Inhibition of Cholinesterases by the Oximes P2AM and Toxogonin

The reversible inhibition of electric eel acetylcholinesterase (EC 3.1.1.7) by P2AM (2-(hydroxyimino)methyl-1-methyl-pyridinium chloride) and Toxogonin (1,1\u27-[oxybis(methylene)] bis(4-(hydroxyimino) methyl-pyridinium) dichloride) was studied using ac.etylthiocholine as substrate. Two techniques were applied for measuring acetylthiocholine hydrolysis, the conventional spectrophotometric and the stopped-flow (at 25 °c in 100 mM phosphate buffer pH = 7.4). The correlation between the degree of inhibition, and acetylthiocholine and oxime concentrations fits a theoretical model which postulates that the substrate and the inhibitor bind to two sites on the enzyme: the catalytic site and an allosteric, substrate-inhibition, site. The calculated dissociation constants for the two sites are: 0.13 and 0.76 mM for P2AM, and 0.16 and 2.0 mM for Toxogonin. The suggested model is an alternative to the hypothesis that two types of binding occur within the catalytic site. Horse serum cholinesterase and bovine erythrocyte acetylcholinesterase are also inhibited by P2AM and Toxogonin to about the same degree as the electric eel enzyme. Acetylthiocholine reacts with P2AM and Toxogonin; assuming that the reaction is bimolecular the corresponding rate constants are 13.4 and 22.4 M-1 min-

Inhibition of Cholinesterases by the Oximes P2AM and Toxogonin

The reversible inhibition of electric eel acetylcholinesterase (EC 3.1.1.7) by P2AM (2-(hydroxyimino)methyl-1-methyl-pyridinium chloride) and Toxogonin (1,1\u27-[oxybis(methylene)] bis(4-(hydroxyimino) methyl-pyridinium) dichloride) was studied using ac.etylthiocholine as substrate. Two techniques were applied for measuring acetylthiocholine hydrolysis, the conventional spectrophotometric and the stopped-flow (at 25 °c in 100 mM phosphate buffer pH = 7.4). The correlation between the degree of inhibition, and acetylthiocholine and oxime concentrations fits a theoretical model which postulates that the substrate and the inhibitor bind to two sites on the enzyme: the catalytic site and an allosteric, substrate-inhibition, site. The calculated dissociation constants for the two sites are: 0.13 and 0.76 mM for P2AM, and 0.16 and 2.0 mM for Toxogonin. The suggested model is an alternative to the hypothesis that two types of binding occur within the catalytic site. Horse serum cholinesterase and bovine erythrocyte acetylcholinesterase are also inhibited by P2AM and Toxogonin to about the same degree as the electric eel enzyme. Acetylthiocholine reacts with P2AM and Toxogonin; assuming that the reaction is bimolecular the corresponding rate constants are 13.4 and 22.4 M-1 min-

Primjena tehnologije rekombinantne DNA za pripravke kolinesteraza kao antidota i detektora organofosfata

To develop new avenues for synthesizing novel antidotes for organophosphate poisoning and for detection of the organophosphates, we have turned to recombinant DNA methods to synthesize cholinesterases with unusual properties. For antidotal therapy we describe mutations of the native mouse and human enzymes that allow for enhanced rates of oxime reactivation. Such enzymes, when localized in the circulation, would enable the circulating cholinesterase to become a catalytic rather than simply a stoichiometric scavenger. Hence, “oxime-assisted catalysis” provides a means for scavenging the organophosphates in the circulation thereby minimizing their tissue penetration and toxicity. Accordingly, the oxime antidote or prophylactic agent has a dual action within the circulation and at the tissue level. Second, through a novel chemistry, termed freeze-frame, click chemistry, we have used organophosphate conjugates of acetylcholinesterase as templates for the synthesis of novel nucleophilic reactivating agents. Finally, acetylcholinesterase can be modified through cysteine substitution mutagenesis and attachment of fluorophores at the substitution positions. When linked at certain locations in the molecule, the attached fluorophore is sensitive to organophosphate conjugation with acetylcholinesterase, and thus the very target of insecticide or nerve agent action becomes a detection molecule for organophosphate exposure.Razvijajući novi pristup sintezi antidota pri otrovanju organofosfatima kao i njihovu detekciju, primijenili smo metode rekombinantne DNA za pripremu kolinesteraza s neuobičajenim svojstvima. Za antidotsku terapiju istražili smo mutacije prirodnih enzima miša i čovjeka koje povećavaju brzine reaktivacije oksimom. Takvi enzimi bi po unosu u cirkulaciju postali katalitički, a ne samo stehiometrijski odstranjivači organofosfata. Na taj način “oksimom potpomognuta kataliza” omogućava čišćenje organofosfata iz cirkulacije umanjujući prodiranje organofosfata u tkiva i njihovu toksičnost. Prema tome, oksim kao antidot ima dvojaku ulogu: u cirkulaciji i na razini tkiva. S druge strane, uporabom novog sintetskog pristupa u oblikovanju biološki aktivnih spojeva poznatog kao “klik kemija” diskretnih proteinskih konformacija, organofosforilirani konjugati acetilkolinesteraze služe kao kalup u sintezi novih nukleofilnih reaktivatora. Naposljetku, acetilkolinesteraza se može mutagenezom modificirati uvo|enjem cisteina na koje se mogu vezati fluorofori. Fluorofori uvedeni na određena mjesta u molekuli acetilkolinesteraze mijenjaju svoja fluorescentna svojstva pri konjugaciji organofosfata s enzimom koji na taj način od objekta djelovanja insekticida i živčanih bojnih otrova postaje molekula za detekciju izloženosti organofosfatima

