bluuebunny/crossref_metadata_2025 huggingface dataset:
A compact time‐of‐flight mass spectrometer for high‐flux cosmic dust analysis
Authors: Austin Daniel E. , Beauchamp J. L. , Manning Heidi L. K. , Bailey Carl L.
Published: June 2004

Time‐of‐flight mass spectrometers on spacecraft are the most direct method for determining chemical composition of cosmic dust grains. Miniaturization of these instruments presents many challenges. Larger space‐charge effects, greater deviations from the paraxial approximation, and various ion‐optical aberrations negatively affect mass resolution in small time‐of‐flight instruments. We report on the building and testing of an instrument design that may reduce these effects. In addition to a linear reflectron, ions pass through a ring aperture that transmits only those ions with transverse velocity components that fall within a specific range. This novel design focuses ions onto a detector with greatly reduced spherical aberration. Space‐charge effects and the effects of impact plate cratering and grid scatter are also reduced using this design. Controlled impacts of iron microparticles at several km/s demonstrate instrument performance. This instrument is suited for characterization of cosmic dust in regions of very high dust flux, such as a comet flyby, and it may also have practical laboratory or field applications.
Photophysics and photochemistry of water ice films on graphite
Authors: Bergeld J. , Gleeson M. , Kasemo B. , Chakarov D.
Published: June 2004

Our model system consists of water ice films with submonolayer to several monolayer thickness grown at ultrahigh vacuum (UHV) and low (from ∼25 K) temperatures on an atomically clean graphite (0001) surface. We characterize them experimentally with temperature programmed desorption‐isothermal desorption spectroscopy (TPD‐ITD) and high‐resolution electron energy loss spectroscopy (HREELS) and provide examples of how ice structure and chemical properties are altered by incoming photons in broad wavelength and photon flux ranges (1.6 × 1016–5.5 × 1024 photons cm−2 s−1 for λ = 220–700 nm). The phenomena of “photon annealing” of pure amorphous films and their structure‐selective laser ablation are discussed. In the case of water ice doped with alkali metals and coadsorbed with NO, we report on the observations of photoreactions taking place on the graphite‐ice interface. Depending on the composition, irradiation leads to formation of H2, CH4, CO, NH3, and CO2. In all examples the attention is on the operating mechanism for the transformations. The role of photo‐excited charge carriers in the graphite substrate is underlined.
Spatial and temporal structure of shear layer turbulence at a stream confluence
Authors: Rhoads Bruce L. , Sukhodolov Alexander N.
Published: June 2004

The shear layer that develops at the interface between converging flows is a prominent but poorly understood hydrodynamic feature at stream confluences. To examine the spatial and temporal characteristics of turbulence and coherent turbulent structures within a shear layer, three‐dimensional velocity measurements were obtained at a small stream confluence in Illinois using two acoustic Doppler velocimeters spaced at various separation distances. Results indicate that coherent structures can be readily identified through cross‐correlation analysis of the velocity time series for different measurement locations. Maximum correlations decrease relatively slowly over short separation distances but decrease rapidly for separation distances greater than about 2 times the flow depth. Convective velocities of coherent structures derived from the correlation analysis differ from local mean velocities and from spatially averaged mean velocities, indicating that coherent structures evolve as they are transported along the shear layer, a finding consistent with dye‐tracing experiments. Despite this evolution, correlation and spectral analyses indicate that the frozen turbulence hypothesis holds for frequencies corresponding to length scales of at least some shear‐generated coherent vortices. A domain of quasi‐two‐dimensional turbulence energy at low frequencies appears to be associated with large‐scale intrusions of fluid from one stream into the other along the mixing interface.
Intensification of the annual cycle in the tropical Pacific due to greenhouse warming
Authors: Timmermann A. , Jin F.‐F. , Collins M.
Published: June 2004

The annual cycle is one of the most important components of the global climate system Yet little attention has been paid to the response of the equatorial annual cycle to anthropogenic climate change. Here we present results from a global climate model with high tropical resolution that simulates a strong intensification of the annual cycle in the tropical Pacific in response to increased greenhouse gas concentrations. This sensitivity of the tropical annual cycle to greenhouse warming can be explained in terms of tropical ocean‐atmosphere feedbacks. A possible amplification of the tropical annual cycle is expected to have also profound ecological and societal impacts.
Anomalous recent growth of part of a large Arctic ice cap: Austfonna, Svalbard
Authors: Bamber Jonathan , Krabill William , Raper Vivienne , Dowdeswell Julian
Published: June 2004

Observations from repeat‐pass airborne laser altimetry, acquired in 1996 and 2002, indicate an anomalous positive ice‐surface elevation change for the central accumulation area of the largest ice cap in the Eurasian Arctic; Austfonna, eastern Svalbard. The increase is equivalent to 35% of the long‐term annual accumulation rate and coincides with the loss of perennial sea ice in the adjacent Barents Sea, which we conclude is the most likely explanation for the increase. Extrapolation of the observed trends in sea ice decline, over the next 50 years, suggests large perturbations in the mass‐balance of other Arctic ice masses may be expected.
Application of the Yin‐Yang grid to a thermal convection of a Boussinesq fluid with infinite Prandtl number in a three‐dimensional spherical shell
Authors: Yoshida Masaki , Kageyama Akira
Published: June 2004

