Burkholderia thailandensis as a microbial cell factory for the bioconversion of used cooking oil to polyhydroxyalkanoates and rhamnolipids.

The present work assessed the feasibility of used cooking oil as a low cost carbon source for rhamnolipid biosurfactant production employing the strain Burkholderia thailandensis. According to the results, B. thailandensis was able to produce rhamnolipids up to 2.2 g/L, with the dominant congener being the di-rhamnolipid Rha-Rha-C14-C14. Rhamnolipids had the ability to reduce the surface tension to 37.7 mN/m and the interfacial tension against benzene and oleic acid to 4.2 and 1.5 mN/m, while emulsification index against kerosene reached up to 64%. The ability of B. thailandensis to accumulate intracellular biopolymers, in the form of polyhydroxyalkanoates (PHA), was also monitored. Polyhydroxybutyrate (PHB) was accumulated simultaneously and consisted of up to 60% of the cell dry weight. PHB was further characterized in terms of its molecular weight and thermal properties. This is the first study reporting the simultaneous production of polyhydroxyalkanoates and rhamnolipids by the non-pathogen rhamnolipid producer B. thailandensis.

Analysis of PEG 400 and 4000 in urine for gut permeability assessment using solid phase extraction and gel permeation chromatography with refractometric detection.

We developed a treatment of urine samples allowing the analysis of two intestinal permeability markers: polyethylene glycol (PEG) 400 (highly diffusible; basal permeability indicator) and PEG 4000 (poorly diffusible; indicator of an abnormal increase of permeability) by a unique gel permeation chromatography (GPC) with refractometric detection. Urinary PEG were extracted using a mixed-bed resin composed of C2 and C18 layers. Permeability mean values determined in 11 human healthy subjects were 24.20 +/- 9.30% and 0.12 +/- 0.08% for, respectively, PEG 400 and 4000. The percentage of the PEG 4000 permeability value to the one of PEG 400 corresponded to an intestinal permeability index (IPI) of 0.52 +/- 0.35 expressing a low diffusion of this poorly permeability marker.

Synthesis and inverse emulsion polymerization of aminated acrylamidodextran.

A chemically modified form of dextran was prepared, having a polymerizable moiety (acrylamide) and a reactive functional group (primary amine). Dextran was activated with 4-nitrophenyl-chloroformate (24 mol per polysaccharide, 9.8 mol per 100 glucose residues); 9.8% glucose residues were converted to aliphatic carbonates and 5.2% were converted to cyclic carbonates. The activated dextran was coupled with trityldiaminoethane (8 mol per 100 glucose residues), reactivated with 4-nitrophenylchloroformate, then coupled with acryloamidodiaminohexane (6.8 mol per 100 glucose residues). The trityl group was removed by hydrolysis with trifluoroacetic acid to yield the required aminated acryloamidodextran. The modified dextran was shown to be polymerizable by inverse emulsion polymerization. Submicron particles were successfully prepared, containing functional amine groups suitable for preparing drug conjugates or for modifying the surface properties of this biomaterial.

[Statement on polyamines]. / Le point sur les polyamines.

Polyamines are ubiquitous substances. Their intracellular concentration is controlled quickly and rigorously by extremely sophisticated systems. It depends on metabolism and cellular permeability. Polyamines act as structural and functional elements in the cell (nucleic acid conformation, cytoskeleton, radioprotection, apoptosis, proliferation and differentiation of cells...). They also play a role in various diseases (origin of food allergy, cancers...). They present a great therapeutic interest (oncology, molecular transfer to cell nucleus, transfer across the blood-brain barrier, parasitosis, effects on NMDA and GABA receptors in the central nervous system...).

[The size of nanoparticles after incorporation into gastroresistant spheroids]. / Etude de la taille des nanoparticules après incorporation dans des sphéroïdes gastrorésistants.

Nanoparticles can be used as an attractive carrier for the controlled delivery of drugs by the oral route. This nanoparticular delivery system can protect an active ingredient from enzymatic degradations and enhance its absorption from the intestinal mucosa. We studied the development of a gastroresistant dosage form for the oral administration of nanoparticles. Fast disintegrating pellets were used for the administration of nanoparticles and for their transit to the site of absorption. A gastroresistant film was applied onto the pellets to protect the nanoparticles and/ or the active substance from the acid gastric environment. The integrity of the nanoparticle size was assessed after two critical manufacturing steps: the extrusion/spheronisation process and the coating process. The determination of the nanoparticles size distribution was performed by photon correlation spectroscopy (PCS). The comparison of the PCS results between the original nanoparticles and the nanoparticles incorporated in the pellets shows a very low aggregation. Moreover the coating process was not a critical step since it was not responsible for an additional agglomeration of the nanoparticles. In conclusion, our manufacturing process allows the redispersion of the nanoparticles with a little change of the size distribution. In a next study we well try to quantify the release of the nanoparticles from the pellets.

Solubilization of sodium channel from human brain.

[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0 degree C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P-40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5.

