Biomedical subjects

V Talesa

Publications and source records attributed to V Talesa.

At least 19 recordsLinked to original sources

PubMed2001

Soluble and membrane-bound acetylcholinesterases in Mytilus galloprovincialis (Pelecypoda: Filibranchia) from the northern Adriatic sea.

Three forms of acetylcholinesterase (AChE) were detected in samples of the bivalve mollusc Mytilus galloprovincialis collected in sites of the Adriatic sea. Apart from the origin of the mussels, two spontaneously soluble (SS) AChE occur in the hemolymph and represent about 80% of total activity, perhaps hydrolyzing metabolism-borne choline esters. These hydrophilic enzymes (forms A and B) copurified by affinity chromatography (procainamide-Sepharose gel) and were separated by sucrose gradient centrifugation. They are, respectively, a globular tetramer (11.0-12.0 S) and a dimer (6.0-7.0 S) of catalytic subunits. The third form, also purified from tissue extracts by the same affinity matrix, proved to be an amphiphilic globular dimer (7.0 S) with a phosphatidylinositol tail giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. Such an AChE is likely functional in cholinergic synapses. All three AChE forms show a good substrate specificity and are inactive on butyrylthiocholine. Studies with inhibitors showed low inhibition by eserine and paraoxon, especially on SS forms, high sensitivity to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284c51) and no inhibition with propoxur and diisopropylfluorophosphate (DFP). The ChE forms in M. galloprovincialis are possibly encoded by different genes. Some kinetic features of these enzymes suggest a genetic polymorphism.

Acetylcholinesterase↗

PubMed2001

Acute toxicity screening of novel AChE inhibitors using neuronal networks on microelectrode arrays.

Spontaneously active neuronal networks grown from embryonic murine frontal cortex on substrate integrated electrode arrays with 64 recording sites were used to assess acute neurobiological and toxic effects of a series of seven symmetrical, bifunctional alkylene-linked bis-thiocarbonate compounds designed to possess anticholinesterase activity. Acute functional neurotoxicity in the absence of cytotoxicity was defined as total collapse of spontaneous activity. All of the compounds were characterized as mixed inhibitors of AChE, with K(i)'s in the 10(-7)-10(-6) M range. The neuronal network assays revealed high repeatability for each compound, but surprisingly diverse effects among these closely related compounds. Six of the seven compounds produced changes in network activity at concentrations of 10-350 microM. Three of the compounds were excitatory, two were biphasic (excitatory at lower concentrations, inhibitory at higher), and one was solely inhibitory. Two of the inhibitory compounds produced irreversible inhibition of activity. Responses of cortical cultures to eserine were compared to the effects produced by the test compounds, with only one of seven providing a close match to the eserine profile. Matching of response patterns allows the classification of new drugs according to their response similarity to well-characterized agents. Spontaneously active neuronal networks reflect the interactions of multiple neurotransmitter and receptor systems, and can reveal unexpected side effects due to secondary binding. Utilizing such networks holds the promise of greater research efficiency through a more rapid recognition of physiological tissue responses.

Animals↗

PubMed2001

Expression of glyoxalase I and II in normal and breast cancer tissues.

The present work aimed to study the activities of glyoxalase system enzymes, glyoxalase I (G I) and glyoxalase II (G II), as well as the expression of their genes in human breast carcinoma. Samples of tumoral tissue and normal counterparts were drawn from several patients during surgery. They served either for preparing extracts to be used in enzyme activity evaluations or for RNA extraction and subsequent northern blot analysis. A far higher activity level of G I and G II occurs in the tumor compared with pair-matched normal tissue, as shown by both spectrophotometrical assay and electrophoretic pattern. Such increased activities of G I and G II likely result from an enhanced enzyme synthesis as a consequence of increased expression of the respective genes in the tumoral tissue, as evidenced by northern blot. The present findings confirm a key-role of glyoxalase system to detoxify cytotoxic methylglyoxal and modulate S-D-lactoylglutathione levels in tumor cells. Moreover, they suggest a possible employment of GI inhibitors as anti-cancer drugs.

Adult↗

PubMed1999

Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate.