A new numerical finite difference code has been developed to solve a thermal convection of a Boussinesq fluid with infinite Prandtl number in a three‐dimensional spherical shell. A kind of the overset (Chimera) grid named “Yin‐Yang grid” is used for the spatial discretization. The grid naturally avoids the pole problems which are inevitable in the latitude‐longitude grids. The code is applied to numerical simulations of mantle convection with uniform and variable viscosity. The validity of the Yin‐Yang grid for the mantle convection simulation is confirmed.
Deliquescence and crystallization of ammonium sulfate‐glutaric acid and sodium chloride‐glutaric acid particles
Authors: Pant Atul , Fok Abel , Parsons Matthew T. , Mak Jackson , Bertram Allan K.
Published: June 2004

In the following, we report the deliquescence relative humidities (DRH) and crystallization relative humidities (CRH) of mixed inorganic‐organic particles, specifically ammonium sulfate‐glutaric acid and sodium chloride‐glutaric acid particles. Knowledge of the DRH and CRH of mixed inorganic‐organic particles is crucial for predicting the role of aerosol particles in the atmosphere. Our DRH results are in good agreement with previous measurements, but our CRH results are significantly lower than some of the previous measurements reported in the literature. Our studies show that the DRH and CRH of ammonium sulfate and sodium chloride only decreased slightly when the mole fraction of the acid was less than 0.4. If other organics in the atmosphere behave in a similar manner, then the DRH and CRH of mixed inorganic‐organic atmospheric particles will only be slightly less than the DRH and CRH of pure inorganic particles when the organic mole fraction is less than 0.4. Our results also show that if the particles contain a significant amount of organics (mole fraction > 0.5) the crystallization relative humidity decreases significantly and the particles are more likely to remain in the liquid state. Further work is needed to determine if other organics species of atmospheric importance have a similar effect.
Effects of microbial processes on electrolytic and interfacial electrical properties of unconsolidated sediments
Authors: Abdel Aal Gamal Z. , Atekwana Estella A. , Slater Lee D. , Atekwana Eliot A.
Published: June 2004

The effect of microbial processes on electrical properties of unconsolidated sediments was investigated in a laboratory experiment consisting of biotic and abiotic sand columns. The biotic column (nutrient, diesel and bacteria) showed (a) temporal increase in the real, imaginary, and surface conductivity, and (b) temporal decrease in the formation factor. The abiotic columns (nutrient; and nutrient and diesel) showed no significant changes. Increase in microbial population numbers, decrease in organic carbon source, nitrate, and sulfate and increase in dissolved inorganic carbon and fluid conductivity were indicative of microbial activity in the biotic column. We also measure relative increase in the interfacial electrical properties that exceed relative increase in the electrolytic conductivity. Thus changes in the real and imaginary conductivity were induced by microbial processes. These results suggest that interpretation of geoelectrical data from near surface environments should consider effects of microbial processes.
The footprint of urban climates on vegetation phenology
Authors: Zhang Xiaoyang , Friedl Mark A. , Schaaf Crystal B. , Strahler Alan H. , Schneider Annemarie
Published: June 2004

Human activity, through changing land use and other activities, is the most fundamental source of environmental change on the Earth. Urbanization and the resultant “urban heat islands” provide a means for evaluating the effect of climate warming on vegetation phenology. Using data from the Moderate Resolution Imaging Spectroradiometer, we analyzed urban‐rural differences in vegetation phenological transition dates and land surface temperatures for urban areas larger than 10 km2 in eastern North America. The results show that the effect of urban climates on vegetation phenology decays exponentially with distance from urban areas with substantial influence up to 10 km beyond the edge of urban land cover, and that the ecological “footprint” of urban climates is about 2.4 times that of urban land use in eastern North America. The net effect is an increase in the growing season by about 15 days in urban areas relative to adjacent unaffected rural areas.
Antibodies: a Paradigm for the Evolution of Molecular Recognition
Authors: Neuberger M. S.
Published: June 2004

Novel proteins have been elaborated over evolutionary time by an iterative alternation of mutation and selection. In a similar way, the humoral immune system also uses an iterative alternation of mutation and selection to generate novel antibodies that display a high affinity for their cognate antigen - but this is achieved in a matter of a days. Gene rearrangement is used to produce a primary repertoire of antibodies and, on entering the body, antigen triggers the clonal expansion of those B lymphocytes that express a cognate antibody, albeit one of low affinity. Rapid and specific affinity maturation is then achieved by subjecting the immunoglobulin genes in the rapidly expanding B cells to a period of intense mutation. The intensity of this mutational assault is tolerated because it is targeted specifically to the immunoglobulin genes, causing relatively little damage to other loci. Antigen-mediated selection then allows the preferential expansion of those mutants expressing antibodies displaying improved binding characteristics. Here, studies are described that have been performed to glean insight into the mechanisms of the hypermutation and selection processes. Experiments are also described in which an attempt has been made to recapitulate aspects of physiological antibody generation in vitro, allowing the development of novel approaches to the generation of proteins with high-affinity binding sites.
Specificity of 14-3-3 isoform dimer interactions and phosphorylation
Authors: Aitken A. , Baxter H. , Dubois T. , Clokie S. , Mackie S. , Mitchell K. , Peden A. , Zemlickova E.
Published: June 2004

