Combined Experimental and Theoretical Studies on Electron Transfer in Potassium Collisions with CCl4.

Negative ion formation in electron transfer experiments from fast neutral potassium (K) atom collisions with neutral tetrachloromethane (CCl4) molecules has been investigated in the laboratory frame range of 8-1000 eV. Comprehensive calculations on the electronic structure were performed for CCl4 in the presence of a potassium atom and used to help analyze the lowest unoccupied molecular orbitals participating in the collision process. Additionally, K+ energy loss produced in the forward direction has served to further our knowledge on the electronic state spectroscopy of CCl4. A vertical electron affinity of -0.79 ± 0.20 eV has been obtained and assigned to a purely repulsive transition from CCl4 ground state to the 2T2 state of the temporary negative ion yielding Cl- formation. Other features in the energy loss spectrum were observed for the first time and related to Cl2-, CCl2-, and CCl3- formation. Special attention is also given to the unresolved feature corresponding to a positive electron affinity of 0.24 ± 0.2 eV, assigned to a vibrationally hot transition from CCl4 ground state into the triply degenerate 2T2 excited state of the negative ion. The combined time-of-flight mass spectrometry together with K+ energy loss data represents the most comprehensive assignment of the tetrachloromethane anion yields and the role of CCl4 electronic states in collision induced dissociation to date.

Profiling of how nociceptor neurons detect danger - new and old foes.

The host evolves redundant mechanisms to preserve physiological processing and homeostasis. These functions range from sensing internal and external threats, creating a memory of the insult and generating reflexes, which aim to resolve inflammation. Impairment in such functioning leads to chronic inflammatory diseases. By interacting through a common language of ligands and receptors, the immune and sensory nervous systems work in concert to accomplish such protective functions. Whilst this bidirectional communication helps to protect from danger, it can contribute to disease pathophysiology. Thus, the somatosensory nervous system is anatomically positioned within primary and secondary lymphoid tissues and mucosa to modulate immunity directly. Upstream of this interplay, neurons detect danger, which prompts the release of neuropeptides initiating (i) defensive reflexes (ranging from withdrawal response to coughing) and (ii) chemotaxis, adhesion and local infiltration of immune cells. The resulting outcome of such neuro-immune interplay is still ill-defined, but consensual findings start to emerge and support neuropeptides not only as blockers of TH 1-mediated immunity but also as drivers of TH 2 immune responses. However, the modalities detected by nociceptors revealed broader than mechanical pressure and temperature sensing and include signals as various as cytokines and pathogens to immunoglobulins and even microRNAs. Along these lines, we aggregated various dorsal root ganglion sensory neuron expression profiling datasets supporting such wide-ranging sensing capabilities to help identifying new danger detection modalities of these cells. Thus, revealing unexpected aspects of nociceptor neuron biology might prompt the identification of novel drivers of immunity, means to resolve inflammation and strategies to safeguard homeostasis.

Study of the one dimensional and transient bioheat transfer equation: multi-layer solution development and applications.

In this work we derive an analytical solution given by Bessel series to the transient and one-dimensional (1D) bioheat transfer equation in a multi-layer region with spatially dependent heat sources. Each region represents an independent biological tissue characterized by temperature-invariant physiological parameters and a linearly temperature dependent metabolic heat generation. Moreover, 1D Cartesian, cylindrical or spherical coordinates are used to define the geometry and temperature boundary conditions of first, second and third kinds are assumed at the inner and outer surfaces. We present two examples of clinical applications for the developed solution. In the first one, we investigate two different heat source terms to simulate the heating in a tumor and its surrounding tissue, induced during a magnetic fluid hyperthermia technique used for cancer treatment. To obtain an accurate analytical solution, we determine the error associated with the truncated Bessel series that defines the transient solution. In the second application, we explore the potential of this model to study the effect of different environmental conditions in a multi-layered human head model (brain, bone and scalp). The convective heat transfer effect of a large blood vessel located inside the brain is also investigated. The results are further compared with a numerical solution obtained by the Finite Element Method and computed with COMSOL Multiphysics v4.1©.

Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation.

Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assembled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after surface immobilization, boophilin was biotinylated after the formation of a boophilin-thrombin complex to minimize the biotinylation of the residues involved in thrombin-boophilin interaction. The extent of boophilin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using ¹²5I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophilin-free surfaces, demonstrating boophilin's potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices.

Use of the reverse transcription polymerase chain reaction (RT-PCR) for the rapid diagnosis of foot and mouth disease in South America.

Foot and mouth disease (FMD) is a limiting factor for the economic progress of the animal industry in South America. The presence of the disease results in the imposition of national and international sanitary barriers to animals and animal products, and, most especially, a reduction in the availability of protein from animal origin and in income. Rapid and accurate identification of infected animals, those with either clinical or subclinical disease as well as with persistent infection, is essential for maintaining an efficient eradication programme. The polymerase chain reaction was used to rapidly identify infected animals. With a primer set that corresponds to a conserved region of the 3D sequence of the viral genome, it was possible to amplify, regardless of the serotype, 116 strains of FMD virus, of which 109 were strains collected from outbreaks of FMD throughout South America from 1945 to the most recent outbreaks in 2000/2001. The PCR technique should be of considerable value in facilitating the diagnosis of FMD in South America. where laboratory resources are limited and a rapid response is needed, particularly in areas where national programmes for controlling or eradicating the disease are being implemented.

New insights into the thermostability of bacterial ferredoxins: high-resolution crystal structure of the seven-iron ferredoxin from Thermus thermophilus.

The crystal structure of the seven-iron ferredoxin from Thermus thermophilus (FdTt) has been determined at 1.64 A resolution, allowing us to unveil the common mechanisms of thermostabilization within "bacterial-type" ferredoxins. FdTt and other homologous thermophilic seven-iron ferredoxins are smaller than their mesophilic counterparts. Thermostabilizing features are optimized in a minimal structural and functional unit, with an extensive cross-linking of secondary structure elements mediated by improved polar and hydrophobic interactions. Most of the potentially stabilizing features are focused on the vicinity of the functional [3Fe-4S] cluster. The structural [4Fe-4S] cluster is shielded in thermophilic FdTt by an increased number of polar interactions involving the two N-terminal residues. Comparisons with the hyperthermostable ferredoxin from Thermotoga maritima reveal that (1) a reduction in the number of non-glycine residues in strained conformations, (2) improved polar interactions within the common iron-sulfur cluster binding (betaalphabeta)2 motif, and (3) an optimized charge distribution at the protein surface, constitute a common strategy for increasing the thermal stability of these ferredoxins.

Activation of human prothrombin by arginine-specific cysteine proteinases (Gingipains R) from porphyromonas gingivalis.

The effect of 95- (HRgpA) and 50-kDa gingipain R (RgpB), arginine-specific cysteine proteinases from periodontopathogenic bacterium Porphyromonas gingivalis on human prothrombin activation was investigated. Each enzyme released thrombin from prothrombin in a dose- and time-dependent manner with the former enzyme, containing adhesion domains, being 17-fold more efficient than the single chain RgpB. A close correlation between the generation of fibrinogen clotting activity and amidolytic activity indicated that alpha-thrombin was produced by gingipains R, and this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin active site labeling, and amino-terminal sequence analysis of prothrombin digestion fragments. Significantly, the catalytic efficiency of HRgpA to generate thrombin (k(cat)/K(m) = 1.2 x 10(6) m(-)1 s(-)1) was 100-fold higher than that of RgpB (k(cat)/K(m) = 1.2 x 10(4) m(-)1 s(-)1). The superior prothrombinase activity of HRgpA over RgpB correlates with the fact that only the former enzyme was able to clot plasma, and kinetic data indicate that prothrombin activation can occur in vivo. At P. gingivalis-infected periodontitis sites HRgpA may be involved in the direct production of thrombin and, therefore, in the generation of prostaglandins and interleukin-1, both have been found to be associated with the development and progression of the disease. Furthermore, by taking into account that the P. gingivalis bacterium has been immunolocalized in carotid atherosclerotic plaques at thrombus formation sites (Chiu, B. (1999) Am. Heart J. 138, S534-S536), our results indicate that bacterial proteinases may potentially participate in the pathogenesis of cardiovascular disease associated with periodontitis.

