Engineering a Virus-like Particle to Display Peptide Insertions Using an Apparent Fitness Landscape.

Peptide insertions in the primary sequence of proteins expand functionality by introducing new binding sequences, chemical handles, or membrane disrupting motifs. With these properties, proteins can be engineered as scaffolds for vaccines or targeted drug delivery vehicles. Virus-like particles (VLPs) are promising platforms for these applications since they are genetically simple, mimic viral structure for cell uptake, and can deliver multiple copies of a therapeutic agent to a given cell. Peptide insertions in the coat protein of VLPs can increase VLP uptake in cells by increasing cell binding, but it is difficult to predict how an insertion affects monomer folding and higher order assembly. To this end, we have engineered the MS2 VLP using a high-throughput technique, called Systematic Mutagenesis and Assembled Particle Selection (SyMAPS). In this work, we applied SyMAPS to investigate a highly mutable loop in the MS2 coat protein to display 9,261 non-native tripeptide insertions. This library generates a discrete map of three amino acid insertions permitted at this location, validates the FG loop as a valuable position for peptide insertion, and illuminates how properties such as charge, flexibility, and hydrogen bonding can interact to preserve or disrupt capsid assembly. Taken together, the results highlight the potential to engineer VLPs in a systematic manner, paving the way to exploring the applications of peptide insertions in biomedically relevant settings.

Mathematical modeling and simulation in animal health. Part III: Using nonlinear mixed-effects to characterize and quantify variability in drug pharmacokinetics.

A common feature of human and veterinary pharmacokinetics is the importance of identifying and quantifying the key determinants of between-patient variability in drug disposition and effects. Some of these attributes are already well known to the field of human pharmacology such as bodyweight, age, or sex, while others are more specific to veterinary medicine, such as species, breed, and social behavior. Identification of these attributes has the potential to allow a better and more tailored use of therapeutic drugs both in companion and food-producing animals. Nonlinear mixed effects (NLME) have been purposely designed to characterize the sources of variability in drug disposition and response. The NLME approach can be used to explore the impact of population-associated variables on the relationship between drug administration, systemic exposure, and the levels of drug residues in tissues. The latter, while different from the method used by the US Food and Drug Administration for setting official withdrawal times (WT) can also be beneficial for estimating WT of approved animal drug products when used in an extralabel manner. Finally, NLME can also prove useful to optimize dosing schedules, or to analyze sparse data collected in situations where intensive blood collection is technically challenging, as in small animal species presenting limited blood volume such as poultry and fish.

Is-it possible to distinguish irritable bowel syndrome with constipation from functional constipation?

BACKGROUND: The Rome III criteria classify patients complaining of constipation into two main groups: patients with functional constipation (FC) and patients with constipation predominant irritable bowel syndrome (IBS-C). The purpose of this study was to identify differences in the intensity of symptoms and total and segmental colonic transit time in these two types of patients. METHODS: We performed a prospective evaluation of 337 outpatients consecutively referred for chronic constipation and classified according to the Rome III criteria as FC or IBS-C. They were asked to report symptom intensity, on a 10-point Likert scale, for diarrhea, constipation, bloating and abdominal pain. Stool form was reported using the Bristol scale, and colonic transit time was measured by using multiple-ingestion single-marker single-film technique. Statistical analysis was completed by a discriminant analysis. RESULTS: Female gender and obstructed defecation was more frequent in IBS-C patients than in FC patients. IBS-C patients reported greater symptom intensity than FC patients, but stool form, and total and segmental colonic transit time were not different between the two groups. Multivariate logistic regression showed that only two parameters, bloating and abdominal pain, were related to the IBS-C or to the FC phenotype, and discriminant analysis showed that these two parameters were sufficient to give a correct classification of 71% of the patients. CONCLUSIONS: Our study suggests that self-evaluation of abdominal pain and bloating is more helpful than colonic transit time in classifying patient as IBS-C or FC.

In vivo characterization of the biodistribution profile of amphipol A8-35.

