Diagnostics hardening for harsh environment in Laser Megajoule (invited).

The diagnostic designs for the Laser Megajoule (LMJ) will require components to operate in environments far more severe than those encountered in present facilities. This harsh environment will be induced by fluxes of neutrons, gamma rays, energetic ions, electromagnetic radiations, and, in some cases, debris and shrapnel, at levels several orders of magnitude higher than those experienced today on existing facilities. The lessons learned about the vulnerabilities of present diagnostic parts fielded mainly on OMEGA for many years, have been very useful guide for the design of future LMJ diagnostics. The present and future LMJ diagnostic designs including this vulnerability approach and their main mitigation techniques will be presented together with the main characteristics of the LMJ facility that provide for diagnostic protection.

Formulation, characterization, and efficacy of an adenosine-containing dissolvable film for a localized anti-wrinkle effect.

A water-dissolvable film was developed to topically deliver adenosine for a localized anti-wrinkle effect. The polymers used to produce the film were cellulose derivatives. An aqueous mixture of the film components was made, coated on a liner, and then dried to form a solid film. No preservatives were added and the film was shown to be stable over time. The film quickly dissolves in water to form a uniform layer at the surface of the skin, as shown by scanning electron microscopy. The film layer can still be visualized on the wrinkle six hours after being applied on the skin. A randomized, placebo-controlled, investigator-blind study was conducted in female volunteers to assess the efficacy of the 1% adenosine-containing dissolvable film. After three weeks and eight weeks, a twice daily application led to a significant decrease in the skin roughness parameters as observed using fast optical in vivo topometry (FOITS). These results demonstrate that water-dissolvable films may be used as novel, preservative-free, cosmetic delivery systems.

Determination of the passive absorption through the rat intestine using chromatographic indices and molar volume.

Immobilized artificial membrane (IAM) chromatography coupled to physicochemical descriptors was evaluated to model the passive intestinal absorption of drugs through rat gut sacs. The chromatographic capacity factors (logk'(IAM)) of 12 structurally diverse compounds were determined on a IAM PC DD2 column. The passive permeabilities (P(a)) of the drugs were determined through rat everted gut sacs or through non-everted sacs for actively transported molecules. Correlation studies between logk'(IAM), physicochemical descriptors and P(a) were conducted by stepwise multiple linear regression (MLR) and back-propagation neural network (BPNN). MLR and BPNN showed that logk'(IAM) was the descriptor which correlated best with P(a). Considering the molar volume as an additional descriptor, the correlation was improved. Retention indices on IAM and the molar volume can be used concurrently to predict passive drug absorption.

Effect of polyoxyl 35 castor oil and Polysorbate 80 on the intestinal absorption of digoxin in vitro.

Surfactants are classically used to improve the solubilization of lipophilic drugs such as digoxin. Polysorbate 80 and Cremophor EL (polyoxyl 35 castor oil) are such surfactants but they may also modulate the action of P-glycoprotein, an energy-dependent "counter-transport" system implicated in the phenomenon of multidrug resistance in cancer cells. P-glycoprotein is also present in the intestine on the apical membrane of mature enterocytes and can potentially reduce the absorption of a wide range of drugs. In this study, using the improved everted gut sac method, the effects of Polysorbate 80, Cremophor EL and cyclosporin on the absorption of digoxin were studied. An increase in the uptake of digoxin in the presence of these three products could be shown with our in vitro model. Cremophor EL and Polysorbate 80 had no toxic effects at the concentrations used. These results suggest that surfactants such as Cremophor EL and Polysorbate 80 should not only support solubilization but can also modulate the P-glycoprotein system to improve the bioavailability of poorly absorbed drugs.

[Hemoglobin alpha chain isoelectric point modification under the action of urea, sodium cyanate, succinic anhydride or diethylene triamine pentaacetic acid anhydride]. / Modifications du point isoélectrique de la chaine-alpha de l'hémoglobine sous l'action de l'urée, du cyanate de sodium, de l'anhydride succinique ou de l'anhydride diéthylène triamine pentaacétique.

