Induction of tumor-specific CTL responses using the C-terminal fragment of Viral protein R as cell penetrating peptide.

The discovery of tumor-associated antigens recognized by T lymphocytes opens the possibility of vaccinating cancer patients with defined antigens. However, one of the major limitation of peptide-based vaccines is the low immunogenicity of antigenic peptides. Interestingly, if these epitopes are directly delivered into the cytoplasm of antigen presenting cells, they can be efficiently presented via the direct MHC class I presentation pathway. To improve antigen entry, one promising approach is the use of cell penetrating peptides (CPPs). However, most studies use a covalent binding of the CPP with the antigen. In the present study, we focused on the C-terminal domain of Vpr which was previously demonstrated to efficiently deliver plasmid DNA into cells. We provide evidence that the peptides Vpr55-91 and Vpr55-82 possess the capacity of delivering proteins and epitopes into cell lines as well as into human primary dendritic cells, without the necessicity for a chemical linkage. Moreover, immunization of HLA-A2 transgenic mice with Vpr55-91 as the sole adjuvant is able to induce antigen-specific cytotoxic T lymphocytes against multiple tumor epitopes.

Growth process of nanosized aluminum thin films by pulsed laser deposition for fluorescence enhancement.

Pulsed laser deposition was used to deposit aluminum thin films of various thicknesses (tAl) ranging from 5 to 40 nm and to investigate their growth process when they are deposited onto SiO2 and Y2O3. Atomic force microscopy and x-ray reflectivity measurements show that the structure of the Al films are related to the wettability properties of the underlaying layer. Onto SiO2, ultra-smooth layers of aluminum are obtained, due to a perfect wetting of SiO2 by Al. In contrast when deposited onto Y2O3, percolated Al layers are observed with apparent pore size decreasing from 200 to 82 nm as t(Al) is increased from 5 to 40 nm, respectively. This particular morphology is related to partial dewetting of Al on Y2O3. These two different growth mechanisms of aluminum depend therefore on the surface properties of SiO2 and Y2O3. The plasmon resonance of such Al nanostructures in the UV region was then analyzed by studying the coupling between Eu(3+) rare earth emitters and Al.

N-3 fatty acid enriched eggs and production of egg yolk powders: an increased risk of lipid oxidation?

Lipid oxidation is generally favoured by thermal processing and long-term storage. Oxidised lipids can alter nutritional and sensorial properties of foods. As eggs are widely used in food industries in dried powder form, our aim was to determine whether compositional or processing parameters have an impact on lipid oxidation from the shell eggs up to the dried powders and subsequent storage. Two batches of shell eggs were processed: one issued from hens fed with a standard diet and another receiving a diet enriched in extruded linseed, rich in linolenic acid. The extent of lipid oxidation was evaluated by quantification of conjugated dienes (CD) and malondialdehyde (MDA), but also by assessment of tocopherols, lutein and zeaxanthin losses. Results highlighted the remarkable oxidative stability of control and enriched yolk powders as revealed by a moderate increase of the quantities of CD and MDA, the lack of oxidised cholesterol and small loss of α-tocopherol.

Synthesis, characterization, and gene transfer application of poly(ethylene glycol-b-ethylenimine) with high molar mass polyamine block.

The study of ethyloxazoline/methyloxazoline (EtOXZ/MeOXZ) copolymerization, initiated by methyl tosylate (MeOTs), showed that (i) incorporation of MeOXZ units into random copolymer becomes effective over DP = 100 and (ii) propagation process proceeds with negligible transfer to monomer up to a DP of 400 despite the presence of MeOXZ in the polymerization medium. These results produced random poly(EtOXZ-co-MeOXZ) copolymers with various molar composition ratios in alkyloxazoline units. The close values found for the comonomer reactivity ratios in acetonitrile (r(1MeOXZ) = 1.18; r(2EtOXZ) = 0.34) implied a random chain organization in short sequences of each repeating unit, which was an important parameter in view of the optimization of their subsequent modification: the alkaline hydrolysis was successfully achieved when the MeOXZ unit content of the polyoxazoline chains reached 75%. Using these results, the diblock copolymer poly(ethylene glycol-b-(ethyloxazoline-co-methyloxazoline)) (poly(EG-b-(EtOXZ-co-MeOXZ))) with high DP was synthesized by cationic copolymerization of EtOXZ/MeOXZ comonomers using CH(3)-PEG(2kDa)-Ts as macroinitiator. The comonomer composition of this new compound was adjusted in order to optimize the hydrolysis step and obtain finally the diblock copolymer poly(ethylene glycol-b-ethylenimine) (poly(EG-b-EI)). The high molar mass of this copolymer was confirmed both by (1)H NMR and SANS measurements. Gene delivery experiments showed that the copolymer has significant DNA transfection capacities.

