V. S. Saakov

Application of high-order derivative spectrophotometry for studying the interaction of calcium ions with various anticoccidial aminoguanidine derivatives.

The recording of second and higher-order derivative spectra in studies on biochemistry, pharmacochemistry, and physiology has yielded a large body of original data, which are impossible to obtain by trivial absorption spectrophotometry [1–4]. This was evident from our analysis of about 3000 references concerning the application of derivative spectrophotometry in biochemistry, pharmacochemistry, physiology, and clinical studies from the database of the National Center of Biotechnological Information (NCBI, United States). To attempt new steps on the road, we studied the specifics of the interaction between anticoccidial benzylidenaminoguanidine derivatives, in particular, bis(4-chlorobenzylidenamino)guanidine (Cl-BAG), and several metals [5, 6]. At the same time, analysis showed that, in the presence of Ca 2+ , nonsubstituted bis(benzylidenamino)guanidine (BAG) is incapable of complexation with Va 2 and fails to exert an anticoccidial effect [7]. A hypothesis was advanced that Cl atoms play a specific role in anticoccidial activity, which is associated with complexation of Cl-BAG with metal ions [5, 6]. The hypothesis was verified and developed in our further works [8, 9].

Alternative pathways of carotenoid biosynthesis in prokaryotes and eukaryotes.

Numerous experimental data published during the past 15 years raise doubts in the generalization of the acetate–mevalonate (AC/MVA) pathway of isoprenoid biosynthesis in different representatives of flora and fauna. However, the experiments performed at the end of 1950s by a well-known expert in carotenoid biosynthesis, Goodwin, raise doubts in his postulate that the AC/MVA pathway of isopentenyl pyrophosphate (IPP) synthesis was the only proper route of synthesis of plant carotenoids (tetraterpenes) [1]. This conviction of Goodwin in unambiguous domination of the AC/MVA pathway of isoprenoid biosynthesis considerably slowed down the studies on the routes of their biosynthesis in different evolutionary groups of plants and bacteria all over the world, including Russia. The experiments of Costes [2, 3] made the researchers reconsider the sequence of synthesis of violoxanthine, lutein epoxide, and zeaxanthine and to put forward the hypothesis on the synthesis of carotene via the reduction of xanthophylls. When studying xanthophylls renovation, we obtained the data indicative of the existence of alternative carotenoid precursors, in which the photosynthetic carbon was incorporated during the first minutes of incubation [4, 5]. Despite the difficulties related with publishing those results, we proposed a hypothesis on the formation of different pools of precursors for epoxy xanthophylls and carotene and existence of a special pathway of synthesis of epoxy xanthophylls, related to the use of primary products of carbon metabolism (phosphoglyceric acid (PGA), phosphoenol pyruvate (PEP), etc.). We also considered the possibility of oxidative reamination of leucine and deamination of glycocol, alanine, and serine with their subsequent decarboxylation to form a pool of acetate . Decarboxylation of phosphotrioses and oxidation decarboxylation of oxaloacetate (OxAc), pyruvate (PYR), and PGA gave a similar result. At that time, we and researchers from other countries sought to substantiate the formation of pools of acetate as the main precursor of acetyl-CoA (ACCoA) during the synthesis of plastidic carotenoids. It was also shown that 14 C-labeled exogenous pyruvate was rapidly incorporated both into extraplastidic isoprenoids and tetraterpenes of chloroplasts [6, 7]. However, because of the common standpoint dominating for almost forty years and excessive carefulness of the expert reviewers of journals, the interpretation of the majority of results reduced to substantiation of the pathways of formation of pools of AC, ACCoA, and MVA as precursors of cytosolic and plastidic IPP. Nevertheless, even 30 year ago we regarded decarboxylation of PYR, PGA, phosphoglycerol aldehyde (PhGA), and OxAc, as well as the route CO 2 → glycolate → glyoxylate → glycine → serine → pyruvate → acetyl-CoA → terpenoids as pathways of isoprenoid synthesis, characteristic of plastids. This also concerns the interpretation of the possibility of another pathway of label migration (ribulose bisphosphate → 3 PGA → 2 PGA → PEP) and the use of the pools of PEP and acetate for the biosynthesis of IPP and then carotenoids [4, 5]. Thus, although it was shown that, in the course of photosynthesis, 14 CO 2 was rapidly incorporated into carotenoids and that MVA was very weakly incorporated into β -carotene (Car) and other plastidic carotenoids but rapidly incorporated into cytoplasmic sterols [1], the idea on the predominant role of the AC/MVA pathway of the synthesis of plastidic carotenoids was leading for many years.