Structure of human biliverdin IXbeta reductase, an early fetal bilirubin IXbeta producing enzyme.

Biliverdin IXbeta reductase (BVR-B) catalyzes the pyridine nucleotide-dependent production of bilirubin-IXbeta, the major heme catabolite during early fetal development. BVR-B displays a preference for biliverdin isomers without propionates straddling the C10 position, in contrast to biliverdin IXalpha reductase (BVR-A), the major form of BVR in adult human liver. In addition to its tetrapyrrole clearance role in the fetus, BVR-B has flavin and ferric reductase activities in the adult. We have solved the structure of human BVR-B in complex with NADP+ at 1.15 A resolution. Human BVR-B is a monomer displaying an alpha/beta dinucleotide binding fold. The structures of ternary complexes with mesobiliverdin IValpha, biliverdin IXalpha, FMN and lumichrome show that human BVR-B has a single substrate binding site, to which substrates and inhibitors bind primarily through hydrophobic interactions, explaining its broad specificity. The reducible atom of both biliverdin and flavin substrates lies above the reactive C4 of the cofactor, an appropriate position for direct hydride transfer. BVR-B discriminates against the biliverdin IXalpha isomer through steric hindrance at the bilatriene side chain binding pockets. The structure also explains the enzyme's preference for NADP(H) and its B-face stereospecificity.

Aprotinin binding to amyloid fibrils.

Different low molecular mass ligands have been used to identify amyloid deposits. Among these markers, the dyes Thioflavin T and Congo Red interact specifically with the beta-sheet structure arranged in a cross-beta conformation, which is characteristic of amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium-99m, the proteinase inhibitor aprotinin has been shown to represent a very important radiopharmaceutical agent for in vivo imaging of extra-abdominal deposition of amyloid in amyloidosis of the immunoglobulin type. However, no information is available as to whether aprotinin binds other types of amyloid fibrils and on the nature and characteristics of the interaction. The present work shows aprotinin binding to insulin, transthyretin, beta-amyloid peptide and immunoglobulin synthetic amyloid fibrils by a specific dot-blot ligand-binding assay. Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 microM-1 for the binding of aprotinin to insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an aprotinin variant, a spermadhesin and the soybean trypsin inhibitor were tested and results suggest that both aprotinin and the spermadhesin interact with amyloid fibrils through pairing of beta-sheets of the ligands with exposed structures of the same type at the surface of amyloid deposits. An electrostatic component may also be involved in the binding of aprotinin to amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in aprotinin; on the other hand beta-sheet containing acidic proteins, such as the soybean trypsin inhibitor, are unable to bind amyloid fibrils.

Intra-sellar salivary gland-like pleomorphic adenoma arising within the wall of a Rathke's cleft cyst.

Salivary gland rests occur in the posterior lobe of the pituitary gland near or often communicating with the Rathke's cleft or its cystic subdivisions, and are usually incidental autopsy findings. They are attributed to the oropharyngeal development of the Rathke's pouch and may rarely give rise to salivary gland-like tumors in the sella. We present a pleomorphic adenoma, a rare tumor of the sellar region, that has not been previously recognized in association with Rathke's cleft cyst. It occurred in a 44-year-old man who presented with hypopituitarism and reduced vision. Magnetic resonance imaging showed a sellar mass with suprasellar extension which was totally removed. It consisted of segments of a cyst wall lined by focally ciliated columnar of cuboid epithelium containing goblet cells. An eosinophilic granular material with cholesterol clefts represented the contents of the cyst. Within its wall there was a tumor with ductal structures and non-ductal varied cellular components including hypercellular areas of spindle and ovoid cells forming interlacing fascicles. Individual cells appeared to float in abundant mucinous material. The appearances were those of a salivary gland pleomorphic adenoma arising within the wall of a Rathke's cleft cyst. The myoepithelial nature of non-ductal tumor cells was confirmed with immunocytochemistry. The existence of seromucous glands communicating with the Rathke's cleft remnants, explains the concomitant occurrence of the tumor and the cyst. This rare neoplasm from salivary gland rest should be considered in the differential diagnosis of unusual sellar and suprasellar tumors.