Proteins that interact with 14-3-3 isoforms are involved in regulation of the cell cycle, intracellular trafficking/targeting, signal transduction, cytoskeletal structure and transcription. Recent novel roles for 14-3-3 isoforms include nuclear trafficking the direct interaction with cruciform DNA and with a number of receptors, small G-proteins and their regulators. Recent findings also show that the mechanism of interaction is also more complex than the initial finding of the novel phosphoserine/threonine motif. Non-phosphorylated binding motifs that can also be of high affinity may show a more isoform-dependent interaction and binding of a protein through two distinct binding motifs to a dimeric 14-3-3 may also be essential for full interaction. Phosphorylation of specific 14-3-3 isoforms can also regulate interactions. In many cases, they show a distinct preference for a particular isoform(s) of 14-3-3. A specific repertoire of dimer formation may influence which of the 14-3-3-interacting proteins could be brought together. Mammalian and yeast 14-3-3 isoforms show a preference for dimerization with specific partners in vivo.
Survival-promoting functions of 14-3-3 proteins
Authors: Masters S. C. , Subramanian R. R. , Truong A. , Yang H. , Fujii K. , Zhang H. , Fu H.
Published: June 2004

The 14-3-3 proteins are a family of phosphoserine/phosphothreonine-binding molecules that control the function of a wide array of cellular proteins. We suggest that one function of 14-3-3 is to support cell survival. 14-3-3 proteins promote survival in part by antagonizing the activity of associated proapoptotic proteins, including Bad and apoptosis signal-regulating kinase 1 (ASK1). Indeed, expression of 14-3-3 inhibitor peptides in cells is sufficient to induce apoptosis. Interestingly, these 14-3-3 antagonist peptides can sensitize cells for effective killing by anticancer agents such as cisplatin. Thus, 14-3-3 may be part of the cellular machinery that maintains cell survival, and targeting 14-3-3-ligand interactions may be a useful strategy to enhance the efficacy of conventional anticancer agents.
Effect of multiple phosphorylation events on the transcription factors FKHR, FKHRLI and AFX
Authors: Woods Y. L. , Rena G.
Published: June 2004

The insulin-stimulated phosphoinositide 3-kinase (PI 3-kinase)/3-phosphoinositide-dependent kinase-1 (PDK1)/protein kinase B (PKB) kinase cascade is believed to play a critical role in metabolic control and cell survival, largely mediated through PKB phosphorylation of many proteins. Recent findings demonstrate that the transcription factors FKHR (forkhead in rhabdomyosarcoma), AFX (ALL1 fused gene from chromosome X) and FKHRL1 (FKHR-like 1; termed FKHR isoforms) are phosphorylated by PKB in cells, leading to their exit from the nucleus. These exciting results suggest that FKHR isoforms may be critical effectors of PI 3-kinase/PDK1/PKB signalling in vivo.
14-3-3s are DNA-replication proteins
Authors: Zannis-Hadjopoulos M. , Novac O. , Alvarez D. , Price G. B.
Published: June 2004

14-3-3 proteins are conserved multifunctional molecules, involved in many biological processes. Several 14-3-3 isoforms were recently shown to be cruciform DNA-binding proteins, which is a new activity ascribed to the 14-3-3 family. As cruciform-binding proteins, 14-3-3 proteins are putatively involved in the regulation of DNA replication. Inverted repeat sequences that are able to extrude into cruciform structures are a common feature of replication origins in both prokaryotes and eukaryotes. The involvement of cruciform structures in the initiation of DNA replication has been demonstrated. A leading model of 14-3-3 function proposes that they facilitate critical protein-protein interactions, thus serving as a central component of a wide variety of cellular processes.
Transgenic 14-3-3 isoforms in plants: the metabolite profiling of repressed 14-3-3 protein synthesis in transgenic potato plants
Authors: Szopa J.
Published: June 2004

