Electric Organ Of Electrophorus Electricus Research Articles

QSAR Study of Some 1,3-Oxazolylphosphonium Derivatives as New Potent Anti-Candida Agents and Their Toxicity Evaluation.

The incidence of invasive fungal infections caused by Candida spp. has increased continuously in recent decades, especially in populations of immunocompromised patients or individuals hospitalized with serious underlying diseases. Therefore, the goal of our study was the search for new potent Candida albicans inhibitors via the development of QSAR models that could speed up this search process. A number of the most promising 1,3-oxazol-4-yltriphenylphosphonium derivatives with predicted activities were synthesized and experimentally tested. Furthermore, the toxicity of the studied compounds was determined in vitro using acetylcholinesterase enzyme as a biological marker. The classification QSAR models were created using Random Forests (WEKA-RF), k-Nearest Neighbors and Associative Neural Networks methods and different combinations of descriptors on the Online Chemical Modeling Environment (OCHEM) platform. Аntifungal properties of the investigated compounds were performed using standard disk diffusion method. The enzyme inhibitory action of the compounds was determined by modified Ellman's method using acetylcholinesterase from the electric organ of Electrophorus electricus. Three classification QSAR models were developed by the WEKA-RF, k-NN and ASNN methods using the ALogPS, E-State indices and Dragon v.7 descriptors. The predictive ability of the models was tested through cross-validation, giving a balanced accuracy BA = 80-91%. All compounds demonstrated good antifungal properties against Candida spp. and slight inhibition of the acetylcholinesterase activity. The high percentage of coincidence between the QSAR predictions and the experimental results confirmed the high predictive power of the developed QSAR models that can be applied as tools for finding new potential inhibitors against Candida spp. Furthermore, 1,3-oxazol-4- yl(triphenyl)phosphonium salts could be considered as promising candidates for the treatment of candidiasis and the disinfection of medical equipment.

Organic Bio-Electronics: Bridging The Gap Between Natural and Artificial Materials for Bio-Electronics Applications

This paper aims to review current research and advancement in technology in organic bioelectronics and to reinvestigate the relationship between organic bioelectronics materials, properties, and application. A comprehensive literature review on organic bioelectronics and its dependent variables created a theoretical foundation for the paper. Using the review-centric theory, a model was developed and presented to encapsulate highly dynamic interaction of organic bioelectronics, synthetic and natural material sources that can be employed in present innovations and its implications on modern technologies. The model highlights the relationship between organic bioelectronics and its four main drivers namely, synthetic material (Piezoelectric energy harvesters), natural materials (electric organs), application (bio-devices, sensors, nanogenerators) and properties. Limitations to this research include; availability of raw materials in appropriate amounts, problems associated with interfacing natural with synthetic materials, maintenance of bioelectronics devices in living organisms and design methods for a variety of specific devices. The empirical data is limited to 4 dependent variables which do not present a conclusive theory about organic bioelectronics. In this review, we explored the possibility of interfacing synthetic material (PVDF) and a natural material (electric organ of Electrophorus electricus) by reviewing their properties, fabrication methods to create a composite that has application in a variety of bioelectronics devices.

Organic Bio-Electronics: Bridging The Gap Between Natural and Artificial Materials for Bio-Electronics Applications

This paper aims to review current research and advancement in technology in organic bioelectronics and to reinvestigate the relationship between organic bioelectronics materials, properties, and application. A comprehensive literature review on organic bioelectronics and its dependent variables created a theoretical foundation for the paper. Using the review-centric theory, a model was developed and presented to encapsulate highly dynamic interaction of organic bioelectronics, synthetic and natural material sources that can be employed in present innovations and its implications on modern technologies. The model highlights the relationship between organic bioelectronics and its four main drivers namely, synthetic material (Piezoelectric energy harvesters), natural materials (electric organs), application (bio-devices, sensors, nanogenerators) and properties. Limitations to this research include; availability of raw materials in appropriate amounts, problems associated with interfacing natural with synthetic materials, maintenance of bioelectronics devices in living organisms and design methods for a variety of specific devices. The empirical data is limited to 4 dependent variables which do not present a conclusive theory about organic bioelectronics. In this review, we explored the possibility of interfacing synthetic material (PVDF) and a natural material (electric organ of Electrophorus electricus) by reviewing their properties, fabrication methods to create a composite that has application in a variety of bioelectronics devices.