Specific inhibition of insect alpha-amylases: yellow meal worm alpha-amylase in complex with the amaranth alpha-amylase inhibitor at 2.0 A resolution.

BACKGROUND: alpha-Amylases constitute a family of enzymes that catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related polysaccharides. The Amaranth alpha-amylase inhibitor (AAI) specifically inhibits alpha-amylases from insects, but not from mammalian sources. AAI is the smallest proteinaceous alpha-amylase inhibitor described so far and has no known homologs in the sequence databases. Its mode of inhibition of alpha-amylases was unknown until now. RESULTS: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with AAI was determined at 2.0 A resolution. The overall fold of AAI, its three-stranded twisted beta sheet and the topology of its disulfide bonds identify it as a knottin-like protein. The inhibitor binds into the active-site groove of TMA, blocking the central four sugar-binding subsites. Residues from two AAI segments target the active-site residues of TMA. A comparison of the TMA-AAI complex with a modeled complex between porcine pancreatic alpha-amylase (PPA) and AAI identified six hydrogen bonds that can be formed only in the TMA-AAI complex. CONCLUSIONS: The binding of AAI to TMA presents a new inhibition mode for alpha-amylases. Due to its unique specificity towards insect alpha-amylases, AAI might represent a valuable tool for protecting crop plants from predatory insects. The close structural homology between AAI and 'knottins' opens new perspectives for the engineering of various novel activities onto the small scaffold of this group of proteins.

The structure of the human betaII-tryptase tetramer: fo(u)r better or worse.

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-A crystal structure of human betaII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 A x 15 A govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrations in vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.

The 2.2 A crystal structure of human chymase in complex with succinyl-Ala-Ala-Pro-Phe-chloromethylketone: structural explanation for its dipeptidyl carboxypeptidase specificity.

Human chymase (HC) is a chymotrypsin-like serine proteinase expressed by mast cells. The 2.2 A crystal structure of HC complexed to the peptidyl inhibitor, succinyl-Ala-Ala-Pro-Phe-chloromethylketone (CMK), was solved and refined to a crystallographic R-factor of 18.4 %. The HC structure exhibits the typical folding pattern of a chymotrypsin-like serine proteinase, and shows particularly similarity to rat chymase 2 (rat mast cell proteinase II) and human cathepsin G. The peptidyl-CMK inhibitor is covalently bound to the active-site residues Ser195 and His57; the peptidyl moiety juxtaposes the S1 entrance frame segment 214-217 by forming a short antiparallel beta-sheet. HC is a highly efficient angiotensin-converting enzyme. Modeling of the chymase-angiotensin I interaction guided by the geometry of the bound chloromethylketone inhibitor indicates that the extended substrate binding site contains features that may generate the dipeptidyl carboxypeptidase-like activity needed for efficient cleavage and activation of the hormone. The C-terminal carboxylate group of angiotensin I docked into the active-site cleft, with the last two residues extending beyond the active site, is perfectly localized to make a favorable hydrogen bond and salt bridge with the amide nitrogen of the Lys40-Phe41 peptide bond and with the epsilon-ammonium group of the Lys40 side-chain. This amide positioning is unique to the chymase-related proteinases, and only chymases from primates possess a Lys residue at position 40. Thus, the structure conveniently explains the preferred conversion of angiotensin I to angiotensin II by human chymase.