14-3-3 proteins are abundant eukaryotic proteins that interact with many other proteins, thereby modulating their function and thus cell metabolism. The data from mRNA analysis confirm the developmental regulation of 14-3-3 isoform expression in potato plants. In order to test whether or not 14-3-3 protein expression affects plant phenotype and metabolism, transgenic potato plants either overexpressing Cucurbita pepo 14-3-3 or underexpressing endogenous 14-3-3 isoforms were analysed. An increase in tuber number and a decrease in tuber size in the over-expressed transformant was observed; the transgenic plants contain more chlorophyll than the control and they lose it more slowly than the control when transferred to the dark. The 14-3-3-repressed transgenic plants showed a decrease in tuber number and an increase in tuber size; an increase in the fresh weight of the transgenic tubers was also detected. The increased catecholamine level was accompanied by an increased ratio of soluble sugars to starch in overexpressed transformant. The opposite effect was detected in 14-3-3-repressed transgenic plants. All the repressed plants showed significant increases in nitrate reductase (NR) activity, suggesting that the regulation of NR occurs in vivo, and is not isoform-dependent. The increase in NR activity resulted in a significant decrease in nitrate level. The level of sucrose phosphate synthase activity was also significantly increased in all 14-3-3-underexpressed transgenes, and remarkably the increase in enzyme activity was accompanied by respective changes in sucrose levels in the tubers. The most intriguing finding was the significant (2–3-fold) increase in ethylene content in all the 14-3-3-repressed transgenic lines, which probably resulted from a methionine level increase. The substantial increase of ethylene level in the repressed forms might explain the significant shortening of the vegetation period of the analysed transgenic plants.
Identification of secretin, vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations
Authors: Waelbroeck M. , Perret J. , Vertongen P. , Van Craenenbroeck M. , Robberecht P.
Published: June 2004

We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln3 side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp3]glucagon as well as by glucagon.
Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs)
Authors: Fischer J. A. , Muff R. , Born W.
Published: June 2004

The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8–32) and not by CGRP-(8–37), and CRLR-RAMP1, antagonized by CGRP-(8–37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.
Interaction of G-protein-coupled receptors with synaptic scaffolding proteins
Authors: Kreienkamp H.-J. , Soltau M. , Richter D. , Böckers T.
Published: June 2004

The calcium-independent receptors for latrotoxin (CIRL1-CIRL3) constitute a family of seven-transmembrane receptors with an unsually large N-terminal extracellular domain which comprises several motifs usually found in cell adhesion molecules. By yeast two-hybrid screening, we have identified the intracellular C-termini of CIRL1 and CIRL2 as interaction partners of the PDZ domain of the proline-rich synapse-associated protein (ProSAP)/somatostatin receptor-interacting protein (SSTRIP) family of postsynaptic proteins (SSTRIP, ProSAP1 and ProSAP2, also known as shank1-shank3 respectively). Overlay assays indicate that the ProSAP1/shank2 PDZ domain in particular interacts strongly with the C-terminus of CIRL1 and CIRL2. Co-immuno-precipitation of ProSAP1 and CIRL1 (but not CIRL2) from rat brain extracts indicates that this interaction also occurs in vivo in rat brain. The known postsynaptic localization of ProSAP1, as well as our observation that CIRL1 (but not CIRL2) is enriched in postsynaptic density preparations from the rat brain, suggests that CIRL1 is localized pre- as well as post-synaptically in the central nervous system.
Development and potential of non-peptide antagonists for calcitonin-generelated peptide (CGRP) receptors: evidence for CGRP receptor heterogeneity
Authors: Wu D. , Doods H. , Arndt K. , Schindler M.
Published: June 2004

Calcitonin-gene-related peptide (CGRP) is a 37-amino-acid vasodilatory peptide, of which two isoforms, αCGRP and βCGRP, have been described. The use of C-terminal fragments of CGRP peptide, such as human αCGRP-(8–37), has led to the pharmacological subdivision of CGRP receptors into CGRP-1 [high potency for binding of human αCGRP-(8–37)] and CGRP-2 (lower potency) receptors. We have recently developed BIBN4096BS, the first non-peptide CGRP antagonist, which has sub-nanomolar affinity for primate CGRP receptors. The use of BIBN4096BS has led to the discovery of further functional CGRP receptor heterogeneity in rat tissues. To further exploit BIBN4096BS as a pharmacological tool, we used BIBN4096BS in pig left anterior descending coronary vessels and cerebral basilar arteries, and compared functional with molecular data, characterizing CGRP receptor components. Our data show that, apart from a subdivision into CGRP-1 and -2 receptors, BIBN4096BS reveals additional functional differences between the actions of αCGRP and βCGRP. However, evidence for CGRP receptor heterogeneity on a molecular level is scarce.
Bioinformatics and type II G-protein-coupled receptors
Authors: Foord S. M. , Jupe S. , Holbrook J.
Published: June 2004

The best known family B, or Type II, G-protein-coupled receptors (GPCRs) recognize peptides as ligands. The receptors for corticotrophin-releasing factor, parathyroid hormone and secretin typify this group. However, there are only 15 such GPCRs. Many other receptors share sequence homology and have been assigned to this family. The ten ‘Frizzled’ and one ‘Smoothened’ receptors show the lowest sequence homology and are not necessarily G-protein coupled. Drosophila genetics have enabled our understanding of their biology. In contrast, relatively little is known about the largest group with family B, the 33 ‘large amino termini’ or large N-terminal family B seven-transmembrane (LNB 7TM) receptors. This review highlights the similarities found between family B receptors and provides a classification of LNB 7TM receptors.
The human IgA-Fc α receptor interaction and its blockade by streptococcal IgA-binding proteins
Authors: Woof J. M.
Published: June 2004