Acetylcholine esterase inhibitors in effluents from oil production platforms in the North Sea

Inhibition of acetylcholine esterase (AChE) activity is a biomarker for the exposure to neurotoxic compounds such as organophosphates and is intimately associated with the toxicity of several pesticides. In the present study, the AChE inhibiting potential of organic extracts of production water (produced water) from oil and gas production platforms in the Norwegian sector of the North Sea was determined in an in vitro bioassay based on commercially available purified AChE from the electric organ of Electrophorus electricus (L.). The results from the studies show that produced water contains a combination of AChE inhibiting compounds and compounds stimulating AChE enzymatic activity. The AChE inhibition was predominantly caused by unidentified aromatic compounds in the oil/particulate fraction of produced water, whereas polar compounds in both the water soluble and oil/particulate fraction of produced water caused an apparent stimulation of AChE activity. Substrate saturation studies with fixed concentrations of produced water extracts confirmed that the inhibition occurred in a non-destructive and competitive manner. The concentrations of AChE inhibitors (7.9–453ng paraoxon-equivalentsL−1, 2.2–178μg dichlorvos-equivalentsL−1) were in many cases found to be several orders of magnitude higher than background levels. The findings demonstrate that produced water contains potentially neurotoxic compounds and suggest that further laboratory studies with fish or field studies in the vicinity of oil production facilities are highly warranted.

Purification and Amino Acid Sequence of Fructose-1,6-bisphosphate Aldolase from the Electric Organ of Electrophorus electricus (L.)

A soluble fructose-1,6-bisphosphate aldolase enzyme has been purified 50.2-fold (2.36%) at the homogeneity from the electric organ of Electrophorus electricus by one step of DEAE-52 anion exchange chromatography followed by Superose-12 gel filtration-FPLC. Like other aldolase enzymes the E. electricus protein is a dimer with two identical subunits of 45 kDa. The N-terminal (20 residues) revealed a high homology with S. aurata (75%, goldfish), R. ratus and M. musculus (mouse, 80%) enzymes.

Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP⇔ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

Purification and partial characterization of creatine kinase from electric organ of Electrophorus electricus (L.)

The present investigation deals with the purification and the partial characterization of the soluble creatine kinase (CK) isoenzyme, isolated from the electric organ electrocyte of Electrophorus electricus (L.). Purification was performed by precipitation of the enzyme in the crude extract with ammonium sulfate (80%). The precipitate obtained was analyzed on an ion exchange column of diethylaminoethyl cellulose-52 (DEAE) followed by gel filtration on Superose 12 in a Fast Protein Liquid Chromatography (FPLC) system. Electrophoretic mobility of the active peak confirmed previous results identifying the hybrid isoenzyme MB in the eletrocyte cytoplasm. Electrocyte CK is a dimeric enzyme with two identical subunits of approximately 40 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The sequence analysis of the N-terminal peptide (14 amino acids) of the 40 kDa subunit showed homology with other CK enzymes from electric fish ( Torpedo) and human muscle type CK.

Purification and partial characterization of glyceraldehyde-phosphate dehydrogenase from electric organ of Electrophorus electricus (L.).

The glyceraldehyde-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified to homogeneity from electric organ of Electrophorus electricus (L.) by a hydrophobic chromatography method on deacetylcolchicine-Sepharose. The purification resulted in a 162 fold increase in specific activity of the GAPDH and final yield was approximately 37%. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. The purity of the colchicine-Sepharose isolated material was analysed by isoelectrophocusing and immunoblotting using a heterologous rabbit serum anti-GAPDH. Sequence analysis of the 40-N-terminal amino acids, determined by Edman degradation, revealed its identity to other GAPDHs proteins being the largest number of identical amino acids to lobster (92.5%), rabbit muscle (85%) and human liver (80%) GAPDH.

Choline Acetyltransferase from the electric organ of Electrophorus electricus (L.)--physicochemical characterization and immunochemical identification.