Human beta-tryptase is a ring-like tetramer with active sites facing a central pore.

Human tryptase, a mast-cell-specific serine proteinase that may be involved in causing asthma and other allergic and inflammatory disorders, is unique in two respects: it is enzymatically active only as a heparin-stabilized tetramer, and it is resistant to all known endogenous proteinase inhibitors. The 3-A crystal structure of human beta-tryptase in a complex with 4-amidinophenyl pyruvic acid shows four quasi-equivalent monomers arranged in a square flat ring of pseudo 222 symmetry. Each monomer contacts its neighbours at two different interfaces through six loop segments. These loops are located around the active site of beta-tryptase and differ considerably in length and conformation from loops of other trypsin-like proteinases. The four active centres of the tetramer are directed towards an oval central pore, restricting access for macromolecular substrates and enzyme inhibitors. Heparin chains might stabilize the complex by binding to an elongated patch of positively charged residues spanning two adjacent monomers. The nature of this unique tetrameric architecture explains many of tryptase's biochemical properties and provides a basis for the rational design of monofunctional and bifunctional tryptase inhibitors.

Caracterización serológica y molecular de cepas del virus de la fiebre aftosa, serotipo O, aisladas a partir de brotes ocurridos entre 1958 y 1983 en Brasil y Argentina

La caracterización genética de cepas del virus de la fiebre aftosa representativas de brotes importantes del serotipo O, ocurridos entre 1958 y 1983 en el sudeste de Brasil y el centro-este de Argentina, fue obtenida usando mapas de oligonucleótidios resistentes a T1. Los resultados obtenidos constituyen la base de un banco de datos para ser aplicado en estudios epidemiológicos.

Genetic characterization of representative foot-and-mouth disease virus strains from major serotype O outbreaks which occurred between 1958 and 1983 in southeastern Brazil and centraleastern Argentina was obtained using T1 maps. The results presented constitute the basis for a data bank to be applied in epidemiological studies.

Caracterización molecular de cepas del virus de la fiebre aftosa usadas para la producción de vacunas en Sudamérica

Las cepas del virus de la fiebre aftosa usadas actualmente para la producción de vacunas en Sudamérica fueron caracterizadas por mapeamiento bidimensional ARNasa T1 fingerprinting y por secuenciamiento de nucleótidos directamente del cADN correspondiente a la región que codifica para la mitad carboxiterminal de la VP1 (aproximadamente 290 nucleótidos). Las secuencias obtenidas fueron comparadas con aquellas determinadas previamente para algunas de estas cepas por otros laboratorios. Los resultados presentados constitueyen la base de un banco de datos para evaluar el grado de homología entre virus de campo y vacunales, y para monitorear la estabilidad genética de cepas durante la producción y control de vacunas.

Desarrollo de un metodo mas simple y rapido para el analisis bidimensional de ologonucleotidos del virus de la fiebre aftosa: minifingerprinting de ARN / Developement of a more rapid and simple procedure for two-dimensional oligonucleotide analysis of foot-and-mouth disease virus: RNA minifingerprinting

Una característica significativa del virus de la fiebre aftosa (VFA) es su alto grado de variabilidad, lo cual constiruye un importante obstáculo para el control de la enfermedad. Actualmente se sispone de varias técnicas bioquímicas como fingerprinting de ARN, ADN recombinante y secuencimiento rápido para estudiar, con significativa precisión, las características genéticas de las cepas virales. La tecnica de fingerprinting resulta muy adecuada para evaluar las relaciones evolutivas entre cepas virales muy relacionadas y en el caso del VFA tambien se ha probado su utilidad para fines de diagnostico. Con la creciente aplicacion de estudios epidemiologicos a nivel molecular que requieren el analisis de un gran numero de muestras, se hace evidente que el empleo de tecnicas mas practicas y rapidas y de menor costo, seria de gran utilidad para la caracterizacion del ARN genomico