IgA plays a key role in immune defence of the mucosal surfaces. IgA can trigger elimination mechanisms against pathogens through the interaction of its Fc region with FcαRs (receptors specific for the Fc region of IgA) present on neutrophils, macrophages, monocytes and eosinophils. The human FcαR (CD89) shares homology with receptors specific for the Fc region of IgG (FcαRs) and IgE (FcαRIs), but is a more distantly related member of the receptor family. CD89 interacts with residues lying at the interface of the two domains of IgA Fc, a site quite distinct from the homologous regions at the top of IgG and IgE Fc recognized by FcαR and FcαRI respectively. Certain pathogenic bacteria express surface proteins that bind to human IgA Fc. Experiments with domain-swap antibodies and mutant IgAs indicate that binding of three such proteins (Sir22 and Arp4 of Streptococcus pyogenes and β protein of group B streptococci) depend on sites in the Fc interdomain region of IgA, the binding region also used by CD89. Further, we have found that the streptococcal proteins can inhibit interaction of IgA with CD89, and have thereby identified a mechanism by which a bacterial IgA-binding protein may modulate IgA effector function.
The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors
Authors: Armour K. L. , Atherton A. , Williamson L. M. , Clark M. R.
Published: June 2004

A virally encoded, high-affinity Fc receptor (FcR) is found on herpes simplex virus type 1 (HSV-1) particles and infected cells where its binding of non-immune IgG protects cells from host-mediated lysis. Whilst mutation or aglycosylation of the IgG CH2 domain reduced binding to human FcR, the interaction with HSV-1 FcR was not affected. However, the HSV-1 FcR, unlike human FcR, discriminates between human IgG1 allotypes, being sensitive to changes at positions 214 (CH1) and 356/358 (CH3), away from its proposed binding site at the CH2-CH3 interface. The biological consequences are not known but this is the first evidence of a major functional difference between IgG1 allotypes.
Troybodies and Pepbodies
Authors: Lunde E. , Lauvrak V. , Rasmussen I. B. , Schjetne K. W. , Thompson K. M. , Michaelsen T. E. , Brekke O. H. , Sollid L. M. , Bogen B. , Sandlie I.
Published: June 2004

All antibodies (Abs) with effector function are produced in mammalian cells, whereas bacterial production is restricted to smaller targeting fragments (scFv and Fab) without effector functions. In this project, we isolated different peptides that bind one of several Ab effector molecules. We have developed bacterial expression vectors for direct cloning of these peptides as fusions to scFv and Fab, and have obtained targeting fragments that also have the ability to bind Ab effector molecules. Some of these fusions (pepbodies) may also initiate Ab effector functions. We have also genetically inserted T-cell epitopes into Abs with specificity for antigen-presenting cell (APC) surface molecules to target the Ab-T-cell epitope fusions (Troybodies) to APCs. The approach is to exchange loops in Ig constant domains with single copies of well-defined T-cell epitopes. We have shown that a number of such T-cell epitopes are loaded on to MHC class II on APCs and are presented to specific T-cells. An increase in T-cell activation of up to four orders of magnitude is achieved compared with synthetic peptide. Our current goal is to identify all the loops in all Ig constant domains that may be loaded with T-cell epitopes to produce a multi-vaccine.
Targeted cytokine delivery to neuroblastoma
Authors: Dehal P. K. , Embleton M. J. , Kemshead J. T. , Hawkins R. E.
Published: June 2004

The aim of this study was to construct a fusion protein from the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) and a single-chain Fv fragment (scFv D29) and to investigate its potential to activate cells of the immune system against neuroblastoma cells expressing neural cell adhesion molecule (NCAM). Mammalian cell expression of the scFv D29-GM-CSF fusion protein was compared using a number of vectors, including retroviral and adenoviral vectors. The resultant fusion protein, expressed by HeLa cells, was found by ELISA to bind immobilized recombinant NCAM. Moreover, FACS analysis confirmed binding to the human neuroblastoma cell line SKNBE and a murine neuroblastoma cell line engineered to express the glycosylphosphatidylinositol form of human NCAM (N2A-rKNIE). The fusion protein was also found to stimulate the proliferation of the FDC-P1 haemopoietic cell line, which is dependent on GM-CSF (or interleukin 3) for continued growth. In vitro clonogenic assays indicated that scFv-GM-CSF could selectively induce growth inhibition of SKNBE cells by murine lymphoid cells.
Examining the structure of the mature amyloid fibril
Authors: Makin O. S. , Serpell L. C.
Published: June 2004

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.
Cholesterol and Alzheimer's disease
Authors: Wolozin B.
Published: June 2004