It is well known that the regulation of choline acetyltransferase (ChAT) activity, under physiological conditions, is important for the development and neuronal activities of cholinergic systems. The purification of ChAT has been obtained from many sources such as electric organs of fishes, Drosophila melonogaster, and mammals. We have prepared choline acetyltransferase from a pool of supernatants obtained by differential centrifugation of electric organ homogenates from Electrophorus electricus (L.) in Tris-phosphate buffer, 0.05 M, pH 7.6. The first step of the enzyme purification was performed by ammonium sulfate precipitation at 40% and 80%. The precipitate at 80% was solubilized with sodium-phosphate buffer 0.05 M, pH 7.6, dialyzed, chromatographed on DEAE-52 column and the active fraction submitted to FPLC system columns (Mono-Q: ion exchange- Superose-12: gel filtration). ChAT activity from the eluates was estimated by Fonnun's method [Fonnun, 1975], with Acetyl-Coenzyme A tritium labelled ([3H]AcCoA) as substrate, and the synthesis of 3HACh formed was measured. The peak from gel filtration showed a relative molecular mass of 80 offkDa with highest activity in the order of 77,42 nmoles ACh/min/mg protein. This fraction was analyzed by SDS-PAGE and a band of 42 kDa was detected with Coomassie blue stain, indicating that the enzyme is formed by two subunits. Employing an antibody, the presence of ChAT was confirmed with the Western blotting technique. Isoelectrofocusing analysis demonstrated two isoforms with pI of 6,49 and 6,56, respectively.

Differences in the isodesmin pattern between the electric organs of Electrophorus electricus L.

Desmin, the intermediate filament protein of muscle, is present in the electric organs of Electrophorus electricus L. as five isovariants, instead of the one to two isovariants found in muscle. We analyzed the isodesmin pattern in the three different electric organs using densitometry of Coomassie blue-stained bands in electrofocusing polyacrylamide gel electrophoresis. We were able to compare the relative amount of each of the five desmin isovariants in an isodesmin pattern characteristic of each electric organ. These patterns proved to be, in some cases, statistically different. Desmin in each electric organ could have slightly different functions in order to correlate with the organ-specific isovariant patterns.

Lipids associated with the (Na+ - K+)ATPase of normal and denervated electric organs of Electrophorus electricus (L.).

The effect of denervation on the lipid metabolism and on the activity of (Na+ - K+)ATPase isoforms from the membrane fraction P3, which corresponds to the innervated electrocyte membrane, was evaluated. On a discontinuous sucrose gradient, normal P3 membranes exhibit a bimodal ("a" and "b bands) distribution of the (Na+ - K+)ATPase activity, which upon denervation changes to an unimodal ("c" band) distribution. Using these fractions, which have a higher (Na+ - K+)ATPase activity, we characterized the lipids at the hydrophobic protein surface boundary, (i.e., the bulk lipids that surround the protein). The results confirm that these lipids consist of phospholipids and cholesterol. The quantitative composition of the phospholipids is similar for both isoform fractions obtained from the discontinuous gradient of normal membranes, with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine representing about 90% of the total phospholipids. Sphingomyelin, phosphatidylinositol, diphosphatidylglycerol and phosphatidic acid were in the minority. However, in the single band obtained after denervation, the three major phospholipid components decreased to 70% of the total, and a significant increase in the other phospholipids and in cholesterol was observed. The high cholesterol content of the denervated fraction may confer membrane stabilization, as it is likely to cause a decrease in the membrane fluidity and consequently in the enzyme activity.

Microheterogeneity of desmin in the electric organ and dorsal muscle of the electric eel Electrophorus electricus

Abstract Two-dimensional gel electrophoresis of purified desmin from the electric organ of Electrophorus electricus exhibited five isoforms as opposed to four from the dorsal muscle of the eel. Differences in isoforms may indicate different degrees of desmin phosphorylation in these tissues. The similarities detected in the two less acidic isodesmin from the electric organ and from the dorsal muscle of Electrophorus electricus suggest that the electric organ has conserved some characteristics seen in the dorsal muscle, favoring previous suggestions that considered the electric tissue as a differentiated form of striated muscle.

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ.

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-->E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s-1.

Solubilization of thiamine triphosphatase from the electric organ of Electrophorus electricus

The membrane-associated, anion-regulated thiamine triphosphatase from Electrophorus electricus electric organ can be solubilized by various neutral detergents. Polyoxyethylene ethers are the most effective. Anionic detergents readily inactivate the enzyme. A 6.4-fold increase in specific activity is obtained by successive treatment of crude membranes with octanoyl-N-methylglucamide, which solubilized other proteins, and Lubrol-PX with releases 60% of the thiamine triphosphatase (TTPase) activity. Solubilization by Lubrol-PX rapidly modifies kinetic parameters. The Km, Vmax and pH optimum are decreased. However, the solubilized TTPase may be kept at 0°C for many hours without further change in specific activity. At 35°C, the half-life is still 83 min at pH 5.0, but denaturation becomes rapid at pH ⩾ 7. Solubilization modifies anion effects on TTPase activity. The activating effect of nitrate is nearly lost, while inhibition by sulfate is no longer time-dependent.

