds exclusively to HCN + NH(2).
RESUMEN
Advances in experimental techniques, especially the development of the CRESU (Cinétique de Réaction en Ecoulement Supersonique Uniforme) method, allow many gas-phase molecular processes to be studied at very low temperatures. This Review focuses on the reactions of molecular and atomic radicals with neutral molecules. Rate constants for almost 50 such reactions have been measured at temperatures as low as 13 K by using the CRESU method. The surprising demonstration that so many reactions between electrically neutral species can be extremely rapid at these very low temperatures has excited interest both from theoreticians and from those seeking to understand the chemistry that gives rise to the 135 or so molecules that are present in low-temperature molecular clouds in the interstellar medium. Theoretical treatments of these reactions are based on the idea that a reaction occurs when the long-range potential between the reagent species brings them into close contact. The astrochemical context, theoretical studies, and the determination of the rate constants of these low-temperature reactions are critically discussed.
RESUMEN
The role of biotic agents in the dieback syndrome Mundulla Yellows (MY) was investigated by analysis of 40 Eucalyptus camaldulensis, E. leucoxylon, or E. cladocalyx trees and soil samples from South Australia and Victoria, Australia. No pathogenic fungi, bacteria, phytoplasmas, or insect pests or vectors were found to be associated with MY. However, nematode analysis identified Merlinius spp. to be associated with soil, but not roots, from symptomatic trees. Interveinal chlorosis symptoms were not transmissible by seed, mechanical inoculation, or grafting using plant material derived from symptomatic trees. Virus-like particles were detected at a single symptomatic study site using transmission electron microscopy. MY symptoms were induced in E. camaldulensis seedlings by sowing seed from asymptomatic trees into sterilized and unsterilized soil collected from underneath symptomatic trees. Significantly, sterilized soil induced more severe symptoms in seedlings than unsterilized soil. Soil collected from under asymptomatic trees did not induce MY symptoms. This preliminary investigation indicates that, with the exception of Merlinius spp., pathogenic organisms and pests were not consistently associated with MY symptoms.
RESUMEN
The role of HO3 as a temporary reservoir of atmospheric OH radicals remains an open question largely because of the considerable uncertainty in the value of the dissociation energy of the HO-O2 bond (D0) or, equivalently, the standard enthalpy of formation of HO3 (Delta(f)H;{\overline{);\circ }}$$). Using a supersonic flow apparatus, we have observed by means of laser-induced fluorescence the decay of OH radicals in the presence of O2 at temperatures between 55.7 and 110.8 kelvin (K). Between 87.4 and 99.8 K, the OH concentration approached a nonzero value at long times, allowing equilibrium constants for the reaction with O2 to be calculated. Using expressions for the equilibrium constant from classical and statistical thermodynamics, and values of partition functions and standard entropies calculated from spectroscopic data, we derived values of D0 = (12.3 +/- 0.3) kilojoules per mole and Delta(f)H;{\overline{);\circ }}$$ (298 K) = (19.3 +/- 0.5) kilojoules per mole. The atmospheric implications of HO3 formation are therefore very slight.
RESUMEN
The rates of chemical reactions and the dependence of their rate constants on temperature are of central importance in chemistry. Advances in the temperature-range and accuracy of kinetic measurements, principally inspired by the need to provide data for models of combustion, atmospheric, and astrophysical chemistry, show up the inadequacy of the venerable Arrhenius equation--at least, over wide ranges of temperature. This critical review will address the question of how to reach an understanding of the factors that control the rates of 'non-Arrhenius' reactions. It makes use of a number of recent kinetic measurements and shows how developments in advanced forms of transition state theory provide satisfactory explanations of complex kinetic behaviour (72 references).
RESUMEN
Rate coefficients (ktot,Ni) are reported (a) for total removal (reactive+inelastic) of CN(X2Sigma+,v=2,Ni) radicals from selected rotational levels (Ni=0, 1, 6, 10, 15, and 20) and (b) for state-to-state rotational energy transfer (ki-->f) between levels Ni and other rotational levels Nf in collisions with C2H2. CN radicals were generated by pulsed laser photolysis of NCNO at 573 nm. A fraction of the radicals was then promoted to a selected rotational level in v=2 using a tunable infrared "pump" laser operating at approximately 2.45 microm, and the subsequent fate of this subset of radicals was monitored using pulsed laser-induced fluorescence (PLIF). Values of ktot,Ni were determined by observing the decay of the PLIF signals as the delay between pump and probe laser pulses was systematically varied. In a second series of experiments, double resonance spectra were recorded at a short delay between the pump and probe laser pulses. Analysis of these spectra yielded state-to-state rate coefficients for rotational energy transfer, ki-->f. The difference between the sum of these rate coefficients, Sigmafki-->f, and the value of ktot,Ni for the same level Ni is attributed to the occurrence of chemical reaction, yielding values of the rotationally selected rate coefficients (kreac,Ni) for reaction of CN from specified rotational levels. These rate coefficients decrease from (7.9+/-2.2)x10(-10) cm3molecule-1 s-1 for Ni=0 to (0.8+/-1.3)x10(-10) cm3 molecule-1 s-1 for Ni=20. The results are briefly discussed in the context of microcanonical transition state theory and the statistical adiabatic channel model.
RESUMEN
A remarkable number of reactions between neutral free radicals and neutral molecules have been shown to remain rapid down to temperatures as low as 20 kelvin. The rate coefficients generally increase as the temperature is lowered. We examined the reasons for this temperature dependence through a combined experimental and theoretical study of the reactions of O(3P) atoms with a range of alkenes. The factors that control the rate coefficients were shown to be rather subtle, but excellent agreement was obtained between the experimental results and microcanonical transition state theory calculations based on ab initio representations of the potential energy surfaces describing the interaction between the reactants.