Interactions of butane, but-2-ene or xylene-like linked bispyridinium para-aldoximes with native and tabun-inhibited human cholinesterases

Kinetic parameters were evaluated for inhibition of native and reactivation of tabun-inhibited human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) and human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) by three bispyridinium para-aldoximes with butane (K074), but-2-ene (K075) or xylene-like linker (K114). Tested aldoximes reversibly inhibited both cholinesterases with the preference for binding to the native AChE. Both cholinesterases showed the highest affinity for K114 (K_i was 0.01 mM for AChE and 0.06 mM for BChE). The reactivation of tabun-inhibited AChE was efficient by K074 and K075. Their overall reactivation rate constants were around 2000 min⁻¹ M⁻¹, which is seven times higher than for the classical bispyridinium para-aldoxime TMB-4. The reactivation of tabun-inhibited AChE assisted by K114 was slow and reached 90% after 20 h. Since the aldoxime binding affinity of tabun-inhibited AChE was similar for all tested aldoximes (and corresponded to their K_i), the rate of the nucleophilic displacement of the phosphoryl-moiety from the active site serine was the limiting factor for AChE reactivation. On the other hand, none of the aldoximes displayed a significant reactivation of tabun-inhibited BChE. Even after 20 h, the reactivation maximum was 60% for 1 mM K074 and K075, and only 20% for 1 mM K114. However, lower BChE affinities for K074 and K075 compared to AChE suggest that the fast tabun-inhibited AChE reactivation by these compounds would not be obstructed by their interactions with BChE in vivo.     

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri , Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii . Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.     

Esthetic reconstruction of teeth in patient with dentinogenesis imperfecta--a case report

Dentinogenesis imperfecta (DI) is the result of a dominant genetic defect and affects both the deciduous and permanent dentitions. It is characterized by opalescent teeth composed of irregularly formed and undemineralized dentin which obliterates pulp chamber and root canal. DI can appear as a separate disorder or with osteogenesis imperfecta (OI). The teeth with DI show a grayish-blue to brown hue with dislodged enamel, dysplastic dentine with irregular dentinal tubules and interglobular dentine, short roots and pulpal obliteration, which all may lead to rapid and extensive attrition which require adequate crown reconstruction. The aim of this study was to show a reconstruction of frontal teeth in upper jaw with direct composite veneers in young adult patient with DI.     

Population dynamics of noctuid moths and damage forecasting in sugar beet

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What stabilizes the 314‐helix in β3‐peptides? A conformational analysis using molecular simulation

β‐Peptides are analogs of natural α‐peptides and form a variety of remarkably stable structures. Having an additional carbon atom in the backbone of each residue, their folded conformation is not only influenced by the side‐chain sequence but also and foremost by their substitution pattern. The precise mechanism by which the side chains interact with the backbone is, however, hitherto not completely known. To unravel the various effects by which the side chains influence the backbone conformation, we quantify to which extent the dihedral angles of a β³‐substited peptide with an additional methyl group on the central C_α‐atom can be regarded as independent degrees of freedom and analyze the distributions of these dihedral angles. We also selectively capture the steric effect of substituents on the C_α‐ and C_β‐atoms of the central residue by alchemically changing them into dummy atoms, which have no nonbonded interactions. We find that the folded state of the β³‐peptide is primarily stabilized by a steric exclusion of large parts of the unfolded state (entropic effect) and only subsequently by mutual dependence of the ψ‐dihedral angles (enthalpic effect). The folded state of β‐peptides is stabilized by a different mechanism than that of α‐peptides. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization,efficient signaling,and interaction with β-arrestin 2

This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and β-arrestin (β-arr 319–418) and by means of cell lines that expressed various endogenous levels of β-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and β-arrestin and even more profoundly in cells lacking both β-arrestin1 and β-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with β-arrestin2. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by the Slovenian Ministry of Higher Education, Science, and Technology (grant number 0406-007) and a Slovenian-Danish collaboration grant (BI-DK/06-07-007).     

Interaction of Human Butyrylcholinesterase Variants with Bambuterol and Terbutaline

Bambuterol, a dimethylcarbamate, carbamoylates butyrylcholinesterase (BChE; EC 3.1.1.8). The carbamoylated enzyme is not very stable and the final product of the two-step hydrolysis is a bronchodilator drug, terbutaline (1-(3,5-dihydroxyphenyl)-2-t-butylaminoethanol sulphate). Both bambuterol and terbutaline inhibit BChE, but their affinities differ in human serum BChE variants (U, A, F, K and S) due to their positive charge. Bambuterol inhibition rate constants for the homozygous usual (UU), Kalow (KK), fluoride-resistant (FF) or atypical (AA) variant ranged from 4.4 to 0.085?min-1?μM-1. Terbutaline showed competitive reversible inhibition for all BChE variants. The dissociation constants for UU, FF and AA homozygotes were 0.18, 0.31 and 3.3?mM, respectively. The inhibition rate or dissociation constants for heterozygotes were distributed between the respective constants for the corresponding homozygotes. A 50-fold difference in inhibition between the UU and AA enzyme might affect terbutaline release in humans. The affinity of all studied BChE variants for terbutaline was low, which suggests that terbutaline originating from bambuterol hydrolysis should not affect the hydrolysis of bambuterol by BChE.     

Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.     

Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers

Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC 3.1.1.8) by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002–250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine ‘slides down’ the enzyme gorge, resulting in interaction of the three rings of ethopropazine through π–π interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme–substrate, enzyme–inhibitor and enzyme–substrate–inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.