The Chelating Ability of the Anticoccidial Drug 1,3-bis(p-chlorobenzilideneamino)guanidine: the Complexes with Ca2+ and La3+

Among the few drugs for treatment and prevention of protozoal diseases manufactured in Russia, Chimkoktsid (1,3bis ( p -chlorobenzilideneamino)guanidinium, Cl-BAG), a highly effective anticoccidial preparation against animal coccidioses and toxoplasmosis, occupies a special place [1]. This preparation is also of interest for medicine as a potential drug that may be used for human toxoplasmosis treatment [1]. However, the molecular mechanism of Cl-BAG action remains obscure. Earlier, it was shown by the method of molecular mechanics that folded conformations of Cl-BAG are the most preferable due to attraction between the benzene rings [2]:

[COMPARATIVE SENSITIVITY OF CHOLINESTERASES IN VERTEBRATES AND INVERTEBRATES TO HIGHLY SPECIFIC ORGANOPHOSPHORUS INHIBITORS DIISOPROPYL FLUOROPHOSPHATE (DFP) AND (2-ETHOXYMETHYL PHOSPHORYL THIOETHYL) ETHYL (METHYL) SULPHONIUM SULPHOMETHYLAT (GD-42)].

The review presents the data on a comparative reactivity of 68 cholinesterase preparations from various organs and tissues in a number of vertebrates and invertebrates based on the sensitivity to the two highly specific and most studied organophosphorus inhibitors, diisopropyl fluorophosphates (DFP) and (2-ethoxymethyl phosphoryl thioethyl) ethyl (methyl) sulphonium sulphomethylate (GD-42). An analysis of the data obtained suggests a great diversity of enzymologic characteristics of cholinesterase preparations in vertebrates and invertebrates observed even in closely related enzymes in animals at virtually the same level of evolutionary development.

High-Temperature Stress-Related Changes in the Harmonics F0, Fm, and Fv of Pulse-Amplitude Modulated Fluorescence Signals: Locating Thermal Damage in Reaction Centers of Photosystem II

The so-called midday depression (a decrease in the CO 2 fixation rate under the high temperature conditions at midday) contributes to a decrease in the plant productivity not only in arid zones, but also in temperate environments during hot summers. Along with structural changes in the photosynthetic apparatus (which are beyond the scope of this study), functional impairments of chloroplasts have been reported. Earlier, we determined the range of temperatures at which photosynthesis was inhibited [1]. Our data were confirmed in later publications by others [2–8]. We estimated the upper limit of the normal temperature range at 35°ë . At this temperature, a decrease in the CO 2 fixation rate became statistically significant. Exposure to high temperatures (39–41 ° C) and their aftereffect decreased the rate of photosynthesis from 60 to 80%. However, 17–19 h after cessation of exposure at 41 ° C, the photosynthetic rate partly restored (only to 70% of the initial level) [1]. The effects of higher temperatures were lethal. Given that temperature-induced uncoupling of the complexes manifests itself in a reduced rate of electron transport and in energy redistribution between PSI and PSII [3], it is likely that acclimation or adaptation to hot environments enhances the reliability of the mechanism whereby light-harvesting pigment complexes interact with reaction centers (RCs) of photosystems I and II (PSI and PSII, respectively). A reduction in the rate of electron transport is detectable at 35 ° C. Photophosphorylation becomes unstable at this temperature and is progressively inhibited as the incubation temperature is raised above 35 ° C [8]. Thus, there is no doubt that a functional impairment in CO 2 fixation at incubation temperatures exceeding 35 ° C develops concurrently with a decrease in the rate of electron transfer in PSII. To answer the question as to the target of thermal stress in the mechanism coupling the operation of the photosystems with ATP synthesis [3], we studied herbaceous and arborous plants that differently tolerate high temperatures in the physiological range. In this work, we used the pulse amplitude-modulated (PAM) fluorescence technique to identify, in in vivo experiments, which components of the electron transport chain (ETC) of the photosystems are impaired by stressors. Note that we do not call into question the fact that, among the isolated components of the photosynthetic apparatus, PSII is the most susceptible to the effects of environmental extremes. It should be emphasized that, in early studies, the stress tolerance of the photosystems was addressed in in vitro experiments on isolated components (such as chloroplast suspensions prepared by centrifugation), rather than in in vivo experiments on whole leaves. In this study, we varied the temperature from 35 to 45 ° C and observed the changes in the harmonics of the PAM fluorescence signal. Parameters F 0 , F m , and F v were recorded using a Walz 101-103 device (Effeltrich, Germany) [9, 10]. Accepted in many laboratories of Europe and the United States, the PAM fluorescence method has proven to be accurate for monitoring quick changes in the parameters of the fluorescence signal from a sample, which can be either a whole leaf or suspensions of chloroplasts or algae. The photochemical quenching coefficient ( q q ), the nonphotochemical quenching coefficient ( q e ), and the coefficients describing other causes of fluorescence quenching ( K f ) were calculated and interpreted as described elsewhere [9, 10]. The photodetector system of this analytical instrument is such that measurements can be taken without protection from ambient light; therefore, the PAM fluorimeter is an instrument recommended for use in field ecological studies. With this instrument, the effects of various anthropogenic and natural environmental stressors (ESs) are easy to compare by measuring even one High-Temperature Stress-Related Changes in the Harmonics F 0 , F m , and F v of Pulse-Amplitude Modulated Fluorescence Signals: Locating Thermal Damage in Reaction Centers of Photosystem II

New Acylate and Thioacylate Effectors of Mammalian Cholinesterases Based on Cyclic Ammonium Alcohols Containing Elements of the Anabasine Structure

We report a pioneering analysis of the interaction between mammalian cholinesterases and 36 acylates and thioacylates of ammonium alcohols with different structure of an alkyl chain between ammonium and etheric atoms and with different structure of a cyclic ammonium group. Among these ethers, which were both substrates and reversible inhibitors of erythrocyte cholinesterase and serum butyrylcholinesterase, specific effectors of both enzymes were identified.