A monomeric protein, the hemoglobin alpha chain, was used to compare four protocols for conjugation with diethylene triamine pentaacetic (DTPA) anhydride. Carbamylation and succinylation were also performed. The isoelectric point (pI) was 7.7 for the native protein versus only 5.5 to 7.3 for the five carbamylated derivatives and 4.0 to 7.0 for the six succinylated derivatives. With carbamylation or succinylation, increasing the molar ratio (agent/protein) was associated with a gradual downward pI shift producing trains of bands. This phenomenon did not occur with DTPA conjugation, whose results varied with the method used; only one derivate (pI 6.7) was produced by all four methods, and multiple fine bands with pH values in the vicinity of 3.6 were seen. For the protein, the pI shift varied with the number of groups inserted on the primary amine residues. Also, the shift was larger if the inserted groups carried electrically-charged moieties.

N-acyl-(alpha, gamma diaminobutyric acid)n hydrazide as an efficient gene transfer vector in mammalian cells in culture.

PURPOSE: This study investigates the structure/activity relationship of a series of N-acyl-peptides (lipopeptides) for the transfection of mammalian cells. METHODS: Lipopeptides comprising 1 to 3 basic amino-acids and a single fatty acid chain were synthesized. Transfecting complexes between lipopeptide, plasmid DNA and dioleoyl phosphatidylethanolamine were prepared and applied on cells in culture. Transfection efficiency was evaluated by measuring beta-galactosidase activity 48 h post-transfection. Lipopeptide-DNA binding was also investigated by physical means and molecular modelling. RESULTS: Besides the length of the fatty acid chain, the nature of the basic amino-acid and the C-terminal group were crucial parameters for high transfection efficiency. The N-acyl-(diaminobutyric acid)n derivatives were the most potent transfecting agents among those tested and induced a beta-galactosidase activity 2 to 20 times higher than the N-acyl-lysine, -ornithine or -diaminopropionic acid derivatives. Furthermore, a hydrazide C-terminal modification greatly enhanced transfection efficiency for all compounds tested. The reason why alpha, gamma-diaminobutyric acid hydrazide-based lipopeptides were the most potent in transfection is not fully understood but could be related to their high DNA binding. CONCLUSIONS: Poly- or oligo-diaminobutyric acid containing or not a hydrazide C-terminus could advantageously be used in peptide-based gene delivery systems.

Gene transfer mediated by cationic lipids: lack of a correlation between lipid mixing and transfection.

Complexes of DNA with cationic lipids are used to transfect eukaryotic cells. The mechanism of transfection is unknown, but it has been suggested that the complexes are taken up into the cell by endocytosis, after which fusion of the cationic lipids with the membranes of intracellular vesicles would allow the DNA to escape into the cytoplasm. Here, we have compared transfection of CHO-K1 cells with lipid mixing measured by fluorescence assays, using liposomes or complexes with plasmid DNA of the cationic lipids 1,2 dioleolyl-3-N, N, N,-trimethylammonium-propane (DOTAP), N-[2,3-(dioleoyloxy)propyl]-N, N, N,-trimethylammonium (DOTMA), or combinations of these lipids with dioleoylphosphatidylethanolamine (DOPE), at various lipid/DNA charge ratios. Mixing of the lipids of the complexes or liposomes with cellular membranes occurred readily at 37 degrees C, and was more efficient with liposomes than with complexes. Lipid mixing was inhibited at low temperatures (0-17 degrees C), by the presence of NH(4)Cl in the medium, and by low extracellular pH, indicating the involvement of the endocytic pathway in entry. In the absence of DOPE, there was no correlation between the efficiency of lipid mixing and the efficiency of transfection. Moreover, although DOPE, which is thought to promote membrane fusion, enhanced transfection, it did not always enhance lipid mixing. Neither the size nor the zeta potential of the complexes were clearly associated with transfection efficiency. Therefore, although fusion between the lipids of the complexes and cellular membranes takes place, a step at a later stage in the transfection process determines the efficiency of transfection.

