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Biomedical subjects

P Marchot

Publications and source records attributed to P Marchot.

At least 37 records · Page 2Linked to original sources
PubMed1995

Structural bases for the specificity of cholinesterase catalysis and inhibition.

The 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.

Animals↗
PubMed1994

Assessment of coagulation factor activation during cardiopulmonary bypass with a new monoclonal antibody.

Antithrombin-III (AT) is a key inhibitor of blood coagulation that neutralizes activated serine esterases by forming covalent modified complexes (ATm). A new monoclonal antibody directed against short-lived AT-activated serine protease complexes provides a means of measuring subclinical coagulation activity during cardiopulmonary bypass (CPB). Twelve patients undergoing CPB for coronary artery bypass grafting were studied and AT, ATm, D-dimers (DD), and several other coagulation and fibrinolytic markers were measured during the surgical procedure. There were decreases in AT, factors V, II, X, IX, protein S (total and free), C4b-binding protein, thrombomodulin, and platelets counts, whereas heparin, ACT, thrombospondin, plasminogen activator inhibitor (PAI-1), and tissue plasminogen activator (tPA) increased. ATm and the percentage of ATm available (ATm/AT) showed a peak during CPB. These results demonstrate that during CPB, the use of heparin produces an equilibrium involving increased coagulation activation and consumption in association with increased fibrinolysis. The equilibrated consumption of both coagulation and fibrinolytic factors leads to low levels of all factors after cardiac surgery. The ATm assay allows assessment of the differential effects of CPB and surgical trauma on coagulation activation. It is speculated that ATm levels may be useful in monitoring the consumption of coagulation factors.

Adult↗
PubMed1993

Binding of 125I-fasciculin to rat brain acetylcholinesterase. The complex still binds diisopropyl fluorophosphate.

Iodination of fasciculin 3 (FAS3) from Dendroaspis viridis venom provided us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are concomitant, as association and inhibition rate constants k1 and ki are identical. The 125I-FAS3.AChE complex dissociates very slowly (t 1/2 = 48 h) and is characterized by a dissociation constant, Kd, of 0.4 pM. All the specific binding of 125I-FAS3 to AChE is prevented by FAS3 as from D. angusticeps venom (Kd = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bind to peripheral anionic sites of AChE, by Ca2+ and Mg2+, known to enhance AChE activity through an allosteric phenomenon and by acetylthiocholine concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorphosphate and paroxon, which inhibit AChE by phosphorylating the catalytic serine, have no effect on either the binding rate or the number of binding sites of 125I-FAS3. O-Ethyl-S2-diisopropylaminoethyl methylphosphonothionate, however, which binds irreversibly to the AChE catalytic site but reversibly to a peripheral site, induces a 130% increase in the binding rate of 125I-FAS3, without changing the total number of 125I-FAS3 binding sites. Our results demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind. Since phosphorylation of the catalytic serine (esteratic subsite) by [1,3-3H]diisopropyl fluorophosphate can still occur on the FAS3.AChE complex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.

Acetylcholinesterase↗
PubMed1992

1.9-A resolution structure of fasciculin 1, an anti-acetylcholinesterase toxin from green mamba snake venom.

The crystal structure of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba snake venom, has been solved by the multiple isomorphous replacement method complemented with anomalous scattering and subsequently refined at 1.9-A resolution. The overall structure of fasciculin is similar to those of the short alpha-neurotoxins and cardiotoxins, with a dense core rich in disulfide bridges and three long loops disposed as the central fingers of a hand. A comparison of these three prototypic toxin types shows that fasciculin 1 has structural features that are intermediate between those of the other two molecules. Its core region, which can be defined as a continuous stretch of conserved residues, is very similar to that of erabutoxin b, whereas the orientation of its long loops resembles that of cardiotoxin VII4. This result introduces a new element in the study of phylogenetic relationships of snake toxins and suggests that, after divergency from an ancestral gene, convergent evolution may have played an important factor in the evolution of these proteins. In fasciculin 1, several arginine and lysine residues are well ordered and relatively exposed to the solvent medium and may play a role in the binding to the peripheral site of acetylcholinesterases.

Amino Acid Sequence↗
PubMed1992

A fibrinogen-clotting serine proteinase from Cerastes cerastes (horned viper) venom with arginine-esterase and amidase activities. Purification, characterization and kinetic parameter determination.