Substrate Specificity of Cholinesterases in Various Representatives of the Animal Kingdom

This review summarizes the literature data as well as experimental results obtained at our Institute over a period of 50 years on the substrate specificity of cholinesterases–acetylcholine acetylhydrolases (EC 3.1.1.7) and acylcholine acylhydrolases (EC 3.1.1.8). The parameters of enzymatic hydrolysis of oxo- and thiocholine and β-methylcholine esters in different organs and tissues were analyzed in 66 animal species including 22 chordate, 20 insect, 1 mite, 17 mollusk, 4 nematode, and 2 flatworm species. Our substrate specificity studies and extensive data on the inhibitory specificity obtained using irreversible organophosphorous inhibitors and reversible effectors unequivocally indicate that the cholinesterase family is characterized by a clear-cut species and tissue specificity.

Substrate–inhibitor specificity of cholinesterase and monoamine oxydase from optic ganglia of the pacific squid Todarodes pacificus and commander squid Berryteuthis magister

A comparative analysis of enzymological characteristics of cholinesterase (ChE) and monoamine oxydase (MAO) from the optic ganglia was performed in the Pacific squid Todarodes pacificus and Commander squid Berryteuthis magister caught in their four habitats across the Pacific Northwest. A substrate–inhibitor analysis revealed a homogeneity of T. pacificus and B. magister ChE preparations as well as homogeneity of T. pacificus vs. heterogeneity of B. magister MAO preparations. In case of thiocholine derivatives, the rate of hydrolysis induced by T. pacificus ChE was practically independent of the structure of the acyl group, whereas in case of B. magister ChE it was found to decrease in this substrate series. It is only T. pacificus MAO that was found to be able to deaminate also a diaminooxydase substrate histamine. ChE activity was higher in T. pacificus than in B. magister for the whole substrate series, while for MAO the same activity pattern was observed for tyramine, tryptamine and serotonin. In both squids, the sensitivity of ChE to organophosphorus inhibitors containing the dimethylbutyl group was by several orders of magnitude higher than that in mammals. The sensitivity of ChE to the siloxane reversible inhibitors was lower in T. pacificus ChE and much lower in B. magister than in mammals.

Activities of Tetramethylene-Bis-Onium Reversible Cholinesterase Inhibitors as Influenced by the Nature of the Onium Atom

Inorganic tetramethylene bis-onium compounds were studied as reversible inhibitors of various cholinesterases (ChE), i.e., human erythrocyte acetyl-ChE, horse blood serum butyryl-ChE, grass frog Rana temporaria brain ChE, and Pacific squid Todarodes pacificus and commander squid Berryteuthis magister visual ganglia ChE from various habitats in the northwest Pacific Ocean, in order to study the influence of the nature of the onium atom on their anti-ChE activities. bis-Phosphonium inhibitors turned out to be significantly more potent effectors than bis-ammonium compounds, which may actually have been due to the significant increases of size and hydrophobicity of the onium groups. The bis-ammonium organosilicon compound and its monoammonium analog turned out to be equally active as reversible ChE inhibitors in mammals. The bis-phenyliodonium derivative was studied for the first 7time, was characterized by significantly increased hydrophobicity due to introduction of F atoms into the tetramethylene spacer in the onium chain, and exhibited marked anti-ChE activity with respect to mammalian ChE.

Tetramethonium derivatives as reversible inhibitors of various cholinesterases

To study the effect of the onium atom nature on anticholinesterase efficiency, we tested elementorganic derivatives of tetramethylenbisonium compounds as reversible inhibitors of the following cholinesterases (ChE): acetyl-ChE from human erythrocytes, butyryl-ChE from horse serum, ChE from the brain of the grass frog Rana temporaria, ChEs from visual ganglia of the Pacific squid Todarodes pacificus, and ChE from visual ganglia of the commander squid Berryteuthis magister from different habitats in the Northwestern Pacific Ocean. Bisphosphonium inhibitors were found to be much stronger effectors than bisammonum compounds, although this may be due to a significantly increased size and hydrophobicity of their onium groups. Bisammonium organosilicon compound and its monoammonium analog were equally active as reversible ChE inhibitors in mammals. The first studied bis(phenyliodonium) derivative, which is characterized by a significantly increased hydrophobicity due to the introduction of fluorine atoms to the interonium tetramethylene chain, also exhibited a pronounced anticholinesterase effect on mammalian ChE.