Accumulation of a 40–42-amino acid peptide, termed amyloid-β peptide (Aβ), is associated with Alzheimer's disease (AD), and identifying medicines that inhibit Aβ could help patients with AD. Recent evidence suggests that a class of medicines that lower cholesterol by blocking the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), termed statins, can inhibit Aβ production. Increasing evidence suggests that the enzymes that generate Aβ function best in a high-cholesterol environment, which might explain why reducing cholesterol would inhibit Aβ production. Studies using both neurons and peripheral cells show that reducing cellular cholesterol levels, by stripping off the cholesterol with methyl-β-cyclodextrin or by treating the cells with HMG-CoA reductase inhibitors, decreases Aβ production. Studies performed on animal models and on humans concur with these results. In humans, lovastatin, an HMG-CoA reductase inhibitor, has been shown to reduce Aβ levels in blood of patients by up to 40%. The putative role of Aβ in AD raises the possibility that treating patients with statins might lower Aβ, and thereby either delay the occurrence of AD or retard the progression of AD. Two large retrospective studies support this hypothesis. Both studies suggest that patients taking statins had an approx. 70% lower risk of developing AD. Since statins are widely used by doctors, their ability to reduce Aβ offers a putative therapeutic strategy for treating AD by using medicines that have already been proved safe to use in humans.
Memapsin 2 (β-secretase) as a therapeutic target
Authors: Hong L. , Turner R. T. , Koelsch G. , Ghosh A. K. , Tang J.
Published: June 2004

As β-secretase, memapsin 2 cleaves amyloid-β precursor protein, which leads ultimately to the onset of Alzheimer's disease. As such, memapsin 2 is an excellent target of inhibitor drugs for the treatment of this disease. Here we describe the tools for memapsin 2 inhibitor design that have been developed and results from the structure-based inhibitor design. Strategy for the design of memapsin 2 inhibitors with pharmaceutical potential is also discussed.
γ-Secretase inhibition
Authors: Beher D. , Shearman M. S.
Published: June 2004

Development of Alzheimer's disease (AD) pathology appears to be causally related to age-dependent changes in the metabolism of the amyloid-β peptide (Aβ), leading to its enhanced aggregation and deposition.γ-Secretase is a crucial enzyme for the generation of Aβ from the amyloid-β precursor protein and thus represents a valid potential therapeutic target for the treatment or prevention of AD. Enzyme activity has been shown to be dependent on the expression of presenilins and the identification of inhibitors containing transition-state analogue mimics, together with mutagenesis and knockout studies, confirms that presenilins may provide at least a component of the catalytic site for this putative aspartyl protease. Considerable effort has been expended to identify compounds which specifically reduceγ-secretase activity in the central nervous system, and those with the appropriate properties are being utilized in on-going proof-of-concept studies in animals and humans, to determine the extent and duration ofγ-secretase inhibition required to elicit therapeutic benefits.γ-Secretase-mediated substrate cleavage appears to fall into the category of ‘regulated intramembrane proteolysis’. By virtue of its mechanistic similarities, the effects ofγ-secretase inhibitors on proteolysis and signalling through other substrates, such as Notch, has to be determined carefully, since this is likely to impact on the clinically safe dose of these compounds.
Inhibition of toxicity and protofibril formation in the amyloid-β peptide β(25–35) using N-Methylated derivatives
Authors: Doig A. J. , Hughes E. , Burke R. M. , Su T. J. , Heenan R. K. , Lu J.
Published: June 2004

β(25–35) is a fragment of β-amyloid that retains its wild-type properties. N-methylated derivatives of β(25–35) can block hydrogen bonding on the outer edge of the assembling amyloid, so preventing the aggregation and inhibiting the toxicity of the wild-type peptide. The effects are assayed by Congo Red and thioflavin T binding, electron microscopy and an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] toxicity assay. N-methyl-Gly-25 has similar properties to the wild-type, while five other methylation sites have varying effects on prefolded fibrils and fibril assembly. In particular, N-methyl-Gly-33 is able to completely prevent fibril assembly and reduces the toxicity of prefolded amyloid. With N-methyl-Leu-34 the fibril morphology is altered and toxicity reduced. A preliminary study of β(25–35) structure in aqueous solution was made by small-angle neutron scattering (SANS). The protofibrillar aggregates are best described as a disc of radius 140 å and height 53 å (1 å = 0.1 nm), though the possibility of polydisperse aggregates cannot be ruled out. No aggregates form in the presence of N-methyl-Gly-33. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity and that SANS is an important technique for the direct observation of protofibril formation and destruction in solution.
Glutamine repeats: structural hypotheses and neurodegeneration
Authors: Masino L. , Pastore A.
Published: June 2004