Torrential floods and town and country planning in Serbia

Torrential floods are the most frequent natural catastrophic events in Serbia, causing the loss of human lives and huge material damage, both in urban and rural areas. The analysis of the intra-annual distribution of maximal discharges aided in noticing that torrential floods have a seasonal character. The erosion and torrent control works (ETCWs) in Serbia began at the end of the 19th century. Effective protection from torrential floods encompasses biotechnical works on the slopes in the watershed and technical works on the torrent beds, within a precisely defined administrative and spatial framework in order to achieve maximal safety for people and their property. Cooperation to overcome the conflicts between the sectors of the water resources management, forestry, agriculture, energetics, environmental protection and local economic development groups is indispensable at the following levels: policy, spatial planning, practice, investments and education. The lowest and most effective level is through the Plans for Announcement of Erosive Regions (PAERs) and the Plans for Protection from Torrential Floods (PPTFs), with Hazard Zones (HZs) and Threatened Areas (TAs) mapping on the basis of the hydrologic, hydraulic and spatial analysis of the factors that are important for the formation of torrential floods. Solutions defined through PAERs and PPTFs have to be integrated into Spatial Plans at local and regional levels

Antimicrobial activity and cytotoxicity of commercial rosemary essential oil (Rosmarinus officinalis L.)

Rosemary is well known as a spice and widely used plant in ethnomedicine worldwide. In this paper, commercial essential oil of rosemary was tested for antimicrobial and cytotoxic activity together with its effect on germination. Antimicrobial activity testing showed moderate effect to both G-positive and Gnegative bacteria. In order to determine its effect to the cell membrane, spectrophotometric analysis was performed. It was determined that rosemary affects the cell membrane of bacteria. Cytotoxic activity of Rosmarinus officinalis essential oil had been evaluated. As a plant object, germinative bulbs of Allium cepa were used. Cytotoxic activity that corresponded to the concentration of essential oil was determined. It had been noticed that rosemary essential oil affected mitotic phase i.e. it significantly slowed down the mitosis. Also, investigation of rosemary essential oil's activity to germination was performed. It was determined that it had high effect to the germination. Concentration of 5 mg/ml completely inhibited the germination of Triticum vulgare

Odnos između plijesni Trichothecium roseum i Aspergillus parasiticus i biosinteza aflatoksina

The relationship between mould biomass and the biosynthesis of aflatoxins B1 and G1 on solid substrates (whole and crushed maize grain) at temperatures from 15-35 °C and water content in the substrate of 20-36% has been investigated. The experiments have been carried out with the aflatoxigenous mould Aspergillus parasiticus NRRL 2999 in pure culture and in mixed culture respectively, the latter with the mould Trichothecium roseum. ZMFT 1226. Mould T. roseum does not produce aflatoxins and chromatographically similar compounds. The amount of biomass was estimated by measuring the chitin content, and the aflatoxin concentration by means of a fluorodensitometer. It has been established that the biosynthesis of the examined aflatoxins and their ratio primarily depend on the temperature of cultivation, rather than on the growth of the mycelium. The biomass of the mixed culture of A. parasiticus and T. roseum after seven weeks of cultivation reduces the amount of aflatoxin B1 by 20-38%, and the amount of aflatoxin G1 by 30-40%. The decrease of concentration of both toxins is more pronounced in the substrate with a higher initial water content and a higher temperature of cultivation.Istražen je kvantitativan odnos biomase plijesni i aflatoksina B1 i G1 na čvrstim supstratima (cijelo i lomljeno zrno kukuruza) pri temperaturi od 15-35 °C i sadržaju vode u supstratu od 20-38%. Pokusi su provedeni s pomoću aflatoksikogene plijesni Aspergillus parasiticus NRRL 2999 u čistoj kulturi i u mješovitoj kulturi s plijesni Trichothecium roseuin ZMTF 1226. U preliminarnim istraživanjima dokazano je da plijesan T. roseum ZMTF 1226 ne sintetizira aflatoksine, niti kromatografski slične spojeve. Rast biomase praćen je mjerenjem sadržaja hitina, a koncentracija aflatoksina određivana je tankoslojnom kromatografijom pomoću fluorodenzitometra. Utvrđeno je da sinteza istraživanih toksina i njihov međusobni odnos prvenstveno ovise, ne o količini biomase plijesni, nego o temperaturi uzgoja. Biomasa mješovite kulture plijesni A. parasiticus i T. roseum reducira, nakon sedam tjedana uzgoja, količinu aflatoksina B1 za 20-38%, a aflatoksina G1 za 30-40%. Smanjenje količine oba istraživana toksina izrazitije je u supstratu s većom početnom količinom vode i pri višoj temperaturi uzgoja

