[Appreciation of the role of regional anaesthesia in managing facial wounds in the emergency unit]. / Appréciation de la place de l'anesthésie locorégionale pour la prise en charge des plaies de la face aux urgences.

OBJECTIVE: Defining the place of regional anaesthesia (RA) for facial wounds in an emergency department. STUDY DESIGN: Prospective observational study conducted in the emergency department of a regional hospital. PATIENTS AND METHODS: Two hundred and forty-six successive patients with one or more facial wounds were included from 1st august 2004 to 31st december 2004. Data on patient, operator, wound (measured by the number of stitches), anaesthetic method (RA, local anaesthesia [LA], or no anaesthesia), method of repairing skin, duration of intervention, operator comfort (verbal numeric scale [VNS] from 0 to 10) and pain felt by the patient (visual analogic scale [VAS] from 0 to 10) in the different stages of care were collected. RESULTS: Compared to the LA, the RA of the face decreased the number of punctures (1.36 vs 4.38 punctures, p<0.001) and the quantity of local anaesthetic injected (2.8 ml vs 5.3 ml, p<0.01) for wounds requiring more than 10 stitches. It has improved operator comfort (VNS = 10 [8-10] vs 8 [6.75-10] (p<0.01)). Its effectiveness during skin repair was equivalent to that of the LA by infiltration (VAS 0 [0-1] vs 0 [0-1]). CONCLUSION: When practicable, the RA of the face is a better technique than the LA for facial wounds treatment.

Type I and type II fatty acid biosynthesis in Eimeria tenella: enoyl reductase activity and structure.

Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

[Split-cord malformation (diastematolmyelia) presenting in two adults: case report and a review of the literature]. / Diastématomyélie révélée chez l'adulte: étude de deux cas et revue de la littérature.

INTRODUCTION: Split cord malformation (SCM) is an uncommon developmental anomaly characterized a cleft spinal cord. In type I, each of the hemicords is contained within an individual dural tube whereas in type II there is a common dural tube housing both hemicords. Commonly diagnosed in childhood, adult presentation is exceptional. METHODS: We report the case of two women whose type II SCM was discovered at the age of 40 and 54 years. RESULTS: The first patient complained of chronic lombar and radicular chronic pain with dysuria. Physical examination revealed a radicular syndrome with abolition of the left Achille reflex and a lombar hair tuft. MRI showed a disc herniation at the L5-S1 level, with a partial SCM at the level of the L2 vertebra, spina bifida and tethered cord. The second patient complained of lombar pain with perineal irradiation for 6 years. Physical examination showed a lombar cutaneous angioma. MRI revealed a thoraco-lombar SCM at the T12 to L1 level, with spina bifida. No spur could not be identified in either patient. No further surgical treatment was given. CONCLUSION: Based on these two observations, we propose a review of literature reporting 90 cases of adult SCM.

Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase.

A Plasmodium falciparum single copy gene predicting a 122 kDa protein belonging to the Ml family of zincmetallopeptidases was previously reported and related to erythrocytic schizont proteins of 96 (p96) and 68 (p68) kDa. By using protease inhibitors during parasite harvest and enzyme preparations, and polyclonal antibodies specific for 2 peptidic domains deduced from the gene, we identified the 120 kDa precursor and demonstrated its processing into p96 and p68. The N-terminal ends of p96 and p68 were mapped between glycine-123 and lysine-163, both proteins thus containing the catalytic domain. The purified enzyme, here named PfA-M1 (p96/p68), displayed strict aminopeptidase activity, optimal at pH 74, with broad substrate spectrum. Its inhibition and reactivation profiles were typical of zinc-metalloaminopeptidases. By Western blotting, PfA-M1 was detected in trophozoites, in addition to schizonts, but not in early rings. PfA-M1 was localized by indirect immunofluorescence confocal microscopy. In trophozoites, the labelling was diffuse in the parasite cytoplasm, with accumulations around the food vacuole. In schizonts, it turned progressively to a vesicle-like pattern, ending as a clear spot in released merozoites. The involvement of PfA-M1 in haemoglobin breakdown and erythrocyte reinvasion is discussed in light of the dual functions recently reported for several P. falciparum proteases.

A Plasmodium falciparum aminopeptidase gene belonging to the M1 family of zinc-metallopeptidases is expressed in erythrocytic stages.

