Praktičan primer povećanja energetske efikasnosti malih pumpnih stanica reprojektovanjem

The suction station "Gnjilan", belonging to the "Water-supply" Pirot municipal enterprise, has been used as an example of small pump stations energy efficiency increasing by replacement of existing old pumps with new ones. It has been shown that it was possible to identify the pipeline characteristic and an optimum system operating point, even in the absence of appropriate flow measuring devices. This has been done using an indirect method of flow estimation by measurement of driving induction motor elecrtical parameters. In accordance with the obtained results, the new pump has been chosen. Huge savings in the electrical energy consumption referred to actual situation will be achieved through the application of the new pump.Na primeru Crpne stanice "Gnjilan" u okviru JP "Vodovod" Pirot, prikazan je način za povećanje energetske efikasnosti malih pumpnih stanica zamenom postojećih pumpnih agregata novim. Pokazano je da je, čak i u situaciji kada ne postoje odgovarajući uređaji za merenje protoka, moguće identifikovati karakteristiku cevovoda i optimalnu radnu tačku sistema primenom indirektne metode merenja električnih veličina pogonskog motora. U skladu sa identifikovanim parametrima, izvršen je izbor novog pumpnog agregata čijom primenom se mogu ostvariti znatne uštede u odnosu na postojeće stanje sistema

Gene transfer of mutant mouse cholinesterase provides high lifetime expression and reduced cocaine responses with no evident toxicity

Gene transfer of a human cocaine hydrolase (hCocH) derived from butyrylcholinesterase (BChE) by 5 mutations (A199S/F227A/S287G/A328W/Y332G) has shown promise in animal studies for treatment of cocaine addiction. To predict the physiological fate and immunogenicity of this enzyme in humans, a comparable enzyme was created and tested in a conspecific host. Thus, similar mutations (A199S/S227A/S287G/A328W/Y332G) were introduced into mouse BChE to obtain a mouse CocH (mCocH). The cDNA was incorporated into viral vectors based on: a) serotype-5 helper-dependent adenovirus (hdAD) with ApoE promoter, and b) serotype-8 adeno-associated virus with CMV promoter (AAV-CMV) or multiple promoter and enhancer elements (AAV-VIP). Experiments on substrate kinetics of purified mCocH expressed in HEK293T cells showed 30-fold higher activity (U/mg) with (3)H-cocaine and 25% lower activity with butyrylthiocholine, compared with wild type BChE. In mice given modest doses of AAV-CMV-mCocH vector (0.7 or 3 × 10(11) particles) plasma hydrolase activity rose 10-fold above control for over one year with no observed immune response. Under the same conditions, transduction of the human counterpart continued less than 2 months and antibodies to hCocH were readily detected. The advanced AAV-VIP-mCocH vector generated a dose-dependent rise in plasma cocaine hydrolase activity from 20-fold (10(10) particles) to 20,000 fold (10(13) particles), while the hdAD vector (1.7 × 10(12) particles) yielded a 300,000-fold increase. Neither vector caused adverse reactions such as motor weakness, elevated liver enzymes, or disturbance in spontaneous activity. Furthermore, treatment with high dose hdAD-ApoE-mCocH vector (1.7 × 10(12) particles) prevented locomotor abnormalities, other behavioral signs, and release of hepatic alanine amino transferase after a cocaine dose fatal to most control mice (120 mg/kg). This outcome suggests that viral gene transfer can yield clinically effective cocaine hydrolase expression for lengthy periods without immune reactions or cholinergic dysfunction, while blocking toxicity from drug overdose

Cytotoxicity of Platinum(Iv) and Palladium(Ii) Complexes with Meso-1,2-Diphenyl-Ethylenediamine-N,N -Di-3-Propanoic Acid. Crystal Structure of [Pd(1,2-Dpheddp)] Complex

The syntheses of tetradentate ligand, meso-1,2-diphenyl-ethylenediamine-N,N-di-3-propanoic acid (H-2-1,2-dpheddp) and corresponding platinum(IV) and palladium(II) complexes are reported here. The spectroscopically predicted structure of the obtained palladium(II) complex was confirmed by X-ray analysis. Singe crystals suitable for X-ray measurements were obtained by slow crystallization from a DMSO-water mixture. Cytotoxic effects of platinum(IV), palladium(II) complexes and cisplatin on the 4T1 and Bl6F1 cell lines were determined using the MTT colorimetric technique. The complexes showed a dose dependence on cytotoxic effect toward both cell lines. Both complexes were less active than cisplatin, the exception was concentrations above 62.5 mu M of platinum(IV) complex in the B16F1 cell line

Shifts in Backbone Conformation of Acetylcholinesterases upon Binding of Covalent Inhibitors, Reversible Ligands and Substrates

