[Dynamic contrast-enhanced MRI of the prostate: comparison of two different post-processing algorithms]. / Dynamische kontrastmittelunterstützte MRT der Prostata: Vergleich von zwei Auswerteverfahren.

PURPOSE: To evaluate the usefulness of a commercially available post-processing software tool for detecting prostate cancer on dynamic contrast-enhanced magnetic resonance imaging (MRI) and to compare the results to those obtained with a custom-made post-processing algorithm already tested under clinical conditions. MATERIALS AND METHODS: Forty-eight patients with proven prostate cancer were examined by standard MRI supplemented by dynamic contrast-enhanced dual susceptibility contrast (DCE-DSC) MRI prior to prostatectomy. A custom-made post-processing algorithm was used to analyze the MRI data sets and the results were compared to those obtained using a post-processing algorithm from In vivo Corporation (Dyna CAD for Prostate) applied to dynamic T 1-weighted images. Histology was used as the gold standard. RESULTS: The sensitivity for prostate cancer detection was 78 % for the custom-made algorithm and 60 % for the commercial algorithm and the specificity was 79 % and 82 %, respectively. The accuracy was 79 % for our algorithm and 77.5 % for the commercial software tool. The chi-square test (McNemar-Bowker test) yielded no significant differences between the two tools (p = 0.06). CONCLUSION: The two investigated post-processing algorithms did not differ in terms of prostate cancer detection. The commercially available software tool allows reliable and fast analysis of dynamic contrast-enhanced MRI for the detection of prostate cancer.

Influence of back mixing on the convective drying of residual sludges in a fixed bed.

The influence of a backmixing operation on the convective belt drying of two wastewater sludges was studied. The expansion of the sludge extrudates bed due to increasing additions of dry product was quantified by using X-ray tomography. This non-invasive technique was used to determine the bed porosity and the total exchange area available for heat and mass transfers, for increasing levels of backmixing. For a same drying flux, the expansion of the drying bed leads to higher drying rates, allowing a reduction of the total drying time. In this context, rheological properties of the sludges are key properties.

Image analysis of X-ray microtomograms of soft materials during convective drying: 3D measurements.

Drying dewatered sludge leads to a complex three-dimensional porous structure. Moreover, this operation is dependent on the way the material is processed. In this study, textural changes of sludge extrudates submitted to convective drying are followed by a 3D image analysis of reconstructed X-ray microtomograms. To achieve this goal, two different wastewater sludges collected in wastewater treatment plant after the thickening step and dewatered in the laboratory are used. It is showed that the evolution of the 3D-crack ratio vs. the residual water content evolves following a hyperbolic law. The 3D opening crack size distribution reveals two different types of pore development, i.e. a continuous pore size evolution for one sludge and the sudden appearance of cracks for the other sludge.

Wastewater sludge convective drying: influence of sludge origin.

This paper deals with thermal drying of wastewater sludges, whose management will become crucial in the forthcoming years. Sludges collected after mechanical dewatering in 5 different WWTPs are submitted to the same convective drying treatment in order to try finding some relations between the drying behaviours, the type of effluent and wastewater treatments. Results show dearly sludge drying remains a particularly complex operation because sludges may exhibit very different behaviours, both from kinetic and texture points of view. Initial moisture content and global composition are not sufficient to explain the different observed behaviours. It is too early to claim one or another type of water treatment has an influence on the drying behaviour. However, the drying rates can be classified in the inverse order of the organic content. Moreover for sludges at the same siccities, the harder the material (rheological properties), the higher the drying rate. Final textural properties can be related to the rheological properties and the internal diffusion limitations lowering the drying intensity.

Image analysis of X-ray microtomograms of soft materials during convective drying.

X-ray microtomography is used to explore the textural evolution that soft materials undergo during a drying treatment. An original image processing algorithm is applied to vertical projections and reconstructed cross-section images in order to quantify the texture at different stages of drying. Measurements are performed both on grey-level and on binary images. It is shown that X-ray microtomography is a very promising tool in the field of drying investigations. It can be used to determine internal moisture profiles, and to follow crack development and shrinkage in an accurate and non-destructive way. This information is crucial to validate drying models. Waste-water sludges are used as test materials to assess the validity of the proposed methodology. The management of these sludges, often including a drying stage, will become a challenge in the forthcoming years in accordance with environmental regulations. Samples collected in two waste-water treatment plants are investigated. Their analysis by X-ray microtomography brings to the fore two different drying behaviours, illustrating that sludge drying is a complex unit operation very sensitive to the way the material is produced.

Immunocytochemical localization and crystal structure of human frequenin (neuronal calcium sensor 1).

Frequenin, a member of a large family of myristoyl-switch calcium-binding proteins, functions as a calcium-ion sensor to modulate synaptic activity and secretion. We show that human frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4Kbeta, the mammalian homolog of the yeast kinase PIK1. In addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 A resolution. The overall fold of frequenin resembles those of neurocalcin and the photoreceptor, recoverin, of the same family, with two pairs of calcium-binding EF hands and three bound calcium ions. Despite the similarities, however, frequenin displays significant structural differences. A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin. This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand.