An enzyme displaying proteolytic activity toward the natural substrate casein as well as clotting activity on fibrinogen was purified to homogeneity from Cerastes cerastes (horned viper) venom and characterized. The enzyme is constituted of two identical subunits of mol. wt 48,500 as determined by SDS-polyacrylamide gel electrophoresis, and has an isoelectric point of 3.75. N-terminal sequencing up to the 33rd residue evidenced a high homology with other snake venom proteinases. The proteinase is of serine-type as indicated by high sensitivity to DFP and shows both arginine-ester hydrolase and amidase activities on synthetic substrates. Both specific activities were 30-fold higher than the respective activities found in the crude venom. The Km value determined for arginine-containing substrate BAEE was 3.0 x 10(-4) M and the Km for chromogenic substrate CBS 34-47 0.65 x 10(-4) M. The Vm/Km ratio, however, was two-fold higher for BAEE than for CBS 34-47; the arginine-esterase activity of this enzyme is thus slightly higher than its amidase activity.

Amidohydrolases↗
PubMed1992

[Sclerectasia in a pig farm in Sotouboua, Togo].

Cases of sclerectasia were described in a pig farm, Sotouboua, Togo. The causative agent may be a pestivirus, on top of a predisposition revealed by consanguinity. Livestock owners should be kept aware.

Animals↗
PubMed1991

[Serological survey on the prevalence of bovine type-4 herpesvirus infection in cattle in the Accra plains of Ghana].

Sera from cattle of the Accra Plains in Ghana were screened for bovine type-4 herpesvirus (BHV-4) antibodies by an indirect immunofluorescence test. Among the 176 screened serums, 14% were found positive at a 1/100 dilution. Further studies are necessary for a better understanding of the relationships between BHV-4 or some other agents and necrotic vaginitis associated with poor fertility observed in cattle in this area.

Animals↗
PubMed1990

[Neurotoxicity in mice due to cysteine-rich parts of visna virus and HIV-1 Tat proteins].

The trans-activating visna virus and HIV-1 Tat proteins share, at their amino-acid sequence level, a significant 60% analogy on 17 consecutive residues. These homologous sequences are also found in a part of the short neurotoxin sequence from snake venom. Synthetic peptides representative of the two analogous viral sequences are, after intracerebroventricular injection at doses of 200 micrograms per 20 g mouse, responsible for the death of the injected animal in few hours. The HIV-1 recombinant Tat protein has the same effect. Such observation suggests a direct role of the Tat lentiviral protein in the origin of the neurologic effects associated with visna and HIV-1 infections.

Animals↗
PubMed1990

[A note on the first case of dry gangrene of the limbs in cattle, caused by Salmonella typhimurium in Ghana].

Ischaemia and terminal tissue dry gangrene are among the sequels of Salmonella infection. Up to now, these lesions had been described in Europe and in Chile, on calf exclusively. This time, the syndrome was observed on adult cattle reared in the Accra Plains (Ghana) and infected by S. typhimurium. This case give evidence of the spreading of salmonellosis on the African continent and should foster the search of adequate control measures.

Animals↗
PubMed1989

Bovine farcy in the Accra Plains of Ghana.

Bovine farcy is described for the first time in Ghana in herds kept in the Aveyime area of the Accra Plains. Reasons why the disease seems confined to a small geographical area are not yet clear. Different breeds of cattle are equally affected. Mineral deficiencies were not observed in the samples taken for analysis (Ca, Mg, P, Na, K, Cl). Limited examination of tick population on cattle showed a predominance of A. variegatum. Factors enhancing the pathogenicity and persistence of the Mycobacterium are still not clear. A study is soon to start.

Animals↗
PubMed1988

Localization of the toxic site of Naja mossambica cardiotoxins: small synthetic peptides express an in vivo lethality.

Cardiotoxins are small basic proteins which cause heart failure when they are injected in vivo. In order to better understand their molecular mode of action, short peptides designed on the model of the first loop of the molecule of cardiotoxin IV from Naja mossambica mossambica venom have been synthetized by the solid-phase procedure of Merrifield. These peptides express lethality in mouse when they are injected intravenously. Taking into account the respective molecular weights, they are 3.5 to 5% as toxic as the cardiotoxin. Furthermore, the symptomatology they induce is undistinguishable from that induced by cardiotoxins. These results strongly support our previous hypothesis that the first loop of the molecule is the toxic site of cardiotoxins.