The influence of ligand binding to human, mouse and Torpedo californica acetylcholinesterase (EC 3.1.1.7; AChE) backbone structures is analyzed in a pairwise fashion by comparison with X-ray structures of unliganded AChEs. Both complexes with reversible ligands (substrates and inhibitors) as well as covalently interacting ligands leading to the formation of covalent AChE conjugates of tetrahedral and of trigonal-planar geometries are considered. The acyl pocket loop (AP loop) in the AChE backbone is recognized as the conformationally most adaptive, but not necessarily sterically exclusive, structural element. Conformational changes of the centrally located AP loop coincide with shifts in C-terminal α-helical positions, revealing interacting components for a potential allosteric interaction within the AChE backbone. The stabilizing power of the aromatic choline binding site, with the potential to attract and pull fitting entities covalently tethered to the active Ser, is recognized. Consequently, the pull can promote catalytic reactions or relieve steric pressure within the impacted space of the AChE active center gorge. These dynamic properties of the AChE backbone inferred from the analysis of static X-ray structures contribute towards a better understanding of the molecular template important in the structure-based design of therapeutically active molecules, including AChE inhibitors as well as reactivators of conjugated, inactive AChE

Substrate and product trafficking through the active center gorge of acetylcholinesterase analyzed by crystallography and equilibrium binding.

Hydrolysis of acetylcholine catalyzed by acetylcholinesterase (AChE), one of the most efficient enzymes in nature, occurs at the base of a deep and narrow active center gorge. At the entrance of the gorge, the peripheral anionic site provides a binding locus for allosteric ligands, including substrates. To date, no structural information on substrate entry to the active center from the peripheral site of AChE or its subsequent egress has been reported. Complementary crystal structures of mouse AChE and an inactive mouse AChE mutant with a substituted catalytic serine (S203A), in various complexes with four substrates (acetylcholine, acetylthiocholine, succinyldicholine, and butyrylthiocholine), two non-hydrolyzable substrate analogues (m-(N,N,N-trimethylammonio)-trifluoroacetophenone and 4-ketoamyltrimethylammonium), and one reaction product (choline) were solved in the 2.05-2.65-A resolution range. These structures, supported by binding and inhibition data obtained on the same complexes, reveal the successive positions and orientations of the substrates bound to the peripheral site and proceeding within the gorge toward the active site, the conformations of the presumed transition state for acylation and the acyl-enzyme intermediate, and the positions and orientations of the dissociating and egressing products. Moreover, the structures of the AChE mutant in complexes with acetylthiocholine and succinyldicholine reveal additional substrate binding sites on the enzyme surface, distal to the gorge entry. Hence, we provide a comprehensive set of structural snapshots of the steps leading to the intermediates of catalysis and the potential regulation by substrate binding to various allosteric sites at the enzyme surface

Structural bases for the specificity of cholinesterase catalysis and inhibition

International audienceThe availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin

Benzyl 2-(benzylsulfanyl)benzoate

In the title compound, C21H18O2S, the central aromatic ring makes dihedral angles of 5.86 (12) and 72.02 (6)° with the rings of the terminal O-benzyl and S-benzyl groups, respectively. The dihedral angle between the peripheral phenyl rings is 66.16 (6)°. In the crystal, molecules are linked by pairs of C - H⋯O hydrogen bonds, forming inversion dimers. These dimers are linked via C - H⋯π interactions, forming a three-dimensional network

Backbone Conformation Shifts in X-ray Structures of Human Acetylcholinesterase upon Covalent Organophosphate Inhibition

Conformations of Cα backbones in X-ray structures of most organophosphate (OP)-inhibited human acetylcholinesterases (hAChEs) have been previously shown to be similar to that of the native hAChE. One of the exceptions is the structure of the diethylphosphoryl-hAChE conjugate, where stabilization of a large ethoxy group into the acyl pocket (AP) of hAChE-triggered notable loop distortions and consequential dissociation of the hAChE homodimer. Recently, six X-ray structures of hAChE conjugated with large OP nerve agents of the A-type, Novichoks, have been deposited to PDB. In this study we analyzed backbone conformation shifts in those structures, as well as in OP-hAChE conjugates formed by Paraoxon, Soman, Tabun, and VX. A Java-based pairwise alpha carbon comparison tool (PACCT 3) was used for analysis. Surprisingly, despite the snug fit of large substituents on phosphorus, inside Novichok-conjugated hAChEs only minor conformational changes were detected in their backbones. Small magnitudes of observed changes were due to a 1.2–2.4 Å shift of the entire conjugated OP away from the AP. It thus appears that the small AP of AChEs can accommodate, without distortion, substituents of the size of ethoxy or butyryl groups, provided that conjugated OP is “pulled” away from the AP. This observation has practical consequences in the structure-based design of nucleophilic reactivation antidotes as well as in the definition of the AChE specificity that relies on the size of its AP

Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation

The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies