a b s t r a c tAmoebiasis is an intestinal infection caused by the human pathogen Entamoeba histolytica and representing the third leading cause of death by parasites in the world. Host-parasite interactions mainly involve anchored glycoconjugates localized in the surface of the parasitic cell. In protozoa, synthesis of structural oligo-and polysaccharides occurs via UDP-glucose, generated in a reaction catalyzed by UDPglucose pyrophosphorylase. We report the molecular cloning of the gene coding for this enzyme from genomic DNA of E. histolytica and its recombinant expression in Escherichia coli cells. The purified enzyme was kinetically characterized, catalyzing UDP-glucose synthesis and pyrophosphorolysis with V max values of 95 U/mg and 3 U/mg, respectively, and affinity for substrates comparable to those found for the enzyme from other sources. Enzyme activity was affected by redox modification of thiol groups. Different oxidants, including diamide, hydrogen peroxide and sodium nitroprusside inactivated the enzyme. The process was completely reverted by reducing agents, mainly cysteine, dithiothreitol, and thioredoxin. Characterization of the enzyme mutants C94S, C108S, C191S, C354S, C378S, C108/378S, M106S and M106C supported a molecular mechanism for the redox regulation. Molecular modeling confirmed the role of specific cysteine and methionine residues as targets for redox modification in the entamoebic enzyme. Our results suggest that UDP-glucose pyrophosphorylase is a regulated enzyme in E. histolytica. Interestingly, results strongly agree with the occurrence of a physiological redox mechanism modulating enzyme activity, which would critically affect carbohydrate metabolism in the protozoon.

The magnetic properties of the Cu(II)-peptide compounds (L-tyrosyl-L-leucinato)Cu(II) and (L-tryptophyl-glycinato)Cu(II), to be identified as Cu(II)Tyr-Leu and Cu(II)Trp-Gly, respectively, have been investigated by specific heat (0.08 < T < 28 K), dc magnetization (2 < T < 80 K, with B(0) = mu(o)H < or = 9 T), and ac magnetic susceptibility (with B(0) = 0 for 0.03 < T < 3 K and B(0) up to 9 T for 2 < T < 80 K) measurements. Above approximately 1 K, the specific heat and magnetization of both compounds display a ferromagnetic (FM) spin chain behavior sustained by syn-anti carboxylate bridges connecting equatorially Cu(II) ions at about 5 A. To model this behavior, we calculated the eigenvalues of Heisenberg chains with up to 20 spins 1/2 and used the method of Bonner and Fisher. A global fit of the model to the specific heat and magnetization data gives 2J(0)/k(B) = 3.60(5) K and 2.59(5) K for the intrachain exchange interactions in Cu(II)Tyr-Leu and Cu(II)Trp-Gly, respectively (H(ex)(i,j) = -2J(0) S(i).S(j)). These values of 2J(0) are discussed in terms of structural properties of the carboxylate bridges in the two compounds. Using the parameters obtained from the global fit, we calculated isothermal susceptibilities in agreement with the ac susceptibilities measured with small applied dc magnetic fields. However, the ac susceptibility measured with applied dc fields larger than 1 T lie between the values calculated for the isothermal and adiabatic susceptibilities. At 0.16 K for Cu(II)Tyr-Leu and 0.53 K for Cu(II)Trp-Gly, the observed specific heat and magnetic susceptibility display peaks associated to three-dimensional magnetic phase transitions. The interchain exchange couplings 2J(1) producing the 3D magnetic order are ferromagnetic and have magnitudes 2J(1)/k(B) approximately 0.015 and 0.073 K for Cu(II)Tyr-Leu and Cu(II)Trp-Gly, respectively.

We have studied the effect of the insertion of spin-labeled molecules n-doxyl-stearic acid (n-SASL, n = 5, 12, 16) on the structure and dynamics of a model lipid bilayer in gel-like phases using molecular dynamics simulations. We have studied the atomic density depth profiles and configurations of the labeled molecules in a host hydrated stearic acid bilayer system. We have found that the 5-SASL label positions its paramagnetic group at the water-lipid interface, and its polar head builds H bonds to neighboring lipids and to the solvent. 16-SASL positions its paramagnetic group at the lipid-lipid interface. The 12-SASL label presents two configurations at high lateral pressure. In one configuration, the doxyl ring lays at the lipid-lipid interface, shifting its polar head toward the bilayer center. The other equilibrium configuration of 12-SASL presents its paramagnetic group laying in the center of the compact hydrophobic region of the layer (erected configuration). It was determined that the coexistence of these two configurations is governed by the polar head-water interaction. We have found that the insertion of the labeled molecules at the concentrations used in the present work (0.36 mol %) do not perturb global properties like area per lipid, tilt angle, or order parameters. Nevertheless, there are local perturbations of the host system that are confined to a 10 angstroms neighboring shell around the spin label molecule. To study the interactions that determine the position of the labeled molecules in the bilayer, we performed simulations at different lateral pressures, which allowed us to extract important conclusions.