Amphipols (APols) are polymeric surfactants that keep membrane proteins (MPs) water-soluble in the absence of detergent, while stabilizing them. They can be used to deliver MPs and other hydrophobic molecules in vivo for therapeutic purposes, e.g., vaccination or targeted delivery of drugs. The biodistribution and elimination of the best characterized APol, a polyacrylate derivative called A8-35, have been examined in mice, using two fluorescent APols, grafted with either Alexa Fluor 647 or rhodamine. Three of the most common injection routes have been used, intravenous (IV), intraperitoneal (IP), and subcutaneous (SC). The biodistribution has been studied by in vivo fluorescence imaging and by determining the concentration of fluorophore in the main organs. Free rhodamine was used as a control. Upon IV injection, A8-35 distributes rapidly throughout the organism and is found in most organs but the brain and spleen, before being slowly eliminated (10-20 days). A similar pattern is observed after IP injection, following a brief latency period during which the polymer remains confined to the peritoneal cavity. Upon SC injection, A8-35 remains essentially confined to the point of injection, from which it is only slowly released. An interesting observation is that A8-35 tends to accumulate in fat pads, suggesting that it could be used to deliver anti-obesity drugs.

Characterization and engineering of the type 3 secretion system needle monomer from Salmonella through the construction and screening of a comprehensive mutagenesis library.

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needle's assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle's mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS's assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

Characterization and Engineering of the Type 3 Secretion System Needle Monomer from Salmonella Through the Construction and Screening of a Comprehensive Mutagenesis Library.

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here we investigated this link with the PrgI protein, the monomer of the secretory channel of the Type 3 Secretion System (T3SS) of Salmonella enterica . Despite detailed knowledge of the PrgI needle's assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape (SFL) to characterize the PrgI needle's mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS's assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes. Importance: Protein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

Fibrin glue for intraperitoneal laparoscopic mesh fixation: a comparative study in a swine model.

BACKGROUND: The classic method of mesh fixation in laparoscopic ventral hernia repair is transfascial sutures with tacks. This method has been associated with low recurrence rates, but yields significant morbidity from pain and bleeding. Fibrin glue has been used successfully in inguinal hernia repair with decreased incidence of chronic pain without an increase in recurrence rates, but its utility for laparoscopic ventral hernia repair is unknown. Our aim is to evaluate the efficacy of fibrin glue for laparoscopic mesh fixation to the anterior abdominal wall compared with other fixation methods. METHODS: Four different laparoscopic mesh fixation methods were randomly assigned to midline positions along the abdominal wall of 12 female pigs and compared: (1) fibrin glue only (GO), (2) transfascial sutures with tacks (ST), (3) fibrin glue with tacks (GT), and (4) tacks only (TO). At 4 weeks post implantation, tensile strength, adhesions, migration, contraction, and buckling/folding were assessed using Kruskal-Wallis one-way analysis by ranks test. RESULTS: There were no significant differences in tensile strength, adhesions or buckling/folding among the four fixation methods. A significant increase in mean migration (3.3 vs. 0.0 mm, p = 0.03) and percentage contraction (28% vs. 14%, p = 0.02) were identified in the GO group when compared with ST (see Table 3). CONCLUSIONS: Mesh fixation using fibrin glue has comparable tensile strength and adhesion rate to sutures with tacks in the swine model. Increased contraction and migration rates associated with fibrin glue alone may be an issue and warrants further study. On the other hand, the GT group showed similar biomechanical characteristics to the other groups and may represent a reasonable alternative to the use of transfascial sutures.

Laparoscopic versus open appendectomy for the obese patient: a subset analysis from a prospective, randomized, double-blind study.

BACKGROUND: The clinical outcomes for patients randomized to either open or laparoscopic appendectomy are comparable. However, it is not known whether this is true in the subset of the adult population with higher body mass indexes (BMIs). This study aimed to compare the outcomes of open versus laparoscopic appendectomy in the obese population. METHODS: A subgroup analysis of a randomized, prospective, double-blind study was conducted at a county academic medical center. Of the 217 randomized patients, 37 had a BMI of 30 kg/m(2) or higher. Open surgery was performed for 14 and laparoscopic surgery for 23 of these patients. The primary outcome measures were the postoperative complication rates. The secondary outcomes were operative time, length of hospital stay, time to resumption of diet, narcotic requirements, and Medical Outcomes Survey Short Form 36 (SF-36) quality-of-life data. RESULTS: No differences in complications between the open and laparoscopic groups were found. Also, no significant differences were seen in any of the secondary outcomes except for a longer operative time among the obese patients. CONCLUSIONS: In this study, laparoscopic appendectomy did not show a benefit over the open approach for obese patients with appendicitis.