A growing number of neurodegenerative diseases are caused by expansion of CAG trinucleotide repeats coding for polyglutamine. The presence of intranuclear inclusions in the affected neuronal cells has suggested a mechanism for pathogenesis based on protein misfolding and aggregation. Detailed understanding of these phenomena is therefore crucial in order to rationalize different phases of the diseases. In the past decade, a few studies have focused on the structural properties of polyglutamine and on the molecular bases of the aggregation process. Most of these studies have been performed on polyglutamine peptides and protein models. Only one report is currently available on the characterization of a full-length polyglutamine protein. The structural hypotheses resulting from these studies are reviewed here.
Amyloid-β oligomers: their production, toxicity and therapeutic inhibition
Authors: Walsh D. M. , Klyubin I. , Fadeeva J. V. , Rowan M. J. , Selkoe D. J.
Published: June 2004

Despite extensive genetic and animal modelling data that support a central role for the amyloid β-protein (Aβ) in the genesis of Alzheimer's disease, the specific form(s) of Aβ which causes injury to neurons in vivo has not been identified. In the present study, we examine the importance of soluble, pre-fibrillar assemblies of Aβ as mediators of neurotoxicity. Specifically, we review the role of cell-derived SDS-stable oligomers, their blocking of hippocampal long-term potentiation in vivo and the finding that this blocking can be prevented by prior treatment of oligomer-producing cells withγ-secretase inhibitors.
Properties of neurotoxic peptides related to the BRI gene
Authors: Austen B. , EI-Agnaf O. , Nagala S. , Patel B. , Gunasekera N. , Lee M. , Lelyveld V.
Published: June 2004

Mutations in the BRI gene are thought to cause dementias in members of families. The clinical symptoms are similar to those of Alzheimer's disease, but with additional ocular and hearing deficits, and spasticity. The mutations lead to the release of the 34-residue peptides, ABri and ADan, in the brains of afflicted individuals. We have synthesized the peptides in their straight-chain and oxidized cyclic forms and shown that the oxidized form of ABri and reduced form of ADan are toxic to human neuronal cell lines in culture. Neurotoxicity correlates with the extent of formation of SDS-stable non-fibrillar low-molecular-mass oligomers (SSNFOs).
Aggregation and neurotoxicity of α-synuclein and related peptides
Authors: EI-Agnaf O. M. A. , Irvine G. B.
Published: June 2004

Fibrillar deposits of α-synuclein occur in several neurodegenerative diseases. Two mutant forms of α-synuclein have been associated with early-onset Parkinson's disease, and a fragment has been identified as the non-amyloid-β peptide component of Alzheimer's disease amyloid (NAC). Upon aging, solutions of α-synuclein and NAC change conformation to β-sheet, detectable by CD spectroscopy, and form oligomers that deposit as amyloid-like fibrils, detectable by electron microscopy. These aged peptides are also neurotoxic. Experiments on fragments of NAC have enabled the region of NAC responsible for its aggregation and toxicity to be identified. NAC(8–18) is the smallest fragment that aggregates, as indicated by the concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. Fragments NAC(8–18) and NAC(8–16) are toxic, whereas NAC(12–18), NAC(9–16) and NAC(8–15) are not. Hence residues 8–16 of NAC comprise the region crucial for toxicity. Toxicity induced by α-synuclein, NAC and NAC(1–18) oligomers occurs via an apoptotic mechanism, possibly initiated by oxidative damage, since these peptides liberate hydroxyl radicals in the presence of iron. Molecules with anti-aggregational and/or antioxidant properties may therefore be potential therapeutic agents.
Factors affecting interactions between prion protein isoforms
Authors: Caughey B. , Baron G. S.
Published: June 2004

Interactions between normal, protease-sensitive prion protein (PrP-sen or PrPc) and its protease-resistant isoform (PrP-res or PrPsc) are critical in transmissible spongiform encephalopathy (TSE) diseases. To investigate the propagation of PrP-res between cells we tested whether PrP-res in scrapie brain microsomes can induce the conversion of PrP-sen to PrP-res if the PrP-sen is bound to uninfected raft membranes. Surprisingly, no conversion was observed unless the microsomal and raft membranes were fused or PrP-sen was released from raft membranes. These results suggest that the propagation of infection between cells requires transfer of PrP-res into the membranes of the recipient cell. To assess potential cofactors in PrP conversion, we used cell-free PrP conversion assays to show that heparan sulphate can stimulate PrP-res formation, supporting the idea that endogenous sulphated glycosaminoglycans can act as important cofactors or modulators of PrP-res formation in vivo. In an effort to develop therapeutics, the antimalarial drug quinacrine was identified as an inhibitor of PrP-res formation in scrapie-infected cell cultures. Confirmation of the latter result by others has led to the initiation of human clinical trials as a treatment for Creutzfeldt-Jakob disease. PrP-res formation can also be inhibited using a variety of other types of small molecule, specific synthetic PrP peptides, and an antiserum directed at the C-terminus of PrP-sen. The latter results help to localize the sites of interaction between PrP-sen and PrP-res. Disruption of those interactions with antibodies or peptidomimetic drugs may be an attractive therapeutic strategy. The likelihood that PrP-res inhibitors can rid TSE-infected tissues of PrP-res would presumably be enhanced if PrP-res formation were reversible. However, our attempts to measure dissociation of PrP-sen from PrP-res have failed under non-denaturing conditions. Finally, we have attempted to induce the spontaneous conversion of PrP-sen into PrP-res using low concentrations of detergents. A conformational conversion from α-helical monomers into high-β-sheet aggregates and fibrils was induced by low concentrations of the detergent sarkosyl; however, the aggregates had neither infectivity nor the characteristic protease-resistance of PrP-res.
Altering prion replication for therapy and diagnosis of transmissible spongiform encephalopathies
Authors: Soto C.
Published: June 2004