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The coefficient for exponential attenuation of the averaged Green function [lims p(G oR(E +i 5) ), "-e ""I is calculated for several infinite lattices in one, two, and three dimensions with a diagonal Lorentzian disorder of site energies (Lloyd model). In the limit of extended states, I =~coincidences with the phase coherence length and with the mean free path associated with~k) states In. the opposite limit, that of strongly localized states, the inequality tt) y is almost satisfied as an equality where y is the inverse localization length. Our results for~are the same as those calculated by Johnston and Kunz who associate their results with y, that is, with the localization length. This leads us to reinterpret their results and to conclude that"when the dimensionality is higher than 2, there is still a strong possibility of a mobility edge in this model.

Detergents are essential tools to study biological membranes, and they are frequently used to solubilize lipids and integral membrane proteins. Particularly the nondenaturing zwitterionic detergent usually named CHAPS was designed for membrane biochemistry and integrates the characteristics of the sulfobetaine-type detergents and bile salts. Despite the available experimental data little is known about the molecular structure of its micelles. In this work, molecular dynamics simulations were performed to study the aggregation in micelles of several numbers of CHAPS (≤ 18) starting from a homogeneous water dilution. The force field parameters to describe the interactions of the molecule were developed and validated. After 50 ns of simulation almost all the systems result in the formation of stable micelles. The molecular shape (gyration radii, volume, surface) and the molecular structure (RDF, salt bridges, H-bonds, SAS) of the micelles were characterized. It was found that the main interactions that lead to the stability of the micelles are the electrostatic ones among the polar groups of the tails and the OH's from the ring moiety. Unlike micelles of other compounds, CHAPS show a grainlike heterogeneity with hydrophobic micropockets. The results are in complete agreement with the available experimental information from NMR, TEM, and SAXS studies, allowing the modeling of the molecular structure of CHAPS micelles. Finally, we hope that the new force field parameters for this detergent will be a significant contribution to the knowledge of such an interesting molecule.

Leptospira interrogans synthesizes a range of mannose-containing glycoconjugates relevant for its virulence. A prerequisite in the synthesis is the availability of the GDP-mannose, produced from mannose-1-phosphate and GTP in a reaction catalyzed by GDP-mannose pyrophosphorylase. The gene coding for a putative enzyme in L. interrogans was expressed in Escherichia coli BL21(DE3). The identity of this enzyme was confirmed by electrospray-mass spectroscopy, Edman sequencing and immunological assays. Gel filtration chromatography showed that the dimeric form of the enzyme is catalytically active and stable. The recombinant protein was characterized as a mannose-1-phosphate guanylyltransferase. S (0.5) for the substrates were determined both in GDP-mannose pyrophosphorolysis: 0.20 mM (GDP-mannose), 0.089 mM (PPi), and 0.47 mM; and in GDP-mannose synthesis: 0.24 mM (GTP), 0.063 mM (mannose-1-phosphate), and 0.45 mM (Mg(2+)). The enzyme was able to produce GDP-mannose, IDP-mannose, UDP-mannose and ADP-glucose. We obtained a structural model of the enzyme using as a template the crystal structure of mannose-1-phosphate guanylyltransferase from Thermus thermophilus HB8. Binding of substrates and cofactor in the model agree with the pyrophosphorylases reaction mechanism. Our studies provide insights into the structure of a novel molecular target, which could be useful for detection of leptospirosis and for the development of anti-leptospiral drugs.

The effects of the insertion of a spin-labeled molecule (10-doxyl-stearic acid) on the structure and dynamics of model lipid bilayers in gel-like as well as in liquid-ordered-like phases are studied using molecular dynamic simulations. The perturbing effects of the labeled molecule on the structure of the bilayers are analyzed. We have also studied the relationship between the structural and dynamic properties of the bilayer phase and those of the labeled molecule. We found that the insertion of the labeled molecule in the bilayer at the concentration considered here (1:70) produces local and global perturbations in the gel-like phase. There is an increase of the area associated with the lipid molecules that produces a larger tilting angle of this condensed phase. In this gel-like phase, we also found that the z component of the order parameter of the labeled molecule associated with the electron paramagnetic resonance (EPR) spectra has the same temperature dependence as the axial correlation times of the lipid molecules. The mechanism by which the doxyl reorientation senses the dynamics of the layers is determined by the correlation between the gauche defect transitions of the labeled alkyl chain and its environment. For the liquid-ordered-like phase, we found that cholesterol molecules play the role of wedges that open free spaces in the lipid structure below the ring position and order the alkyl chains at the depths of the rings, leading to small inclination angles. The doxyl ring of the labeled molecule is located just below the cholesterol ring moiety, having fewer gauche defects than in the case of the gel-like phase. The change in depth of the doxyl ring causes a reorientation of this group that leads to an increase of the order parameter as the temperature rises.