Synthesis and antigenic analysis of the BclA glycoprotein oligosaccharide from the Bacillus anthracis exosporium

The glycoprotein BclA is an important constituent of the exosporium of Bacillus anthracis spores. This glycoprotein is substituted with an oligosaccharide composed of a beta-L-rhamnoside substituted with the previously unknown terminal saccharide, 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-D-glucopyranose, also referred to as anthrose. Anthrose has not been found in spores of B. cereus and B. thuringiensis, making it a potential species-specific marker for B. anthracis. In order to study the antigenicity of anthrose, efficient syntheses of an anthrose-containing trisaccharide and a series of structurally related analogues were developed. The analogues lacked either the methyl ether at C-2 or contained modified C-4 amino functionalities of anthrose. The synthetic compounds were equipped with an aminopropyl spacer to facilitate conjugation to the carrier proteins mariculture Keyhole Limpet Hemocyanin (mcKLH) and bovine serum albumin (BSA). Serum antibodies of rabbits immunized with live or irradiated spores of B. anthracis Sterne 34F(2) were able to recognize the synthetic trisaccharide-mcKLH conjugate. The specificity of the interaction was confirmed by competitive inhibition with the free- and BSA-conjugated trisaccharides. Inhibition using the trisaccharide analogues demonstrated that the isovaleric acid moiety of anthrose is an important structural motif for antibody recognition. These data demonstrate that 1) anthrose is a specific antigenic determinant of the B. anthracis Sterne spore; 2) this antigen is presented to the immune system of rabbits receiving the anthrax live-spore vaccine; 3) synthetic analogues of the oligosaccharide retain the antigenic structure; and 4) the antigenic region is localized to specific terminal groups of the oligosaccharide. Collectively these data provide an important proof-of-concept step in the synthesis and development of spore-specific reagents for detection and targeting of non-protein structures in B. anthracis.     

Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study

In a previous communication, kinetic β-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic β-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 ± 0.03, (D3)k(cat) = 1.12 ± 0.02 and (D3)βk(cat) = 0.97 ± 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.     

Simultaneous ion transfer across the microhole ITIES: an example of ternary electrodiffusion

Ternary electrodiffusion at the immiscible liquid liquid interface (ITIES) is considered in the case where a univalent cation is transferring from the water to the organic phase through a microhole, and simultaneously a univalent anion is transferring in the opposite direction. The process is assumed to take place at steady-state. If the separation between the formal transfer potentials of these ions is large enough, a limiting current corresponding to the electrodiffusion of the ion with the lower formal transfer potential is observed. Thereafter, the current begins to rise again as a linear function of the cell potential because the Galvani potential across the interface has reached its limiting value and the potential is expended in the form of ohmic loss in the diffusion boundary layers. The Galvani potential across the interface cannot overcome the average value of the formal transfer potentials due to the electroneutrality condition. The present case is analogous to a redox reaction at the ultramicroelectrode with sign reversal (K.B. Oldham, J. Electroanal. Chem. 337 (1992) 91) but no suitable experimental system has yet been found with which to test this prediction. Experiments without supporting electrolytes seem to have only little value in quantitative analysis of ion transfer.     

Thermally-induced structural and electrical effects in GeTe-based amorphous alloys

The thermal, structural electrical properties of bulk glasses based on GeTe compositions near the binary eutectic, Ge₁₅Te₈₅, are studied. Information regarding the non-crystalline state and the transformation from the non-crystalline to the crystalline state is reported. The particular alloys studied represent binary (Ge₁₇Te₈₃), ternary (Ge₁₅Te₈₀As₅) and quaternary (Ge₁₅Te₈₁Sb₂S₂) compositions. Structural information is obtained using X-ray diffraction techniques and density measurements. Thermal data are reported from differential scanning calorimetry (DSC), thermogravimetry (TGA) and mass spectrometry results. The electrical conductivity is measured as a function of temperature and, on the ‘as-prepared’ glasses, shows semi-conducting behavior with activation energies, E, of 0.43–0.48 eV. DSC, TGA and X-ray powder diffraction patterns indicate the samples crystallize as Te and GeTe in a two-step process, and melt at the binary eutectic temperature. The binary vaporizes as Te and GeTe in a two-step process. GeTeAs and GeTeSbS vaporize by essentially the same mechanism, with As evaporating (<300°C) before the Te, and Sb and S evaporating (420–480°C) after the Te but before GeTe. The results show that the properties of the bulk ‘as-prepared’ glasses are strikingly similar. Thermally-induced changes in the structural and electrical properties of bulk samples have been examined following a series of anneals (5 h, vacuum) at temperatures from 111°C to 190°C (glass transition temperature $\text{?125?133°}\text{C}$; crystallization temperature $\text{?206?228°}\text{C}$ as determined by DSC). DSC, TGA and mass spectrometry results have been correlated to electrical and structural changes. Results show that crystalline Te nucleates at the surface and forms a conductive surface layer. The conductivity of this surface layer is nearly temperature independent with E ≈ 10^?2 eV for all three alloys. Crystallization and the associated electrically conductive regions extend into the bulk material with further annealing. In these disordered alloys the additives As and Sb + S apparently do not act as electrical dopants in the sense of affecting the conductivity activation energy. The additives Sb + S however do retard crystallization of GeTe. The secondary crystallization product, GeTe, apparently changes the conduction mechanism to either a metallic or degenerate semiconductor type behavior.     

Long‐lived intermediates in reversible addition–fragmentation chain‐transfer (RAFT) polymerization generated by γ radiation

A novel experimental procedure is presented that allowed probing of reversible addition–fragmentation chain‐transfer (RAFT) free‐radical polymerizations for long‐lived species. The new experimental sequence consisted of gamma irradiation of a mixture of initial RAFT agent (cumyl dithiobenzoate) and monomer at ambient temperature, a subsequent predetermined waiting period without initiation source also at ambient temperature, and then heating of the reaction mixture to a significantly higher temperature. After each sequence step, the monomer conversion and molecular weight distribution were determined, indicating that controlled polymer formation occurs only during the heating period. The results indicated that stable intermediates (either radical or nonradical in nature) are present in such experiments because thermal self‐initiation of the monomer can be excluded as the reason for polymer formation. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1058–1063, 2002     

Ends of maps,II

Versions of the finiteness obstruction and simple homotopy theory within overX are developed. This provides a setting for obstructions to the map analogs of the end ands-cobordism theorems for manifolds. These are applied to study equivariant mapping cylinder neighborhoods in topological group actions, triangulations of locally triangulable spaces, and block bundle structures on approximate fibrations.