Acetylcholinesterase cDNA was cloned by screening a library from Loligo opalescens optic lobes; cDNA sequence analysis revealed an open reading frame coding for a protein of 610 amino acids that showed 20-41% amino acid identity with the acetylcholinesterases studied so far. The characteristic structure of cholinesterase (the choline binding site, the catalytic triad, and six cysteines that form three intrachain disulfide bonds) was conserved in the protein. The heterologous expression of acetylcholinesterase in COS cells gave a recovery of acetylcholinesterase activity 20-fold higher than in controls. The enzyme, partially purified by affinity chromatography, showed molecular and kinetic features indistinguishable from those of acetylcholinesterase expressed in vivo, which displays a high catalytic efficiency. Both enzymes are true acetylcholinesterase corresponding to phosphatidylinositol-anchored G2a dimers of class I, with a marked substrate specificity for acetylthiocholine. The deduced amino acid sequence may explain some particular kinetic characteristics of Loligo acetylcholinesterase, because the presence of a polar amino acid residue (S313) instead of a nonpolar one [F(288) in Torpedo] in the acyl pocket of the active site could justify the high substrate specificity of the enzyme, the absence of hydrolysis with butyrylthiocholine, and the poor inhibition by the organophosphate diisopropyl fluorophosphate.

Acetylcholinesterase↗

PubMed1998

Acetylcholinesterase at high catalytic efficiency and substrate specificity in the optic lobe of Eledone moschata (Cephalopoda: Octopoda): biochemical characterization and histochemical localization.

In the optic lobe of the cephalopod mollusc Eledone moschata, two acetylcholinesterase forms I and II were detected, both showing a marked active site specificity with differently sized substrates. Catalytic efficiency (kcat/Km) of the prevailing form II is similar to that of acetylcholinesterases from vertebrate nervous system. Enzyme forms I and II were co-purified from a high-salt-Triton X-100 soluble extract of optic lobe by consecutive affinity chromatographies on procainamide- and concanavalin A-Sepharose columns and then separately obtained by preparative density gradient centrifugation. According to gel-filtration chromatography, sedimentation analysis and SDS-PAGE, the major form II is an amphiphilic globular dimer (135-136 kDa, 6.3-7.4 S) of monomers (66 kDa) S-S linked between terminal segments. Phosphatidylinositol anchors give cell membrane insertion, self-aggregation and detergent (Triton X-100, Brij 97) interaction. Form I, characterized only in part owing to its small amount, showed molecular size (129 kDa) and sedimentation coefficient (7.5 S) similar to those of form II; it is likely to be attached to the cell membrane by electrostatic interactions. Both forms behaved similarly with various inhibitors and underwent excess-substrate inhibition. The results obtained suggest a common origin of both form I and II from a single gene. The former could be a degradation product of the prevailing one (II), which is likely to be functional in cholinergic synapses.

Acetylcholinesterase↗

PubMed1997

Synthesis and study of thiocarbonate derivatives of choline as potential inhibitors of acetylcholinesterase.

Fourteen alkyl and aryl thiocarbonate derivatives of choline were synthesized and studied as potential inhibitors of acetylcholinesterase (AChE). Twelve of the compounds inhibited AChEs derived from calf forebrain, human red blood cells, and octopus brain ranging from low to moderately high inhibition potency. The concentration of each inhibitory compound giving 50% inhibition of enzyme activity (IC50 values, which ranged from 1 x 10(-2) to 8 x 10(-7) M) was determined and is reported; inhibitor constants (Ki values) for the most inhibitory compounds, (1-pentylthiocarbonyl)choline chloride and (1-heptylthiocarbonyl)choline chloride, were calculated from kinetic data and are also reported. The inhibitors are competitive with substrate, and they are not hydrolyzed by the AChE activities. Certain of these new compounds may provide direction for the development of new drugs that have anticholinesterase activity and may be used for the treatment of Alzheimer's disease.

Acetylcholinesterase↗

PubMed1997

Acetylcholinesterase in Spirographis spallanzanii (Polychaeta: Sedentaria): presence of two dimeric membrane-bound forms.

In the annelid polychaete Spirographis spallanzanii two acetylcholinesterases, named DS and HSDS, were detected. They differ in relative amount, membrane anchoring and pharmacological properties. Studies with inhibitors evidenced complete inhibition of both acetylcholinesterases by 10(-3) M eserine and different sensitivities for edrophonium or procainamide. Both enzymes, sensitive to BW284c51, were unaffected by iso-OMPA; at variance, only the HSDS form underwent excess-substrate inhibition. DS and HSDS enzymes were solubilized by homogenization in a low-salt or high-salt-Triton X-100 buffer and then purified by affinity chromatography on edrophonium- or procainamide-Sepharose column respectively. According to gel-filtration chromatography, sedimentation analysis and SDS-PAGE, the least represented (30%) DS form is a G2 amphiphilic globular dimer (124-130 kDa, 6.0-7.0S) with S-S linked monomers (66 kDa). Phosphatidylinositol anchors give cell membrane insertion, self-aggregation and detergent (Triton X-100, Brij 97) interaction. The prevailing (70%) HSDS acetylcholinesterase is once again a G2 form similar to DS enzyme in its molecular size (117-125 kDa), sedimentation coefficient (6.0S) of the native form and presence of S-S linked subunits (66 kDa). However, it is likely attached to the cell membrane by involvement of strong electrostatic interactions. DS acetylcholinesterase displays moderate active site specificity with differently sized substrates. The HSDS form is inactive on butyrylthiocholine. DS and HSDS forms show a comparable catalytic efficiency (kcat/K(m)) approaching that of other invertebrate enzymes. The results suggest that DS and HSDS enzymes, likely encoded by distinct genes, are both functional in cholinergic synapses.