Thiamine triphosphatase in the membranes of the main electric organ of Electrophorus electricus: substrate-enzyme interactions.

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

Synthesis of [gamma-32P]thiamine triphosphate.

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and thiamine diphosphate in the presence of ethyl chloroformate. After purification by two ion-exchange purification steps, the thiamine derivative has a specific radioactivity of 10 Ci/mmol. The average final yield synthesis is about 10%.

[Use of biodegradable microspheres in experimental anticancer therapy. Chronopharmacological application]. / Utilisation des microsphères biodégradables en thérapie anti-cancéreuse expérimentale. Application chronopharmacologique.

We describe the initial experimentations which show that is possible to chronomodulate the cisplatin liberation out of some microspheres. The goal is to generate, inside one tumeur embolished by those cisplatin loaded microspheres, some concentration peaks at the best tolerance time. The cancer is than more hit, by the high local anticancer drug concentration, and doses chronomodulations preserve the patient by following his tolerance. The experimentation on cancerous mice show that this technique could lead to great survival increases. Such a protocol might usefuly improve the anticancer therapy.

Binding of [3H]ethylenediamine di-tetrodotoxin to its solubilized receptor from excitable tissues. Binding measurements by rapid gel-filtration and receptor stabilization by phosphatidylcholine.

The molecular study of bioelectrogenesis requires the purification of the membrane proteins involved in the Na-channel electrical activity. This complex biological structure contains various binding sites for different classes of neurotoxins. Labelled forms of the blocking agent, tetrodotoxin, are used to identified and quantified the solubilized membrane proteins during the purification. Such a specific probe was synthetized in our laboratory and this work reports the experimental set-up of the binding technique. A fast-gel-filtration method has been optimized with respect to column design, centrifugation time and speed and, delay between sample application and column centrifugation.

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

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

Diamine oxidase and disaccharidase activities in small intestinal biopsies of children.

A relationship between disaccharidase and diamine oxidase (DAO) activities was looked for by measuring these enzyme activities in histologically normal small intestinal biopsies of 18 children. The range for disaccharidase activities expressed in U g-1 wet weight was 0.1-5.7, 7.1-36.7, and 2.3-8.5 for lactase, maltase, and sucrase, respectively. The range for DAO activities expressed in nmol h-1 g-1 wet weight was 202-974. Significant correlations were found between disaccharidase and DAO activities (lactase versus DAO: n = 17, r = 0.80, P less than 0.001; maltase versus DAO: n = 18, r = 0.70, P less than 0.001; sucrase versus DAO: n = 18, r = 0.55, P less than 0.05). Our results further support the hypothesis that DAO is a marker of small intestinal functional integrity in children.

Preparation of poly (D,L) lactide microspheres by emulsion-solvent evaporation, and their clinical applications as a convenient embolic material.

The aim of preoperative embolization is to facilitate surgical removal by reducing tumor volume and vascularity, thereby decreasing blood loss during surgery. In a search for a better embolic material, compared to heterogeneous commercial products, we describe here in detail the preparation of poly (D,L) lactide microspheres by an emulsion-solvent evaporation process. The size distribution of the microparticles and their aggregation state--critical parameters in view of such application--have been investigated. Their effectiveness, as an embolic material, has been evaluated by some preliminary experiments undertaken on humans. The results were assessed on clinical and histological grounds.

Diamine oxidase in serum and small intestinal biopsy tissue in childhood celiac disease.

Diamine oxidase (DAO) activities were measured in small intestinal biopsies and in serum from control children and children with florid celiac disease. Mucosal DAO activities were found to be significantly higher in control children when compared with children with celiac disease (mean +/- SEM: 486 +/- 39 versus 121 +/- 22.1 nmol h-1 g-1 wet weight, p less than 0.001). Similarly, serum DAO activities were significantly higher in control children when compared with children with celiac disease (mean +/- SEM: 39 +/- 12 versus 13.6 +/- 2 pmol h-1 ml-1 serum, p less than 0.002). The lower serum activities associated with low mucosal values support the hypothesis that serum DAO activity reflects small bowel integrity.

Lactase insufficiency revisited.

The definition of "insufficient" small bowel lactase activity varies greatly among authors. The present study is aimed at redefining lactase insufficiency by comparing intestinal lactase activity and results of the lactose breath hydrogen test. Primary "insufficient" lactase activity was considered to be present when a child with a normal small bowel histology showed lactose malabsorption as measured by the lactose breath hydrogen test. The lactase activity of 22 "normal" children ranged from 0.77 to 4.57 U/g wet weight, while five children showed primary lactase insufficiency as defined above. Small bowel lactase activity in the latter patients was less than 0.74 U/g wet weight. Sucrase and maltase activities were similar in both groups of patients. We conclude that children with a normal small bowel histology should be considered to have primary lactase insufficiency when small bowel lactase activity is below 0.75 U/g wet weight.

High-yield synthesis of a [3H]ethylenediamine ditetrodotoxin derivative.

[3H]Tetrodotoxin [( 3H]TTX) and a [3H]ethylenediamine derivative of TTX are the most widely used ligands for the study of the Na channel. The former ligand presents a low specific radioactivity (1 Ci/mmol) while the latter is highly labeled (30 Ci/mmol). However, its two-step synthesis, i.e., mild oxidation followed by coupling of [3H]ethylenediamine, has been described with a low overall yield of 1.7%. In this work, more favorable experimental conditions are defined for the limiting reaction, i.e., the oxidation step, using [14C]testosterone as a model molecule. Applied to the oxidation of tetrodotoxin, this procedure produces yield values of 30-50%, as determined by high-performance liquid chromatography. Moreover, two oxidized TTX molecules appear to be covalently linked to [3H]ethylenediamine, yielding a new labeled tetrodotoxin derivative with a specific radioactivity of 45 Ci/mmol and a dissociation constant of 0.6 nM for electroplax membranes.

Are milk polyamines preventive agents against food allergy?

Insufficient polyamine intake could play a role in the induction of sensitization to dietary allergens. This proposal is based essentially on investigations made in sucking rats and in children. In sucking rats it has been established that oral administration of spermine can induce all the modifications occurring in the digestive tract at weaning. In the intestine events occur in two phases. The early event consists of desquamation of the epithelium resulting from an activation of apoptosis. The late event appears to involve an hormonal cascade in which adrenocorticotropic hormone, cytokines, bombesin and corticosterone are included. Observations in human subjects show that: (1) the spermine and spermidine concentrations are generally lower in infant formulas than in human breast milk. Mothers seem consistently to have relatively high or relatively low concentrations of spermine and spermidine in their milk. These individual variations may be due to diet, lifestyle or genetic background; (2) the probability of developing allergy can reach 80 % if the mean spermine concentration in the milk is lower than 2 nmol/ml milk. It is approximately 0 % if the mean spermine concentration is higher than 13 nmol/ml milk; (3) preliminary results show that the intestinal permeability to macromolecules differs in premature babies when they are fed on breast milk compared with infant formulas (J Senterre, J Rigo, G Forget, G Dandrifosse and N Romain, unpublished results). This difference does not seem to be present when powdered milk is supplemented with polyamines at the concentration found in breast milk; (4) spermine increases proliferation and differentiation of lymphocytes isolated from the tonsils of children.

Polylactide macroporous biodegradable implants for cell transplantation. II. Preparation of polylactide foams by liquid-liquid phase separation.

Potential of thermally induced phase separation as a porogen technique has been studied in an effort to produce a surgical implant suitable for cell transplantation. Emphasis has been placed on the liquid-liquid phase separation of solutions of amorphous poly DL-lactide and semicrystalline poly L-lactide in an 87/13 dioxane/water mixture. The related temperature/composition phase diagrams have been set up by turbidimetry, and the possible occurrence of a gel has been discussed. Freeze-drying of some phase-separated polylactide solutions can produce flexible and tough foams with an isotropic morphology. Interconnected pores of 1-10 microns in diameter are expected to result from the spinodal decomposition of the polylactide solutions with formation of co-continuous phases. Thermodynamics of the polymer/solvent pair has a decisive effect on the final macroporous foams, as shown by the dependence of their porosity, density, porous morphology, and mechanical behavior on molecular weight and crystallinity of polylactide and concentration of the original solutions. On the basis of the foam characteristics, potential of the liquid-liquid phase separation (spinodal decomposition) has been compared with the solid/liquid phase separation (solvent crystallization) as a porogen technique.

Polylactide microparticles prepared by double emulsion/evaporation technique. I. Effect of primary emulsion stability.

The process of microencapsulation of proteins by double emulsion/evaporation in a matrix of polylactide (PLA) can be divided into three successive steps: first, an aqueous solution of the active compound is emulsified into an organic solution of the hydrophobic coating polymer; second, this primary water-in-oil emulsion (w/o) is dispersed in water with formation of a double water-oil-water emulsion (w/o/w); third, the organic solvent is removed with formation of solid microparticles. This paper focuses on the effect of primary emulsion stability on the morphology and properties of polylactide microparticles loaded with bovine serum albumin (BSA) used as model drug. Depending on the stability of the primary emulsion, the internal structure of microparticles can be changed from a multivesicular to a matrix-like structure. Similarly, the average porosity can be controlled in a range from a few tenths of a micron to ca. 20 to 30 microns. This morphology control could find potential applications not only for the controlled drug delivery but also for the production of microporous particles intended for some specific applications, such as cell culture supports and chromatographic matrices. Although, the interplay of several processing parameters (polymer precipitation rate, polymer coprecipitation with interfacial compounds such as protein or surfactant, stirring rate...) may not be disregarded, this study also indicated that a high loading of a hydrophilic drug can only be expected from a stable primary emulsion. When the stability of the primary emulsion is such as to prevent formation of macropores (> 10 microns), the total pore volume is close to that of the originally dispersed aqueous drug solution.