Dioleoylmelittin as a novel serum-insensitive reagent for efficient transfection of mammalian cells.

Amphipathic peptides can be useful effectors to enhance gene delivery. However, peptide/DNA complexes usually require additional effectors, such as fusogenic lipids, to mediate efficient transfection. Due to weak and/or multiple interactions between the various components of the system, the transfecting complexes are often heterogeneous and unstable in biological fluids. Accordingly, a hybrid molecule resulting from the covalent coupling of an amphipathic, membrane-disturbing peptide to a lipid moiety might create a stable and efficient peptide-based gene transfer system. The present work describes such a novel hybrid molecule, dioleoylmelittin, resulting from the conjugation of dioleoylphosphatidylethanolamine-N-[3-(2-pyridyldithio)propionate] with [Cys1]melittin. Dioleoylmelittin had a lower hemolytic and membrane-disturbing activity than melittin. Size and zeta potential measurements, DNA gel electrophoresis, and electron microscopy showed that dioleoylmelittin, unlike melittin, was able to complex plasmid DNA to form spherical particles with a net positive charge and a diameter between 50 and 250 nm. These particles, prepared at an optimal 10/1 dioleoylmelittin/DNA ratio (w/w), mediated efficient transient transfection of reporter genes in cultured mammalian cells including primary cells. The luciferase activity induced by the dioleoylmelittin/DNA complex was 5-500-fold higher than that induced by a cationic lipid/DNA complex, depending on the cationic lipid and the cell-line. Surprisingly, the presence of 10-50% fetal calf serum during dioleoylmelittin-mediated transfection enhanced 1.5-3-fold gene expression. Dioleoylmelittin represents a new class of efficient peptide-based transfection reagents, especially suited for serum-sensitive cells.

Gene targeting of CFTR DNA in CF epithelial cells.

A goal of cystic fibrosis (CF) gene therapy is correction of the mutant CF transmembrane conductance regulator (CFTR) gene with wild-type (wt) DNA sequences to restore normal CFTR protein and function. Experiments with wtCFTR cDNA expression vectors have shown that the Cl ion transport phenotype associated with CF can be corrected to resemble that in normal cells. An alternative to cDNA-based gene therapy strategies is one that corrects endogenous mutant sequences by targeted replacement with the wt homologue. To test whether such a strategy was feasible, a small fragment homologous replacement (SFHR) strategy was used to replace specific genomic sequences in human epithelial cells. Small fragments of genomic wtCFTR DNA were transfected into transformed CF epithelial cells. Replacement by exogenous CFTR DNA at the appropriate genomic locus and its expression as mRNA was indicated by: (1) allele-specific polymerase chain reaction (PCR) amplification of genomic DNA and mRNA-derived cDNA; and (2) hybridization of PCR products with allele-specific probes. In addition, the functional activity of CFTR protein was determined by whole cell patch clamp. Southern hybridization and patch clamp analyses suggested that approximately 1 in 100 CF cells underwent a homologous replacement event that resulted in intact Cl transport.

Short-chain phospholipids enhance amphipathic peptide-mediated gene transfer.

Addition of short-chain phospholipids to the gramicidin S-DNA-dioleoyl phosphatidylethanolamine complex enhanced up to 6-fold beta-galactosidase expression in several cell-lines in vitro. Among the compounds tested, the most potent in enhancing transfection were the dicapryl- and the dicapryloyl phosphatidylcholine. In contrast, no significant enhancement of transfection was seen when short-chain phospholipids were mixed with cationic lipids. Short-chain phospholipid and gramicidin S may act simultaneously on the cell membrane to enhance gene transfer, yet resulting in no toxicity.

Dose optimization of gallium chloride, orally administered, in combination with platinum compounds.