Acetylcholinesterase↗

PubMed1997

Expression of a single dimeric membrane-bound acetylcholinesterase in Parascaris equorum.

A single form of cholinesterase was detected in the parasitic nematode Parascaris equorum and purified from a low-salt Triton X-100 extract of whole animals by affinity chromatography on an edrophonium-Sepharose matrix. Based on gel-filtration chromatography, sedimentation analysis and SDS-PAGE, such a cholinesterase is an amphiphilic globular (G2) dimer (125-129 kDa, 6.1 S). It includes some hydrophobic domain other than phosphatidylinositol, which gives autoaggregation, detergent interaction and also anchors the molecule to the cell membrane. The enzyme, probably functional in cholinergic neurotransmission, is an acetylcholinesterase showing a fairly low substrate specificity with thiocholine esters. Electrostatic interactions seem to play a major role in the catalytic activity. Studies with inhibitors gave complete inhibition with 1 mM eserine, low sensitivity for procainamide and for tetra(monoisopropyl)pyrophosphortetramide as well as higher inhibition with edrophonium chloride and 1,5-bis(4allyldimethylammoniumphenyl)-pentan-3-one dibromide. The enzyme also showed excess-substrate inhibition with acetylthiocholine. No cross-hybridization occurred between the gene(s) encoding acetylcholinesterase in P. equorum and ace-1 from the free-living nematode Caenorhabditis elegans. The expression of a single cholinesterase form in P. equorum, unusual in free-living nematodes, could be due to parasitic life adaptation with resulting reduction of locomotor activity.

Acetylcholinesterase↗

PubMed1996

Acetylcholinesterase in Dendrobaena veneta (Oligochaeta: Opisthopora) is present with forms sensitive and insensitive to phosphatidylinositol phospholipase C. Biochemical characterization and histochemical localization in the nervous system.

Three distinct acetylcholinesterases were detected in the annelid oligochaete Dendrobaena veneta. Two enzymes (alpha, beta), copurified from a Triton-X-100-soluble extract of whole animals by affinity (edrophonium-Sepharose) chromatography, were separately eluted from a Sephadex G-200 column. Gel-filtration chromatography, sedimentation analysis and SDS/PAGE showed the alpha and beta forms to be a globular dimer (110 kDa, 7.0 S) and a hydrophilic monomer (58 kDa, 5.0 S) respectively, both weakly linked to the cell membrane. The third form (gamma), also purified to homogeneity by slower filtration through an edrophonium-Sepharose matrix, proved to be an amphiphilic globular dimer (133 kDa, 7.0 S) with a phosphatidylinositol anchor giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. The alpha acetylcholinesterase showed a fairly low substrate specificity: the beta form hydrolyzed propionylthiocholine at the highest rate and was inactive on butyrylthiocholine; the gamma acetylcholinesterase, showing a marked active-site specificity with differently sized substrates, was likely functional in cholinergic synapses. Studies with inhibitors showed incomplete inhibition of all three acetylcholinesterase by 1 mM eserine and different sensitivity for edrophonium or procainamide. The alpha and beta forms, sensitive to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, were unaffected by tetra(monoisopropyl)-pyrophosphortetramide, while both these agents inhibited the gamma enzyme. All three forms showed excess-substrate inhibition by acetylthiocholine. Enzyme activity was histochemically localized in the nerve ring and its minor branches. Monomeric acetylcholinesterase (beta) is likely the only form present in the ganglionic glial framework.

Acetylcholinesterase↗

PubMed1995

Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis).

Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M.

Acetylcholinesterase↗

PubMed1995

Characterization of a null mutation in ace-1, the gene encoding class A acetylcholinesterase in the nematode Caenorhabditis elegans.