The influence of adding of flaxseed oil to sunflower oil on the content of tocopherols and carotenoids in blended edible oils

Blending vegetable oils of different composition and properties is one of the simplest methods for creating new specific products with the desired properties, which increases their commercial application and improves their nutritional quality. The effect of blending vegetable oils on tocopherols and carotenoids content was examined. Refined sunflower seed oil (S) and cold pressed flaxseed oil (F) were used in the experiment. These oils are blended in three different content of mass: sample 70S:30F (70% S and 30% F), sample 50S:50F (50% S and 50% F) and sample 30S:70F (30% S and 70% F). The results showed significant differences in the content of total tocopherols and total carotenoids between the two oils used for the preparation of three blended oils. Refined sunflower seed oil contains higher amounts of tocopherols and fewer amounts of carotenoids compared to cold pressed flaxseed oil in which the content of tocopherols is lower and the content of carotenoids is higher. In the obtained blends of edible vegetable oils, the content of total tocopherols ranged from 387.96 to 447.83 mg/kg while the determined total carotenoids content (as B-caroten) ranged from 3.11 to 5.63 mg/kg. By blending refined oil of sunflower seed and cold pressed oil of flax-seed, the balance of the parameters studied is contributed. The research in the work showed that the blending of vegetable oils provides the possibility of modulating their composition, and therefore of nutritive quality

The determination of patulin in apple juice

Istraživana je mogućnost kvantitativnog određivanja patulina tu jabučinom soku. Postupak je uključivao ekstrakciju patulina iz soka s pomoću etil-acetata, pročišćavanje ekstrakata kromatografijom na stupcu silikagela te kvalitativno i kvantitativno određivanje s pomoću tankoslojne kromatografijc. Izlaganjem ploča za tankoslojnu kromatografiju koncentriranim parama amonijaka dobiveni su derivati s intenzivnijom fluorescencijom, što je olakšalo kvantitativno fluorodenzitometrijsko određivanje patulina. Granična koncentracija, što se s pomoću upotrijebljene metode mogla odrediti, bila je 200 ng čistog patulina i 100 µg patulina na litru soka. Djelotvomost postupka bila je od 78 do 110%, a prosječna vrijednost djelotvornosti bila je 98%.A quantitative method for the determination of patulin in apple juice was examined. The procedure involved patulin extraction from apple juice with ethyl acetate, clean-up with column chromatography and preparative thin-layer chromatography. Fluorescent derivatives, obtained by exposure of patulin on chromatographic plates to concentrated ammonia fumes, permitted a convenient quantitative fluorodensitometric assay of patulin by means of the fluorescence quenching method. The detection limits were 200 ng of pure patulin and 100 µg of patulin per litre of apple juice. The recoveries of added patulin ranged from 78 to 110.4 per cent, with a mean recovery of 97.8 per cent

Artemia salina larvae as a test organism in research on mycotoxin synergism

lstraživan je utjecaj koncentracije i vremena djelovanja aflatoksina B1 i diacetoksiscirpenola na larve račića Artemia solina, u rasponu temperature od 20 do 35°C. Određeno je toksičko djelovanje za svaki mikotoksin zasebno i u kombinaciji različitih koncentracija. Nakon što su određene LC50 i T50 vrijednosti, dokazan je sinergistički odziv na osnovi smanjenja koncentracija za podjednak toksički učinak.The effect of concentration and contact time of two mycotoxins, aflatoxin 81 and diacetoxyscirpenol was studied on the larvae of Artemia salina. These organisms are sensitive to sub-microgram quantities of the toxins and can be used as bioassay test organisms. In a study of aflatoxin 81 and diacetoxys-cirpcnol synergism in acute toxicity, the LC50 values of toxin pairs exhibited a distinct increase in toxicity