Expression and localization in the developing cerebellum of the carbohydrate epitopes revealed by Elec-39, an IGM monoclonal antibody related to HNK-1

The immunochemical and immunocytochemical reactivity of an anti-carbohydrate monoclonal antibody (Elec-39), obtained against acetylcholinesterase from Electrophorus electricus electric organ, was followed during the postnatal development of the rat cerebellum. The specificity of this antibody resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1 and NSP-4, as well as IgMs that occur in some human neuropathies. As revealed by immunoblotting techniques, the reactivity of Elec-39 is maximum around postnatal days 10–12. At this age, the antibody reveals eight major proteins of mol. wt ranging between 14 and 150 kDa. Some of them (with mol. wts of 14, 18, 28 and 31 kDa) are transiently expressed. They correspond to previously identified glycoproteins binding to the plant lectin concanavalin A and binding also to the endogenous mannose-binding lectin CSL and endogenous membrane-bound mannose-binding lectin. In young animals, an important staining with the Elec-39 antibody can be observed on postmitotic precursors of granule cells, on astrocyte processes in the external granular layer, on newly formed parallel fibres and on unmyelinated axons of the white matter. In adult animals, the labelling is localized essentially in myelin and also in the cytoplasm of astrocytes. These results are discussed in relation to ontogenetic phenomena occurring during cerebellar development and the potential role of the carbohydrate epitope revealed with Elec-39 as a determinant in cell adhesion processes.

Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus electricus: Substrate‐Enzyme Interactions

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent Km is 1.8 mM and the pH optimum is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Km (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO3- (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the Km is 1.1 mM; at pH 7.8, NO3- increases the Vmax 24-fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10(-5)-10(-6) M) substrate concentrations, Lineweaver-Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [gamma-32P]TTP. Our data suggest the existence of a high-affinity binding site (KD of approximately 0.5 microM) for the Mg-TTP complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmembrane flux and receptor desensitization measured with membrane vesicles. Homogeneity of vesicles investigated by computer simulation

The use of membrane vesicles to make quantitative studies of transmembrane transport and exchange processes involves an assumption of homogeneity of the membrane vesicles. In studies of 86Rb+ exchange mediated by acetylcholine receptor from the electric organ of Electrophorus electricus and of 36Cl- exchange mediated by GABA receptor from rat brain, measurements of ion exchange and receptor desensitization precisely followed first order kinetics in support of this assumption. In other measurements a biphasic decay of receptor activity was seen. To elucidate the molecular properties of receptors from such measurements it is important to appreciate what the requirements of vesicle monodispersity are for meaningful results and what the effect of vesicle heterogeneity would be. The experiments were simulated with single vesicle populations with variable defined size distributions as well as with mixtures of different populations of vesicles. The properties of the receptors and their density in the membrane could be varied. Different receptors could be present on the same or different membrane vesicles. The simulated measurements were not very sensitive to size dispersity. A very broad size distribution of a single vesicle population was necessary to give rise to detectable deviations from first order kinetics or errors in the determined kinetic constants. Errors could become significant with mixtures of different vesicle populations, where the dispersity in initial ion exchange rate constant, proportional to the receptor concentration per internal volume, became large. In this case the apparent rate of receptor desensitization would diverge in opposite directions from the input value when measured by two different methods, suggesting an experimental test for such kinetic heterogeneity. A biphasic decrease of receptor activity could not be attributed to vesicle heterogeneity and must be due to desensitization processes with different rates. Significant errors would not arise from the size dispersity apparent in subpopulations of vesicles seen by imaging techniques in membrane preparations.

Desmin heterogeneity in the main electric organ of Electrophorus electricus

Desmin, the muscle-specific intermediate filament protein was purified from the main electric organ of Electrophorus electricus. It is shown that pure desmin can be separated into 5 isoforms presenting different isoelectric points. These isoforms have similar molecular weight, react with an antibody directed against desmin and generate identical peptides after digestion with protease V8 from Staphylococcus aureus.

Thiamine triphosphate and membrane-associated thiamine phosphatases in the electric organ of Electrophorus electricus.

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.

Grasping for calcium binding sites in sodium channels with an EF hand

Amino acid sequences near the carboxy terminal end of the Electrophorus electricus electric organ and rat brain sodium channel polypeptides were discovered to be putative EF hand calcium binding sites. This conclusion was made using the following criteria: the Tufty-Kretsinger and Szebenyi-Moffat EF hand tests, a computer generated analysis, the revised guidelines of Chou & Fasman, and sequence comparisons with other published EF hand calcium binding regions. These results suggest that the sodium channel may be a calcium binding protein.