Two genes (ace-1 and ace-2) encode two major classes (A and B) of acetylcholinesterase (AChE) in the nematode Caenorhabditis elegans. A null mutation in ace-1 (allele p1000) suppresses all acetylcholinesterase activity of class A. We have identified an opal mutation TGG (W99)-->TGA (Stop) as the only alteration in the mutated gene. This leads to a truncated protein (98 instead of 620 amino acids) with no enzymatic activity. The mutation also reduces the level of ace-1 transcripts to only 10% of that in wild-type animals. This most likely results from a destabilization of mRNA containing the nonsense message. In contrast, compensation of class B by class A AChE in the null mutant strain ace-2 takes place with unchanged ace-1 mRNA level and enzymatic activity similar to class A AChE.

Acetylcholinesterase↗

PubMed1995

Acetylcholinesterase in tentacles of Octopus vulgaris (Cephalopoda). Histochemical localization and characterization of a specific high salt-soluble and heparin-soluble fraction of globular forms.

Transverse sections of Octopus tentacles were stained for acetylcholinesterase (AChE) activity. An intense staining, that was suppressed by preincubation in 10(-5) M eserine, was detected in a number of neuronal cells, nerve fibres and neuromuscular junctions of intrinsic muscles of the arm. Octopus acetylcholinesterase was found as two molecular forms: an amphiphilic dimeric form (G2) sensitive to phosphatidylinositol phospholipase C and a hydrophilic tetrameric (G4) form. Sequential solubilization revealed that a significant portion of both G2 and G4 forms was recovered only in a high salt-soluble fraction (1 M NaCl, no detergent), Heparin (2 mg/ml) was able to solubilize G2 and G4 forms with the same efficiency than 1 M NaCl. The solubilizing effect of heparin was concentration-dependent and was reduced by protamine (2 mg/ml). This suggests that heparin operates through the dissociation of ionic interactions existing in situ between globular forms of AChE and cellular or extracellular polyanionic components. Interaction of AChE molecular forms with heparin has been reported so far in only a few instances and its physiological meaning is uncertain. G2 and G4 forms, interacting or not with heparin, all belong to a single pharmacological class of AChE. This suggests the existence of a single AChE gene. Amphiphilic and hydrophilic subunits thus likely result either from the processing of a single AChE transcript by alternative splicing (as in vertebrate AChE) or from a post-translation modification of a single catalytic peptide.

Acetylcholinesterase↗

PubMed1994

Acetylcholinesterase and butyrylcholinesterase expression in adult rabbit tissues and during development.

A large cDNA fragment covering the complete sequence of the mature catalytic subunit of rabbit acetylcholinesterase (AChE) has been cloned and sequenced. This sequence was compared to that of rabbit butyrylcholinesterase [BChE; Jbilo, O. & Chatonnet, A. (1990) Nucleic Acids Res. 18, 3990]. Amino acid sequences of AChE and BChE have 51% identity. They both possessed a choline-binding site W84, a catalytic triad S200-H440-E327 and six cysteine residues (positions 67-94, 254-265, 402-521) in conserved sequence positions to those that form three intrachain disulfide bonds in all cholinesterases (by convention, numbering of amino acids is that used for Torpedo AChE). Rabbit AChE had a larger number of aromatic residues lining the active-site gorge than rabbit BChE (14 compared to 8, respectively) and a smaller number of potential N-glycosylation sites (3 compared to 8, respectively). Both catalytic subunits have a hydrophilic C-terminus (catalytic subunits of type T). Expression of acetylcholinesterase and butyrylcholinesterase genes (ACHE and BCHE) was studied in rabbit tissues and during development by a correlation of Northern-blot analysis and enzymic activities. This correlation was rendered difficult by the presence of an eserine-resistant esterase active on butyrylthiocholine in serum, liver and lung. When the contribution of this carboxylesterase was taken into account, brain was found as the richest source of BChE followed by lung and heart. Rabbit liver had a very low content of BChE that correlated with the low BChE activity in plasma. During development, BCHE transcripts were detected as early as day 10 post coitum, whereas ACHE transcripts appeared only on day 12.

Acetylcholinesterase↗

PubMed1993

Dimeric forms of cholinesterase in Sipunculus nudus.