[A severe G6PD deficiency revealed during a chemotherapy protocol including rasburicase]. / Un déficit sévère en G6PD découvert au décours d'une chimiothérapie avec utilisation de rasburicase.

We present here the case-report of a man with a severe G6PD deficiency revealed after the use of rasburicase (uricolytic drug) during a chemotherapy protocol. The genotypic analysis done to confirm the biochemical measurement revealed the 'Mediterranean mutation' at the hemizygous state (G6PD gene is located on chromosome X). Consequently to this diagnose, a search for G6PD deficiency has been performed (at the biochemical and genotypic levels) for the 9 children (7 daughters and 2 sons) of the proband. Surprisingly, one of his son was found to be hemizygous for the mediterranean mutation and one of his daughter appeared homozygous for this same mutation. This implies that the proband's wife (not studied) is certainly heterozygous for the mediterranean mutation, as it is very unlikely that this mutation had appeared de novo for two children of this couple.

[Bilateral diaphragmatic palsy due to Lyme neuroborreliosis]. / Une paralysie diaphragmatique bilatérale révélant une neuroborréliose de Lyme.

INTRODUCTION: Lyme disease is not uncommon and can sometimes progress to neurological complications. We report here an unusual case of bilateral diaphragmatic paralysis secondary to Lyme neuroborreliosis. CASE REPORT: A 79-year-old man was admitted to the intensive care unit for acute respiratory distress requiring intubation and the long-term use of nocturnal non-invasive ventilation. Three months beforehand he had been bitten by a tick and developed erythema migrans which was treated with Doxycycline for 10 days. This clinical presentation became complicated a few days later by the progressive onset of severe dyspnoea. At admission, chest radiography revealed bilateral elevation of the diaphragm. Pulmonary function tests revealed a severe restrictive disorder aggravated by decubitus. A diaphragmatic electromyogram showed bilateral axonal polyneuropathy of the phrenic nerves. IgG and IgM antibodies to Borrelia burgdorferi were detectable in serum and cerebrospinal fluid, leading to the diagnosis of Lyme disease. He was treated with intravenous ceftriaxone 2g per day for 21 days, leading to a substantial improvement in symptoms. CONCLUSION: In the presence of unilateral or bilateral diaphragmatic paralysis of undetermined aetiology, it seems relevant to perform Lyme serology in the blood and, in positive cases, to follow up with a lumbar puncture in order to detect intrathecal IgG synthesis.

[Feto-maternal metabolism in human normal pregnancies: study of 73 cases]. / Métabolisme foeto-maternel au cours de grossesses humaines normales: étude de 73 cas.