An individual dose adaptation for cisplatin (CDDP), etoposide and gallium chloride (GaCl3) was proposed to improve the efficacy of this combination chemotherapy and avoid its toxicity. A clinical study was performed in 28 non small cell lung cancer patients, to verify this hypothesis. CDDP and etoposide were administered as continuous infusions every 3 weeks and GaCl3 orally during and between the CDDP-etoposide sequential infusions. CDDP doses were adjusted to achieve, during each 5 day infusion, an area under the total plasma platinum concentrations versus time curve (AUC Pt 0-120) ranging between 80,000 and 100,000 micrograms/l.h. Etoposide dosages were 120 mg/24 h during days 1-3 of the CDDP infusion. GaCl3 dosages were adjusted to obtain plasma gallium (Ga) concentrations ranging between 200 and 400 micrograms/l. The proposed methods of adaptation were successful from a pharmacokinetic point of view as AUC Pt 0-120 were respectively 81351 +/- 4788, 88268 +/- 8451 and 88331 +/- 8778 micrograms/l.h during the first 3 courses, and plasma Ga concentrations, determined during the 2nd and 3rd CDDP courses, 16 hours after the beginning of the CDDP infusion, were respectively 264 +/- 127 and 313 +/- 186 micrograms/l. However, these results were not pharmacodynamically successful and the therapeutic window was not confirmed. Past clinical trials with GaCl3 will be reviewed, as well as the factors which modify the pharmacokinetics or the pharmacodynamic effects of CDDP and GaCl3. From this review, an optimal dosage of 400 mg GaCl3 could be proposed to potentiate a combination chemotherapy with a platinum compound. The target AUC of the platinum compound should be the AUC avoiding its cumulative toxicity.

Efficient phosphate binding using a combination of gluconolactate and carbonate calcium salts.

Although renal-failure-related hyperphosphataemia can be corrected by various phosphate binders, there remains a need for safer and more efficient formulations to precipitate phosphate. This work describes both a theoretical approach and a phosphate precipitation test in order to design efficient binding calcium salts formulations. The results show that the combination of a soluble calcium salt (the gluconolactate) and a proton-consuming calcium salt (the carbonate) can precipitate phosphate effectively. Furthermore, the theoretical computations correlate well with the ability of the salt to bind phosphate in vitro.

[Changes in isoelectric points of the monoclonal antibody OC 125 during indium 111 radiolabeling]. / Modifications des points isoélectriques de l'anticorps monoclonal OC 125 lors de son radiomarquage par l'indium 111.

Isoelectrofocusing analysis of the murine monoclonal antibody OC 125 (IgG1) revealed four bands at pI (6.6-7.2). After pepsin digestion, f(ab')2 pIs were increased by 0.6 pH units and two additional bands were visible. DTPA conjugation decreased pI (by up to -2 units) and increased heterogeneity (10 bands or more). With In3+ at saturation level, the conjugate was slightly modified. Autoradiography revealed pI heterogeneity of OC 125-f(ab')2-DTPA-In-111. These data show that isoelectrofocusing enables accurate monitoring of monoclonal antibody changes during In-111 labeling.

Cyclic amphipathic peptide-DNA complexes mediate high-efficiency transfection of adherent mammalian cells.

A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic peptide gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of beta-galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phase-competent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.

Predicting the impact sensitivity of explosive molecules using neuromimetic networks.

A new method for predicting the impact sensitivity of explosive molecules is presented. This method makes use of a network of formal neurons. The experiment uses 124 molecules belonging to different families. The molecular descriptors taken into account are the molecule's oxygen balance and the enumeration of certain groups. The results obtained are satisfactory: 80% of the molecules are correctly classed on a scale of four sensitivities. Comparison with a multivariate linear regression analysis gives a slight advantage to the neural network method.

Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes.