In developing a research on the cholinesterase (ChE) evolution in Invertebrata, this enzyme was studied in the unsegmented marine worm Sipunculus nudus. ChE activity was solubilized through three successive steps of extraction. These fractions are noted as low-salt (LSS), detergent (DS) and high-salt soluble (HSS) and represent 27%, 68% and 5% of total activity, respectively. LSS and DS ChE were purified to homogeneity by affinity chromatography on edrophonium-Sepharose gel. Purification factors of 1700 (LSS) and 1090 (DS) were obtained. The small amount of HSS ChE prevented a similar purification and an extensive characterization. Based on SDS/PAGE and density-gradient centrifugation, both LSS and DS enzymes show a M(r) value of about 130,000 and are likely G2 globular dimers of a 67,000 subunit. Moreover, LSS ChE seems to be an amphiphilic form including a hydrophobic domain, while DS ChE is probably linked to the cell membrane by a phosphatidylinositol anchor. Both LSS and DS enzymes hydrolyze at the highest rate propionylthiocholine. However, they also show a fairly high catalytic efficiency with other thiocholine esters as substrates, thus suggesting a wide and little-specialized conformation of the active site. Based on immunological cross-reactivity trials, LSS and DS ChE from S. nudus show a reduced structural affinity with a molluscan (Murex brandaris) enzyme. HSS ChE, an acetylcholinesterase, is also solubilized by heparin, like typical vertebrate HSS asymmetric enzymes. However, it lacks fast-sedimenting forms and an enzyme-anchoring collagenous structure.

Animals↗

PubMed1990

Glyoxalase I and glyoxalase II from Aloe vera: purification, characterization and comparison with animal glyoxalases.

Glyoxalase I and glyoxalase II from the outer green rind of Aloe vera leaves were purified by (matrix) affinity ligand-enzyme binding methods. The purified enzymes exhibited single protein bands on SDS-PAGE electrophoresis, with MW values of approximately 44,000 and 27,000 for glyoxalase I and glyoxalase II, respectively. The glyoxalase I is a basic protein (pI 7.8), while the glyoxalase II (3 protein bands) is acidic (pI 4.7, 4.8 [prevalent form], and 5.0). The kinetic constants, Km and Vmax, and Ki values for certain inhibitors are reported for both glyoxalase I and glyoxalase II. The glyoxalase enzymes from Aloe vera were compared with reported animal and plant glyoxalases.

Aloe↗

PubMed1990

Presence of glyoxalase II in mitochondria from spinach leaves: comparison with the enzyme from cytosol.

Glyoxalase II has been purified from cytosol and mitochondria of spinach leaves. Electrophoresis and isoelectric focussing have resolved cytosolic and mitochondrial glyoxalase II in multiple forms: pl 5.3, 5.8 and 6.2 (cytosol) and pl 4.8 (mitochondria). The enzyme of both localizations is a monomer showing a relative molecular mass of about 26 kDa. The values of kinetic constants using several glutathione thiolesters as substrates, are similar for the enzymes from cytosol and mitochondria. These results extend also to plant the presence in mitochondria of peculiar forms of glyoxalase II, likewise recently demonstrated in mammalians.

Cytosol↗

PubMed1990

Presence of a plant-like glyoxalase II in Candida albicans.

Glyoxalase II from Candida albicans was purified by affinity chromatography on S-carbobenzoxyglutathione-Affi Gel 10. The enzyme was characterized and compared with the glyoxalases II from animal and plant sources. The relative molecular mass is 29 kDa, and the isoelectric point (pI) is 6.0. The acidic pI value appears to be typical for plant glyoxalase II, in contrast to the uniformly basic glyoxalase II pI values from animals. S-D-Lactoylglutathione and S-acetoacetylglutathione are the best substrates, and S-carbobenzoxyglutathione is the best inhibitor of the yeast enzyme. Glutathione derivatives with a thioether bond are not inhibitory. Glyoxalase II from Candida albicans is compared either with animal and plant enzymes.

Animals↗

PubMed1990

Isolation of glyoxalase II from bovine liver mitochondria.

Bovine liver mitochondria contain about 10% of the total glyoxalase II activity in the homogenate. Electrophoresis and isoelectric focussing of either crude mitochondrial extract or the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms (pl 6.3, 6.7, 7.1, 7.7, and 7.9). Since bovine liver cytosol contains a single form of glyoxalase II (pl 7.5), at least four forms are exclusively mitochondrial with no counterpart in the cytosol. The relative molecular mass of mitochondrial glyoxalase II is about 23-24 kDa, similar to the cytosolic form. The kinetic constants obtained using S-D-lactoyl, S-acetyl-, S-acetoacetyl-, and S-succinyl-glutathione as substrates are similar to those reported for glyoxalase II from rat liver mitochondria. S-D-Lactoyl- and S-acetoacetyl-glutathione are the best substrates. S-Acetylglutathione is the poorest substrate with respect to both Vmax and Km values.

Animals↗