Amino Acid Sequence↗
PubMed1988

Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration.

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.

Animals↗
PubMed1988

Selective loss of binding sites for the iodinated alpha-neurotoxin I from Naja mossambica mossambica venom upon enzymatic deglycosylation of Torpedo electric organ membranes.

Removal of asparagine-linked carbohydrate chains from Torpedo marmorata electric organ membranes was found to inhibit the binding of the iodinated alpha-neurotoxin I from Naja mossambica mossambica snake venom to its receptor. Optimal deglycosylation of membranes by endoglycosidase F resulted in a 55% inhibition of alpha-neurotoxin-I-saturable binding. Under these conditions, up to 70% of concanavalin A binding was also lost, indicating an efficient removal of mannose-rich carbohydrate chains. Saturation binding experiments at equilibrium on membranes incubated in the absence of endoglycosidase F indicated, when analyzed by Scatchard plots, the presence of two classes of high-affinity binding sites for alpha-neurotoxin I (kd = 9 pM and 68 pM respectively) with capacities of 24 and 14 pmol/mg membrane proteins, respectively. After endoglycosidase F treatment, only the former class of binding sites (Kd = 11 pM) was recovered together with a 45% reduction in the number of total binding sites. Dissociation experiments further confirmed the presence of two types of toxin-receptor complexes in control membranes and the selective loss of the rapidly dissociating component upon deglycosylation. The binding of alpha-neurotoxin I to its receptor, deglycosylated or not, was totally inhibited by carbamoylcholine, d-tubocurarine or alpha-bungarotoxin. These findings show that the neurotoxin binding sites present on the acetylcholine receptor can be discriminated on the basis of their differential susceptibility to the removal of asparagine-linked carbohydrate chains.

Animals↗
PubMed1987

Monitoring the purification by high-performance liquid chromatography of cardiotoxins from Naja mossambica mossambica using phase-sensitive two-dimensional nuclear magnetic resonance.

High-resolution phase-sensitive two-dimensional proton nuclear magnetic resonance was used to monitor the preparation by high-performance liquid chromatography of homogeneous proteins from the venom of Naja mossambica mossambica. This resulted in the characterization of a heterogeneous protein preparation VII2, which had been used in earlier structural studies by NMR, as well as a homogeneous protein CTXIIb and a nearly homogeneous protein fraction CTXIIa, which are now both subject to further investigations of their solution conformations.

Amino Acids↗
PubMed1987

In vivo synergy of cardiotoxin and phospholipase A2 from the elapid snake Naja mossambica mossambica.

The lethality of elapid snake venoms is due to toxic polypeptides which are alpha-neurotoxins, phospholipases A2 and cardiotoxins. In contrast to alpha-neurotoxins, less is known about the mode of action of phospholipases A2 and cardiotoxins at a cellular and molecular level, although it has been demonstrated that the hemolytic effect of cardiotoxin and its action on muscle can be accelerated in vitro by phospholipase A2. Here we show that when mice are injected i.v. with phospholipase A2, a significant decrease in survival time is observed if a sub-LD50 amount of cardiotoxin is injected simultaneously. Furthermore, the survival time is drastically reduced when phospholipase A2 (2.3 LD50) is injected first, followed 15 min later by doses of cardiotoxin as small as 0.18 LD50. These results strongly suggest a synergy between the two polypeptides.

Animals↗
PubMed1987

Use of high performance liquid chromatography to demonstrate quantitative variation in components of venom from the scorpion Androctonus australis Hector.

Using reverse-phase high performance liquid chromatography (RP-HPLC), to resolve less than 1 mg of scorpion venom, quantitative variations in protein components were demonstrated in Androctonus australis Hector venoms obtained either by electric or manual stimulation. The results support polymorphism of scorpion venom components at an individual level.

Animals↗
PubMed1987

The resistance to dermatophilosis of Dinka cattle breed, Dinka crossbred and Boran, Friesian, Jersey, Sahiwal crossbreds.

After an outbreak of dermatophilosis in Southern Sudan, the disease resistance has been studied from animals belonging to the Dinka cattle breed, 50% Dinka, 25% Dinka and imported crossbred animals obtained from Boran, Friesian, Jersey and Sahiwal. The results indicate that Dinka cattle is resistant to the disease; this resistance is 66% for 50% Dinka, 25% for 25% Dinka and only 7% for imported crossbreds.

Actinomycetales Infections↗