We compare the transfection efficiency of plasmid DNA encoding either luciferase or beta-galactosidase encapsulated in pH-sensitive liposomes or non-pH-sensitive liposomes or DNA complexed with cationic liposomes composed of dioleoyloxypropyl-trimethylammonium:dioleoylphosphatidyl-eth anolamine (1:1, w/w) (Lipofectin) and delivered into various mammalian cell lines. Cationic liposomes mediate the highest transient transfection level in all cell-lines examined. pH-sensitive liposomes, composed of cholestryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2:1 molar ratio, mediate gene transfer with efficiencies that are 1 to 30% of that obtained with cationic liposomes, while non-pH-sensitive liposome compositions do not induce any detectable transfection. Cationic liposomes mediate a more rapid uptake of plasmid DNA, to about an eightfold greater level than that obtained with pH-sensitive liposomes. The higher uptake of DNA mediated by Lipofectin accounts for part of its high transfection efficiency. Treatment of cells with chloroquine, ammonium chloride, or monensin decreases (threefold) transfection using pH-sensitive liposomes and either has no effect on or enhances cationic liposome-mediated transfection. Therefore plasma membrane fusion is not the only mechanism available to cationic liposomes; in certain cell lines DNA delivery via endocytosis is a possible parallel pathway and could augment the superior transfection efficiency observed with cationic liposomes.

[Urinary iodide determination by X-ray fluorescence]. / Dosage des iodures urinaires par fluorescence X.

Urinary iodide measurement has been carried out by X-ray-fluorescence, either directly on urinary solution, or after matrix concentration. Proportionality between emitted XK alpha rays of iodine and iodide mass in standards has been observed on a large scale, ranging up to 400 micrograms. With an exciting-1.11 GBq (241(95) Am)-radioactive source, 0.44 microgram are detected for solid matrix, and 0.9 microgram/ml for iodide in solution for 10 mn measuring time. So direct measurement on solution can be applied only to high excreted iodide. For normal range iodide determination is performed after anionic resin concentration (on 100 ml or 200 ml). For tracing, Na I131 is employed. The binding ratio is strongly depending on flow, resin weight, and associate urinary anionic components. On 20 healthy subjects, normal range value is 53 +/- 22 micrograms/l (m +/- s.d.). Comparative study with an electrochemical method showed fluorescence iodide values are lower than the former. The proposed method is very simple, one or two steps (function of iodide content). As no interfered Rx has been observed in the Rx iodide region, the authors can ascertain that accurate values are observed by X-ray fluorescence. In case of high iodide content, this methods allows to distinguish urinary iodide versus total urinary iodine, when performing solution and matrix concentration studies on the urinary batch.

Purification and characterization of calpain from human polymorphonuclear leukocytes.

Recent studies have demonstrated that a calcium-sensitive protease converts Ca2+/phospholipid-dependent protein kinase C to a Ca2+/phospholipid-independent form during the activation of human neutrophils. In this paper, the results of the purification and characterization of a calcium-dependent cytosolic protease from neutrophils is reported. Calcium-dependent protease has been purified 1062-fold from human neutrophils and behaves as a single species on native polyacrylamide gels. The protease is active in the neutral pH range with no observable activity amide gels. The protease is active in the neutral pH range with no observable activity at pH values greater than 8.0, has an absolute requirement for calcium for expression of activity with half-maximal activity observed at 12 microM free calcium, and has an apparent molecular weight of 110,000 based on gel filtration. The protease requires the presence of dithiothreitol for activity and is inhibited by sulfhydryl inhibitors, leupeptin, and antipain but not by serine protease inhibitors, pepstatin, or orthophenanthroline. The protease is also susceptible to inactivation by autoproteolysis. Based on the similarities of this calcium-dependent protease with calpains from a variety of other mammalian tissues, the protease isolated from human neutrophils appears to be a calpain I.

F.p.l.c. analysis of the leakage products of proteins crosslinked on Dacron vascular prostheses.

The haemocompatibility of vascular, Dacron prostheses was improved by coating with albumin and/or collagen crosslinked with glutaraldehyde (GTA) or carbodiimide (CDI). F.p.l.c. (fast protein liquid chromatography) analysis of the products desorbed from polymeric matrices incubated in physiological conditions for periods extended up to 10 d did not detect the monomers or polymers of collagen and albumin but small amounts of degradation products, the molecular weights of which were less than 45,000, thus minimizing an eventual immunogenic response after implantation. However GTA and CDI matrices required extensive washing to neutralize the cytotoxic effect of GTA and achieve the release of CDI from protein complexes.