A new single copy gene has been isolated from Plasmodium falciparum, by immunoscreening a genomic DNA expression library. The gene appears devoid of introns, displays the classical A + T richness and codon usage of P. falciparum genes, and is transcribed into a 4 kb mRNA in erythrocytic stages. The deduced amino acid sequence corresponds to a 1056 residue protein (122 kDa) containing the canonical HExxHx18E signature of zinc-metallopeptidase active sites of the M1 family at position 467-490, a downstream conserved tyrosine residue involved in catalysis in position 551, and the GAMEN conserved motif characteristic of aminopeptidases in the M1 family, at position 431-435. The greatest similarities were found with aminopeptidases N of Escherichia coli and Haemophilius influenza (more than 80% identical residues in the canonical signature of the active site) but significant similarities centred on the active site region exist with all other members of the M1 family such as other prokaryotic aminopeptidases, eukaryotic aminopeptidases A and N and leukotriene A4 hydrolases (40-50% identical residues in the canonical signature of the active site). A polyclonal serum raised to a synthetic peptide deduced from the gene labelled schizont proteins of 96 and 68 kDa purified to homogeneity and both displaying aminopeptidase activity, as well as cytoplasmic structures in schizont stages.

Properties of red blood cell concentrates stored in PAGGS-sorbitol.

The physiological, biochemical, morphological and rheological characteristics of RBC resuspended in PAGGS-Sorbitol were studied over, and beyond, 49 days. The levels of P50 and 2,3-DPG, as well as the concentration of ATP, stayed above acceptable values for longer periods than those described with other Optional Additive Solutions (OAS). Spontaneous hemolysis was kept below 1%, cell morphology, viscosity, deformability and other factors such as pH, the ratio of glucose consumption to lactate formation, the total adenylates and energetic charge indicate that good properties should be seen after 49 days' storage. In vivo survival studies using a double isotope technique showed that at least 75% of those RBC stored in this OAS for 49 days were still in circulation 24 hours after injection. Therefore RBC stored in PAGGS-S can be kept for 49 days with excellent in vivo survival, as well as maintaining DPG levels for up to 3 weeks. Furthermore, a clinical trial in routine conditions for use has been carried out with surgical and medical patients as well as with hemodialysed patients. The results obtained are those that could at least be expected of normal stored blood. As regards RBC/PAGGS-Sorbitol, it would be reasonable to propose a storage period of 49 days in the liquid state.

Transbilayer mobility and distribution of red cell phospholipids during storage.

We studied phospholipid topology and transbilayer mobility in red cells during blood storage. The distribution of phospholipids was determined by measuring the reactivity of phosphatidylethanolamine with fluorescamine and the degradation of phospholipids by phospholipase A2 and sphingomyelinase C. Phospholipid mobility was measured by determining transbilayer movements of spin-labeled phospholipids. We were unable to detect a change in the distribution of endogenous membrane phospholipids in stored red cells even after 2-mo storage. The rate of inward movement of spin-labeled phosphatidylethanolamine and phosphatidylserine was progressively reduced, whereas that for phosphatidylcholine was increased. These changes in phospholipid translocation correlated with a fall in cellular ATP. However, following restoration of ATP, neither the rate of aminophospholipid translocation nor the transbilayer movement of phosphatidylcholine were completely corrected. Taken together, our findings demonstrate that red cell storage alters the kinetics of transbilayer mobility of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine, the activity of the aminophospholipid translocase, but not the asymmetric distribution of endogenous membrane phospholipids, at least at a level detectable with phospholipases. Thus, if phosphatidylserine appearance on the outer monolayer is a signal for red cell elimination, the amount that triggers macrophage recognition is below the level of detection upon using the phospholipase technique.

[Removal of plasma lipids by selective filtration]. / Elimination des lipides plasmatiques par filtration sélective.

We herein describe a new depth filter media which exhibits selective adsorption properties for lipids of plasma on Zeta Plus Del I (Cuno Europe). Lipids plug chromatographic columns and filter during plasma fractionnation and cause solution instability for the final product. Several parameters which could affect the lipid removal efficiency on Zeta Plus Del I have been investigated: prefiltration, contact time, ionic strength, pH, and temperature. The maximum percentage of total lipids eliminated, in the better operating conditions was 68%. This method has proven to be efficient for the treatment of plasmatic lipids and can be easily incorporated in an industrial process.

Large scale production of human albumin: three years experience of an affinity chromatography process.

Immobilized Cibacron Blue is a well-known tool for the separation of a number of proteins and enzymes. Among them, human plasma albumin was easily purified from crude plasma and consequently, this approach represents an interesting way for a production scale. Our data describes about three years of experience in the production of human albumin using large columns of immobilized Cibacron Blue. The amount of albumin produced per cycle was about 250 g on a column of about 50 l. Over 500 cycles the final purity of albumin was very high and constant (98-100%). The albumin yield of the chromatographic fractionation was approximately 82%. The column performance remained acceptable when regeneration operations were applied to the sorbent. The long life of the sorbent renders this approach very attractive and economically acceptable for large scale applications.

[Isolation of human thrombin by affinity chromatography with silicate to be used in the preparation of biological glue]. / Isolement par chromatographie d'affinité, sur support de silice, de la thrombine humaine en vue de son utilisation dans les préparations de colle biologique.