From 73 normal pregnancies of gestational age between 17 and 41 weeks of gestation (WG), the concentrations of glucose, pyruvate and lactate, free fatty acids, ketone bodies (aceto-acetate and beta-hydroxybutyrate) and cholesterol were assessed on maternal venous blood (MVB) and umbilical venous blood (UVB), sampled by cordocentesis. The objective of this work was to study feto-maternal metabolism, as well as nutritional exchange between maternal blood and fetal blood during the second and third trimesters of pregnancy. Maternal and fetal glycemias, as well as maternal-fetal glucose concentration gradient, were found stable during the studied gestational period; maternal glucose is always higher than fetal glucose, with a mean concentration delta of 0.69+/-0.34 mmol/L. Maternal lactate level (1.26+/-0.38 mmol/L) is lower than fetal lactate level (1.48+/-0.46 mmol/L), whereas maternal blood pyruvate concentration (0.042+/-0.020 mmol/L) is higher than fetal blood pyruvate concentration (0.025+/-0.010 mmol/L). Consequently, mean lactate / pyruvate ratio is found twice lower in maternal blood (31.77+/-9.89) than in fetal blood (64.10+/-17.12). Free fatty acids concentration is approximately three times higher in maternal blood than in fetal blood (respectively 0.435+/-0.247 mmol/L and 0.125+/-0.046 mmol/L). Maternal venous aceto-acetate (0.051+/-0.042 mmol/L) and beta-hydroxybutyrate (0.232+/-0.270 mmol/L) concentrations are significantly lower than those in UVB (respectively 0.111+/-0.058 and 0.324+/-0.246 mmol/L) and the beta-hydroxybutyrate/aceto-acetate ratio is on average 1.7 times higher in MVB (4.75+/-2.5) than in UVB (2.82+/-1.18). Cholesterol concentration is significantly higher in maternal blood (6.26+/-1.40 mmol/L) than in fetal blood (1.66+/-0.34 mmol/L). Our results show the characteristics of oxidative metabolism of the fetus compared with that of the adult. Blood concentration in energy substrates, measured with glucose and free fatty acids levels, is low in UVB and suggests increased energy needs of the growing fetus. Mean high concentrations in aceto-acetate and beta-hydroxybutyrate in UVB, indicate probably fetal ketogenesis. UVB low cholesterolemia suggests high cholesterol consumption in the fetal compartment for cellular membrane synthesis and steroid biosynthesis.

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases.

BACKGROUND: Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. RESULTS: The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. CONCLUSIONS: We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.

[Amniotic fluid embolism: a review]. / Embolie de liquide amniotique: mise au point.

Amniotic fluid embolism occurs rarely but is a leading cause of maternal mortality. It is a difficult and somewhat intangible diagnosis that warrants a high index of suspicion by physicians. AFE is an unpredictable, unpreventable, and, for the most part, an untreatable obstetric emergency. Management of this condition includes prompt recognition of the signs and symptoms, aggressive resuscitation efforts, and supportive therapy. Any delays in diagnosis and treatment can result in increased maternal and/or foetal impairment or death. Whereas once the invariable outcome of AFE was death of the mother, today the prognosis is somewhat brighter thanks to increased awareness of the syndrome and advances in intensive care medicine. No laboratory test is specific to attest the diagnosis and autopsy must to be realised in case of maternal death. Although non-specific, the diagnosis of AFE could be supported by the observation of amniotic fluid in the central venous blood as well as in the bronchoalveolar fluid. This easy and quick test will be helpful in decision-making. Prompt and aggressive supportive treatment is required to lessen an otherwise dismal outcome, which may include death and permanent disability. This article provides an account of the protean clinical features, pathogenesis, and principles involved in treatment.

Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids.

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.

Analysis of cDNAs encoding the two subunits of crotoxin, a phospholipase A2 neurotoxin from rattlesnake venom: the acidic non enzymatic subunit derives from a phospholipase A2-like precursor.

We report the sequences of three cDNAs encoding the two subunits (CA and CB) of crotoxin, a neurotoxic phospholipase A2 from the venom of the South-American rattlesnake Crotalus durissus terrificus. CB is a basic and toxic phospholipase A2 and CA is an acidic, non toxic and non enzymatic three chain containing protein which enhances the lethal potency of CB. Two cDNAs encoding precursors of CB isoforms have been isolated from a cDNA library prepared from one venom gland. Both precursors are made of the same 16 residues signal peptide followed by a polypeptide of 122 amino acid residues. The two mature sequences differ from each other at eight positions and are in good agreement with the previous polypeptide sequence reported for CB. In the case of CA, the cDNA encodes a signal peptide identical to those found in CB precursors, followed by a polypeptide of 122 amino acids clearly homologous to phospholipases A2 and including three regions which correspond to the three chains of mature CA. This demonstrates that CA is generated from a phospholipase A2-like precursor, called pro-CA, by the removal of three peptides, leaving unchanged the molecule core cross-linked by disulfide bridges. The 5'-untranslated tracts of cDNAs encoding CA and CB are nearly identical and the 3'-untranslated tracts are very similar, suggesting that the mRNAs encoding the two crotoxin subunits may result from the alternative splicing of a single gene or from the existence of a recent gene conversion. Data have been analysed in light of recent results on other phospholipases A2 from different origins.

Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes.

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.

Platelet secretory phospholipase A2 fails to induce rabbit platelet activation and to release arachidonic acid in contrast with venom phospholipases A2.

The ability of platelet secretory phospholipase A2 (sPLA2) to induce platelet activation was investigated. sPLA2 (group II) contained in an activated platelet supernatant, as well as high concentrations of purified recombinant platelet sPLA2, failed to induce platelet activation. Furthermore, sPLA2 did not modify platelet activation induced by various agonists. The possible relationship between the failure of this enzyme to induce platelet activation and its origin (mammalian) or its structural group (group II) was then investigated, using pancreatic PLA2s (group I) and venom PLA2s from groups I, II and III. All venom PLA2s induced platelet activation that was accompanied by the liberation of arachidonic acid and was abolished by aspirin. In contrast, as observed for platelet sPLA2, enzymes from hog or bovine pancreas were unable to induce platelet activation even when used at high concentrations. Interestingly, PLA2 able to induce platelet activation efficiently hydrolyse phosphatidylcholine, while those inactive on platelets did not. Taken together, these results suggest that the catalytic activity of added PLA2 is necessary but not sufficient to induce platelet activation. Moreover, the ability of PLA2 to induce platelet activation is not related to its structural group (I, II, III) but rather to its origin (venom vs. mammalian) and capacity to hydrolyse phosphatidylcholine, the major phospholipid of the outer leaflet of the plasma membrane.

Thermal stability of acetylcholinesterase from Bungarus fasciatus venom as investigated by capillary electrophoresis.

Previous studies on the conformation of the monomeric acetylcholinesterase (AChE) from the krait (Bungarus fasciatus) venom showed that the protein possesses a large permanent dipole moment. These studies predicted that thermal irreversible denaturation must occur via partially unfolded states. The thermal stability of Bungarus AChE was determined using capillary electrophoresis (CE) with optimized conditions. Runs performed at convenient temperature scanning rates provided evidence for an irreversible denaturation process according to the Lumry and Eyring model. The mid-transition temperature, T(m), and the effective enthalpy change, DeltaH(m) were determined at different pH. The temperature dependence of the free energy, DeltaG, of Bungarus AChE unfolding was drawn using values of T(m), DeltaH(m) and DeltaC(p) determined by CE. The thermodynamic parameters for the thermal denaturation of the monomeric snake enzyme were compared with those of different dimeric and tetrameric ChEs. It was shown that the changes in the ratio of DeltaH(cal/)DeltaH(vH) and DeltaC(p) reflect the oligomerization state of these proteins. All these results indicate that wild-type monomeric Bungarus AChE is a stable enzyme under standard conditions. However, designed mutants of this enzyme capable of degrading organophosphates have to be engineered to enhance their thermostability.

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization.

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.

Immunochemical analysis of a snake venom phospholipase A2 neurotoxin, crotoxin, with monoclonal antibodies.

Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus. The crotoxin molecule is composed of two subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit called component-A (CA). Crotoxin exists as a mixture of several isoforms (or variants) resulting from the association of several subunit isoforms. We prepared monoclonal antibodies (MAbs) against each isolated subunit. Six anti-CA MAbs and eight anti-CB MAbs were tested for their cross-reactivities with each subunit and with other toxic and nontoxic PLA2s. Four of the six anti-CA MAbs cross-reacted with CB, whereas only one of the eight anti-CB MAbs cross-reacted with CA. Two anti-CB MAbs were found to cross-react with agkistrodotoxin, a single chain neurotoxic PLA2 purified from the venom of Agkistrodon blomhoffii brevicaudus. We determined the dissociation constants of each MAb for CA and CB isoforms and their capacities to neutralize the lethality and to inhibit the catalytic activity of crotoxin. We defined three epitopic regions on CA and four on CB, and used a schematic representation of the two subunits to characterize these epitopic regions with respect to: (1) the "toxic" and the "catalytic" sites of CB, and (2) the zone of interaction between the two subunits. We propose three-dimensional structures of the crotoxin subunits in which we localize amino acid residues that might be involved in the epitopic regions described here.