On the kaliotoxin and dendrotoxin binding sites on rat brain synaptosomes.

The toxic polypeptides alpha-, beta-, gamma- and delta-dendrotoxin (DTX), known to be potent blockers of voltage-dependent potassium channels of the Kv1 family, were purified from the venom of the green mamba Dendroaspis angusticeps. Their binding behaviour to synaptosomal membranes of rat brain was analysed and compared with that of kaliotoxin (KTX), in a competition assay using [(125)I] KTX. alpha-DTX and delta-DTX were found to compete with radioiodinated-KTX (IC(50) of 8 pM and 0.2 nM respectively), whereas gamma-DTX did not. Several minor components that competed with radioiodinated-KTX binding were identified and characterised chemically and biologically.

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.

Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site.

Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6, 6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim.

Crystal structure of mouse acetylcholinesterase. A peripheral site-occluding loop in a tetrameric assembly.

The crystal structure of mouse acetylcholinesterase at 2.9-A resolution reveals a tetrameric assembly of subunits with an antiparallel alignment of two canonical homodimers assembled through four-helix bundles. In the tetramer, a short Omega loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking alpha-helices, associates with the peripheral anionic site of the facing subunit and sterically occludes the entrance of the gorge leading to the active center. The inverse loop-peripheral site interaction occurs within the second pair of subunits, but the peripheral sites on the two loop-donor subunits remain freely accessible to the solvent. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of the peptidic inhibitor fasciculin bound to mouse acetylcholinesterase. Tetrameric forms of cholinesterases are widely distributed in nature and predominate in mammalian brain. This structure reveals a likely mode of subunit arrangement and suggests that the peripheral site, located near the rim of the gorge, is a site for association of neighboring subunits or heterologous proteins with interactive surface loops.

[The fasciculin-acetylcholinesterase interaction]. / L'interaction fasciculine-acétylcholinestérase.

Acetylcholinesterase (AChE) terminates the action of the neurotransmitter acetylcholine at cholinergic synapses in the central and peripheral nervous systems. Fasciculins, which belong to the family of "three-fingered" snake toxins, selectively inhibit mammalian AChEs with Ki values in the picomolar range. In solution, the cationic fasciculin appears to bind to the enzyme's peripheral anionic site, located near the mouth of the gorge leading to the active center, to inhibit catalysis either allosterically or by creating an electrostatic barrier at the gorge entry (or both). Yet the crystal structure of the fasciculin-mouse AChE complex, which shows that the central loop of fasciculin fits snugly at the entrance of the gorge, suggests that the mode of action of fasciculin is steric occlusion of substrate access to the active center. Mutagenesis of the fasciculin molecule, undertaken to establish a functional map of the binding surfaces, identified determinants common to those identified by the structural approach and revealed that only a few of the many fasciculin residues residing at the complex interface provide the strong contacts required for high affinity binding and enzyme inhibition. However, it did not reconcile the disparity between the kinetic and structural data. Finally, the crystal structure of mouse AChE without bound fasciculin shows a tetrameric assembly of subunits; within the tetramer, a short loop at the surface of a subunit associates with the peripheral site of a facing subunit and sterically occludes the entrance of the active center gorge. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of fasciculin bound to AChE. This suggests not only that the peripheral site of AChE is a site for association of heterologous proteins with interactive surface loops, but also that endogenous peptidic ligands of AChE sharing structural features with the fasciculin molecule might exist.

Inhibition of mouse acetylcholinesterase by fasciculin: crystal structure of the complex and mutagenesis of fasciculin.

Fasciculins are members of the superfamily of three-fingered peptidic toxins from Elapidae venoms. They selectively inhibit mammalian and electric fish acetylcholinesterases (AChE) with Ki values in the pico- to nanomolar range. Kinetic studies performed in solution indicate that fasciculin does not totally occlude ligand access to the active site of AChE, but rather binds to a peripheral site of the enzyme to inhibit catalysis, perhaps allosterically. The crystal structure of the Fas2-mouse AChE complex delineated a large contact area consistent with the low dissociation constant of the complex; the Fas2 and AChE residues participating in the binding interface were unambiguously established, and major hydrophobic interactions were identified. The structure however suggests that fasciculin totally occludes substrate entry into the catalytic site of AChE, and does not reveal to what extent each contact between Fas2 and AChE contributes to the overall binding energy. New probes, designed to delineate the individual contributions of the fasciculin residues to the complex formation and conformation, were generated by site-directed mutagenesis of a synthetic Fas2 gene. A fully processed recombinant fasciculin, rFas2, that is undistinguishable from the natural, venom-derived Fas2, was expressed in a mammalian system; fourteen mutants, encompassing 16 amino acid residues distributed among the three loops (fingers) of Fas2, were developed from both the kinetic and structural data and analyzed for inhibition of mouse AChE. The determinants identified by the structural and the functional approaches do coincide. However, only a few of the many residues which make up the overall interactive site of the Fas2 molecule provide the strong interactions required for high affinity binding and enzyme inhibition. Potential drug design from the fasciculin molecule is discussed.

Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure.

The pentameric structure of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits and their ligand specificity characteristics. This problem has been approached through the study of assembly of subunits and binding characteristics of selective peptide toxins. The receptor, with its circular order of homologous subunits (alpha gamma alpha delta beta), assembles in only one arrangement, and through mutagenesis, the residues governing assembly can be ascertained. Selectivity of certain toxins is sufficient to readily distinguish between sites at the alpha gamma and alpha delta interfaces. By interchanging residues on the gamma and delta subunits, and ascertaining how they interact with the alpha-subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins, which contain two disulfide loops and 12-14 amino acids, show a 10,000-fold preference for the alpha delta over the alpha gamma subunit interface with alpha epsilon falling between the two. The waglerins, as 22-24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for alpha epsilon over the alpha gamma and alpha delta interfaces. Finally, the 6700 Da short alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alpha gamma and alpha delta interfaces over alpha epsilon. Selective mutagenesis enables one to also distinguish alpha-neurotoxin binding at the alpha gamma and alpha delta subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.

Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding.

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.

Expression and activity of mutants of fasciculin, a peptidic acetylcholinesterase inhibitor from mamba venom.

Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, DeltaPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.

Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.

Structure of fasciculin 2 from green mamba snake venom: evidence for unusual loop flexibility.

The crystal structure of the snake toxin fasciculin 2, a potent acetylcholinesterase inhibitor from the venom of the green mamba (Dendroaspis angusticeps), has been determined by the molecular-replacement method, using the fasciculin 1 model and refined to 2.0 A resolution. The introduction of an overall anisotropic temperature factor improved significantly the quality of the electron-density map. It suggests, as it was also indicated by the packing, that the thermal motion along the unique axis direction is less pronounced than on the (ab) plane. The final crystallographic R factor is 0.188 for a model having r.m.s. deviations from ideality of 0.016 A for bond lengths and 2.01 degrees for bond angles. As fasciculin 1, fasciculin 2 belongs to the three-finger class of Elapidae toxins, a structural group that also contains the alpha-neurotoxins and the cardiotoxins. Although the two fasciculins have, overall, closely related structures, the conformation of loop I differs appreciably in the two molecules. The presence of detergent in crystallization medium in the case of fasciculin 2 appears to be responsible for the displacement of the loop containing Thr9. This conformational change also results in the formation of a crystallographic dimer that displays extensive intermolecular interactions.

Structural bases for the specificity of cholinesterase catalysis and inhibition.

The availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin.

Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex.

The crystal structure of the snake toxin fasciculin, bound to mouse acetylcholinesterase (mAChE), at 3.2 A resolution reveals a synergistic three-point anchorage consistent with the picomolar dissociation constant of the complex. Loop II of fasciculin contains a cluster of hydrophobic residues that interact with the peripheral anionic site of the enzyme and sterically occlude substrate access to the catalytic site. Loop I fits in a crevice near the lip of the gorge to maximize the surface area of contact of loop II at the gorge entry. The fasciculin core surrounds a protruding loop on the enzyme surface and stabilizes the whole assembly. Upon binding of fasciculin, subtle structural rearrangements of AChE occur that could explain the observed residual catalytic activity of the fasciculin-enzyme complex.

Afaâcytin, an alpha beta-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified factor X and induces serotonin release from human blood platelets.

Afaâcytin, a proteinase with caseinolytic, arginine-esterase and amidase activities, was purified from the venom of Cerastes cerastes (horned viper) in two steps by gel filtration through Sephadex G75, then HPLC on carboxymethyl-cellulose. Afaâcytin has an isoelectric point of 6.25, and consists of two subunits, alpha and beta, which have the same apparent molecular mass (40,000) and are indistinguishable in the absence of reduction or/and deglycosylation. Subunit beta is constituted of two disulfide-linked polypeptidic chains, beta and beta'. The respective apparent molecular mass of the chains are 43,000 (alpha), 35,500 (beta) and 10,200 (beta') as determined by SDS/PAGE under reducing conditions. Both chains alpha and beta are N-glycosylated. The two chains have the same N-terminal sequence (20 residues) which is similar to those of other proteinases from snake venom. Susceptibility of afaâcytin to diisopropyl fluorophosphate and benzamidine indicates the presence of a serine and an aspartic (or glutamic) acid residues in the catalytic site. Ca2+ appears to be required for structural cohesion of the afaâcytin molecule. Afaâcytin exhibits alpha beta-fibrinogenase and alpha-fibrinase properties. It replaces missing factors VIII and IX in deficient plasmas, and activates purified human factor X into factor Xa. It releases serotonin from platelets and directly aggregates human (but not rabbit) blood platelets. Despite its thrombin-like characteristics, however, afaâcytin is not inhibited by plasmatic thrombin inhibitors. The procoagulant properties of afaâcytin therefore have potential clinical applications.