At present the preparations of fibrin glue used a thrombin from equine or bovine origin. In order to remove the problems of antigenicity we developed a method of purification from acidified plasma. We used one affinity chromatography on benzamidine-Spherodex and compared three methods of elution: non specific (sodium chloride gradient) and biospecific competitors (arginin methylester or benzamidin). The yield is evaluated between 64 and 84% and the purification factor close to 160. The obtained thrombin is better than animal thrombin in the preparations of fibrin glue.

[Purification of human thrombin by affinity chromatography for its use in preparations for biological coagulation]. / Purification de la thrombine humaine par chromatographie d'affinité en vue de son utilisation dans les préparations de colle biologique.

Biological glue is obtained by mixing different specific plasma proteins including a serine protease, thrombin. Surprisingly at present the thrombin used in such a mixture is from equine or bovine origin while all other components are from human. In this paper we described a particular efficient and specific chromatographic method for the purification of human thrombin usable as a serine protease in the preparation of biological glue. A pure and active thrombin is obtained from a plasma fraction after adsorption on benzamidine-Spherodex followed by an elution with non specific (sodium chloride gradient) or biospecific competitors (arginine methylester or benzamidine). The obtained thrombin with a yield close to 80% and a purification factor close to 160, showed good properties in the replacement of animal thrombin in the condition of biological glue.

Blocking activity of mite-specific IgG antibodies studied by skin tests.

The aim of the study was to determine whether IgG antibodies from patients on specific immunotherapy could inhibit skin test reactivity of allergens when mixed with them prior to testing. In a preliminary double-blind study, mite extracts were incubated with glycero-saline solution or non-specific IgG and used in testing 15 patients. Wheal and flare diameters produced with the two mite extracts did not differ significantly. In a second double-blind study with 15 other patients, skin tests using mite extracts incubated with either non-specific IgG or specific IgG showed significantly decreased diameters when mite extracts were preincubated with specific IgG. This result, in vivo, confirms the antigen neutralizing capacity of specific IgG demonstrated in vitro.

[Optimization of the purification of human plasma fibronectin by double affinity chromatography using gelatin and heparin Trisacryl LS]. / Optimisation de la purification de la fibronectine plasmatique humaine par double chromatographie d'affinité sur gélatine et héparine Trisacryl LS.

The authors described an optimized method of preparation to obtain pure human plasma fibronectin. The new performances related to the chromatographic and concentration steps, where the saving of time is about 40% and the lyophilization step where the saving of energy is also about 40%. The final product is without any less of either physical and biological activities. The size of the columns, the volumes of the chromatographic supports (gelatin and heparin-Trisacryl LS) and the quantity of the treated plasma are very much important and also the quantities of the final product are very increased.

The RBC morphological dependence of the RBC disaggregability.

The aim of this study was to determine the red blood cell (RBC) disaggregability dependence upon the RBC shape. The study concentrated on stored blood during bank storage and on suspensions of artificially induced echinocytes. Measurements was performed in autologous plasma of hematocrit 0.45 and at constant plasmatic content. Rheological studies using stationary viscometry, nonstationary viscometry and rheoscopy were made in order to assess different stages of the disaggregability process. Whatever the method of measurement used, the morphological interpretation of the results reveal that beyond 75% of echinocytes within the sample, the disaggregation process is altered. The shear stresses required to dissociate the echinocyte aggregates are significantly higher than those required to disaggregate normal RBC rouleaux.

[Functional adjuvant for stored red blood cells: ascorbate or oxalate?]. / Adjuvant fonctionnel pour les globules rouges conservés: ascorbate ou oxalate?

In stored blood cells the p50 and the 2,3-DPG concentration drop rapidly. To compensate the functional resultant weakness, many different additives have been proposed, in particular ascorbate, which is very unstable, and ascorbate-2-phosphate (AsP), which is a little more stable. We have studied the effect of two commercially available AsP whose impurity content was different. It was found that the more the substance was impure the more it was effective. This led as to doubt the effectiveness of ascorbate itself and to try the impurity (oxalate) with which we have obtained the results published with AsP.

[Purification of human plasma fibronectin by double affinity chromatography on gelatin and heparin-Trisacryl]. / Purification de la fibronectine plasmatique humaine par double chromatographie d'affinité sur gélatine et héparine-trisacryl.

In this work the human plasma fibronectin was purified by affinity chromatography using a tandem column system. The first affinity column was filled with gelatin-Trisacryl whereas the second one contained heparin-Trisacryl. This double affinity chromatography demonstrated its high efficiency in term of purity and yield. Several analytical methods (electrophoresis, immunoelectrophoresis, F.P.L.C. and adhesion assay on cultured eucaryotic cells) evidenced in fact the high purity of the preparation as well as its biological behaviour in term of cell adhesion and spreading. The performances of the sorbents used facilitate the scaling up when large quantities of FNP are needed.