Transmissible spongiform encephalopathies, also known as prion-related diseases, are a group of fatal neurodegenerative disorders associated with the misfolding of the prion protein. In this article, the potential use of the principles of prion replication to develop novel therapeutic and diagnostic strategies for these devastating diseases are described.
Structure and function of glutamyl-tRNA reductase involved in 5-aminolaevulinic acid formation
Authors: Moser J. , Schubert W.-D. , Heinz D. W. , Jahn D.
Published: June 2004

In most bacteria, in archaea and in plants, the general precursor of all tetrapyrroles, 5-amino-laevulinic acid, is formed by two enzymes. The initial substrate, glutamyl-tRNA, is reduced by NADPH-dependent glutamyl-tRNA reductase to form glutamate 1-semialdehyde. The aldehyde is subsequently transaminated by glutamate-1-semi-aldehyde 2,1-aminomutase to yield 5-amino-laevulinic acid. The enzymic mechanism and the solved crystal structure of Methanopyrrus kandleri glutamyl-tRNA reductase are described. A pathway for metabolic channelling of the reactive aldehyde between glutamyl-tRNA reductase and the aminomutase is proposed.
5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism
Authors: Shoolingin-Jordan P. M. , Spencer P. , Sarwar M. , Erskine P. E. , Cheung K.-M. , Cooper J. B. , Norton E. B.
Published: June 2004

5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase. Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes. Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc. Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential. Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity. The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography. These inhibitors reacted with both active-site lysine residues. The role of these two lysine residues in the enzyme mechanism is discussed.
Site-directed mutagenesis of Tyr-189 and Lys-193 in NADPH: protochlorophyllide oxidoreductase from Synechocystis
Authors: Heyes D.J. , Hunter C. N.
Published: June 2004

NADPH: protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. Sequence comparisons have revealed that POR is a member of the short-chain alcohol dehydrogenase family of enzymes. A tyrosine and a lysine residue are conserved throughout all members of this family, and are proposed to be within the active site. This present study describes how site-directed mutagenesis has been used to change Tyr-189 to Phe and Lys-193 to Arg in the Synechocystis POR enzyme. The mutant enzymes were produced with a His tag in Escherichia coli and subsequently purified on a Ni2+-affinity column. The two mutations resulted in inactive enzymes, indicating that both residues are crucial for activity. The Kd value for NADPH binding to the K193R mutant was significantly higher than for the wild-type enzyme, suggesting that the affinity for NADPH has also been reduced.
Production of cobalamin and sirohaem in Bacillus megaterium: an investigation into the role of the branchpoint chelatases sirohydrochlorin ferrochelatase (SirB) and sirohydrochlorin cobalt chelatase (CbiX)
Authors: Leech H. K. , Raux-Deery E. , Heathcote P. , Warren M. J.
Published: June 2004

One of the four operons required for cobalamin biosynthesis in Bacillus megaterium is also associated with sirohaem synthesis, and contains three genes, sirA, sirB and sirC. By undertaking functional complementation experiments and in vitro assays using recombinantly produced enzymes, we have been able to demonstrate that (1) SirA acts as a uroporphyrinogen III methyltransferase, transforming uroporphyrinogen III into precorrin-2, (2) SirC acts as an NAD+ dehydrogenase, responsible for the oxidation of precorrin-2 into sirohydrochlorin, and (3) SirB acts as a ferrochelatase, responsible for the insertion of a ferrous ion into sirohydrochlorin to give sirohaem. Comparative sequence analysis reveals that the primary structure of SirB is highly similar to that of the cobalt chelatase involved in cobalamin biosynthesis in Bacillus megaterium, CbiX, with the exception that CbiX contains a C-terminal histidine-rich motif. Surprisingly, CbiX has been shown (using EPR) to contain a 4Fe-4S centre, a redox centre that is absent from SirB.
Unravelling chlorophyll catabolism in higher plants
Authors: Kräutler B.
Published: June 2004

Chlorophyll metabolism is probably the most visible manifestation of life. In spite of this, chlorophyll catabolism has remained something of a mystery until about 10 years ago. At that time, the first non-green tetrapyrrolic chlorophyll breakdown products from higher plants were discovered, and the structure of the first one of them was elucidated by modern spectroscopic methods. In the meantime, the essential structural features of chlorophyll catabolites and some of the biochemistry of chlorophyll breakdown in higher plants have been uncovered, as outlined in this article.