Strategies and results of field emission scanning electron microscopy (FE-SEM) in the study of parasitic protozoa

Field emission scanning electron microscopy (FE-SEM) provides a range of strategies for investigating the structural organization of biological systems, varying from isolated macromolecules to tissue organization and whole organisms. This review covers some of the results so far obtained using FE-SEM observation and various protocols of sample fixation to analyze the structural organization of parasitic protozoa and their interaction with host cells. The employment of FE-SEM can be broadened through the use of gold-labeled molecules or tracers, gradual extraction by detergents, and cleavage techniques. These analyses provide significant contributions to the characterization of these organisms concerning ultrastructure, cytoskeleton, motility and intracellular behavior.     

Nitrogen nuclear magnetic resonance spectroscopy. Nitrogen-15 and proton chemical shifts of methylanilines and methylanilinium ions

Nitrogen and carbon electron densities of the toluidines and xylidines have been recalculated by the INDO method; previously published errors have been corrected. Although the nitrogen-15 chemical shifts of these compounds still display the earlier suggested correlation with σ and total electron densities, the calculated inverse correlation with proton electron densities has been shown to be incorrect. Methyl proton chemical shifts of these compounds display no meaningful correlation with the nitrogen shifts. The nitrogen chemical shifts of the toluidinium and xylidinium ions correlate moderately well with the ¹³C chemical shifts of the analogous di- and tri-methylbenzenes.     

The Influence of β- and γ-Cyclodextrin Cavity Size on the Association Constant with Decanoate and Octanoate Anions

This work evaluates the influence of the - and -cyclodextrin (CD) cavity size on the association constant (K_CDA) with decanoate (C₁₀) and octanoate (C₈) anions. The spectral displacement technique with phenolphthalein was used to obtain the 1:1 association constant (K_CDA) in NaHCO₃/NaOH buffer pH 10.5 at 25 °C. The K_CDA value obtained were 2.6 (±0.2) × 10³, 2.5 (±0.5) × 10², for beta;CD–C₁₀ and CD–C₁₀ inclusion complexes, and 5.1 (±0.2) × 10² and 4.7 (±0.2) × 10¹ for CD–C₈ and CD–C₈ inclusion complexes, respectively. The K_CDA values of either acid with CD is approximately 10 times higher than for the same acid with CD, where as for the same cyclodextrin, the K_CDA value is 5 times higher for the C₁₀ association than for the C₈. The data demonstrate that the cyclodextrin cavity size exerts a greater influence on the association constant than the chain length of the acid for these compounds. ¹H NMR studies show that fatty acid protonation has a distinct effect on the chemical shift of CD protons.     

Pi-halogen dimer interactions and the inclusion chemistry of a new tetrahalo aryl host

The preparation of 1,4,8,11-tetrabromo-5b[small alpha],6,12b[small alpha],13-tetrahydropentaleno[1,2-b:4,5-b[prime or minute]]diquinoline is described. This is a further member of the tetrahalo aryl host family, and forms crystalline lattice inclusion compounds with many guests. The X-ray structures of the allyl cyanide, 1,2,3-trichloropropane, chlorobenzene, toluene, benzene-water, methyl chloroform and carbon tetrachloride inclusion compounds are described, and compared with that of the solvent-free apohost. Although several different structural types are produced, the recently reported pi-halogen dimer (PHD) interaction plays an important role in all of these, except for that of pure (where the packing energy is the least favourable of the series).     

Comparison of the structures of 1,5-dichloro- and 1,5-dibromonaphthalene-2,6-diol and their co-crystalline compounds with dioxane

The 1,5-dichloro-(1) and 1,5-dibromo-(2) naphthalene-2,6-diols form isostructural lattices incorporating ...O–H...O–H...O–H... hydrogen bonding surrounding 2₁ screw axes. Each phenolic hydroxy group participates in one donor and one acceptor hydrogen bond. When crystallised from dioxane, both compounds form new 1:1 co-crystalline materials whose lattices are closely related but not isostructural. All inter-phenolic hydrogen bonding is now absent with each hydroxy group acting instead as a donor to a dioxane oxygen atom. In consequence, the hydrogen bonded layer structures of pure1 or2 are now replaced by hydrogen bonded chain arrangements. All four crystal structures are analysed and compared in crystal engineering terms through discussion of the various types of other weak intermolecular attractions involved in their lattice constructions. Supplementary Data relating to this article are deposited with the British Library as supplementary publication No. SUP 82202 (19 pages).     

Hybrid boundary element and finite difference method for solving the nonlinear Poisson-Boltzmann equation

A hybrid approach for solving the nonlinear Poisson-Boltzmann equation (PBE) is presented. Under this approach, the electrostatic potential is separated into (1) a linear component satisfying the linear PBE and solved using a fast boundary element method and (2) a correction term accounting for nonlinear effects and optionally, the presence of an ion-exclusion layer. Because the correction potential contains no singularities (in particular, it is smooth at charge sites) it can be accurately and efficiently solved using a finite difference method. The motivation for and formulation of such a decomposition are presented together with the numerical method for calculating the linear and correction potentials. For comparison, we also develop an integral equation representation of the solution to the nonlinear PBE. When implemented upon regular lattice grids, the hybrid scheme is found to outperform the integral equation method when treating nonlinear PBE problems. Results are presented for a spherical cavity containing a central charge, where the objective is to compare computed 1D nonlinear PBE solutions against ones obtained with alternate numerical solution methods. This is followed by examination of the electrostatic properties of nucleic acid structures.     

Translocation of a beta-peptide across cell membranes.

Short cationic peptides derived from DNA-binding proteins, of which HIV Tat is a prototype, can cross the membranes of living cells, and they can bring covalently attached moieties (proteins, drugs) along with them. We show that a beta-amino acid analogue of Tat 47-57 enters HeLa cells with comparable efficiency to Tat 47-57 itself (YGRKKRRQRRR). The beta-peptide is comprised of residues that bear the appropriate side chain at the beta-carbon. Both the alpha- and the beta-peptide were conjugated to fluorescein at the N terminus, and cell penetration was monitored by confocal fluorescence microscopy. Deletion of the three C-terminal arginine residues from the alpha-peptide abolished translocation activity, consistent with prior reports, and deletion of the three C-terminal beta3-homoarginine residues from the beta-peptide had a similarly adverse effect. Thus, alpha- and beta-peptide translocation processes show similar length/charge dependence. The beta-peptide appeared to be largely unfolded in water, which is consistent with the behavior of short Tat-derived alpha-peptides, but in methanol the beta-peptide adopted a helical conformation, in contrast to short Tat-derived alpha-peptides. Our results show that neither altering the oligomeric backbone (amide group spacing) nor increasing the intrinsic propensity to adopt a specific secondary structure affects translocation activity.