Linear topology confers in vivo gene transfer activity to polyethylenimines.

Although polyethylenimines (PEIs) are frequently used transfection agents, it is still unclear which of their properties are required for efficient gene delivery. This is even more striking when working in vivo since some PEIs are able to generate significant gene expression, whereas others are not. To facilitate a rational development of compounds with improved transfection activities, studies aimed at identifying the properties involved in the transfection process seem indispensable. In the present work, we investigated how transfection with linear PEI of 22 kDa allows for high reporter gene expression in lungs after intravenous injection, whereas the branched PEI of 25 kDa does not. To this end, we synthesized L-PEI derivatives that are intermediates between linear and branched PEIs. Our results show that the topology plays a crucial role in obtaining in vivo reporter gene expression, whereas the content of primary, secondary, and tertiary amines is only of minor importance.

DNA compaction into new DNA vectors based on cyclodextrin polymer: surface enhanced Raman spectroscopy characterization.

The ability of DNA to bind polycation yielding polyplexes is widely used in nonviral gene delivery. The aim of the present study was to evaluate the DNA compaction with a new DNA vector using Raman spectroscopy. The polyplexes result from an association of a beta-cyclodextrin polymer (polybeta-CD), an amphiphilic cationic connector (DC-Chol or adamantane derivative Ada2), and DNA. The charge of the polymeric vector is effectively controlled by simple addition of cationic connector in the medium. We used surface enhanced Raman spectroscopy (SERS) to characterize this ternary complex, monitoring the accessibility of adenyl residues to silver colloids. The first experiments were performed using model systems based on polyA (polyadenosine monophosphate) well characterized by SERS. This model was then extended to plasmid DNA to study polybeta-CD/Ada2/DNA and polybeta-CD/DC-Chol/DNA polyplexes. The SERS spectra show a decrease of signal intensity when the vector/DNA charge ratio (Z+/-) increases. At the highest ratio (Z+/- = 10) the signal is 6-fold and 3-fold less intense than the DNA reference signal for Ada2 and DC-Chol polyplexes, respectively. Thus adenyl residues have a reduced accessibility as DNA is bound to the vector. Moreover, the SERS intensity variations are in agreement with gel electrophoresis and zeta potential experiments on the same systems. The overall study clearly demonstrates that the cationic charges neutralizing the negative charges of DNA result in the formation of stable polyplexes. In vitro transfection efficiency of those DNA vectors are also presented and compared to the classical DC-Chol lipoplexes (DC-Chol/DNA). The results show an increase of the transfection efficiency 2-fold higher with our vector based on polybeta-CD.

Polyethylenimine-mediated gene delivery: a mechanistic study.

BACKGROUND: Ethylenimine polymers (PEIs) belong to one of the most efficient family of cationic compounds for delivery of plasmid DNA into mammalian cells. The high transfection efficiencies are obtained even in the absence of endosomolytic agents such as fusogenic peptides or chloroquine, which is in contrast to most of the other cationic polymers. It has been hypothesized that the efficiency of PEI is due to its capacity to buffer the endosomes. METHODS: To investigate the importance of the acidification of endosomes during PEI-mediated DNA transfer we used proton pump inhibitors such as bafilomycin A1 and concanamycin A. Moreover, we tested whether PEI is able to destabilize natural membranes per se at neutral or acidic pH by performing erythrocyte lysis assays. RESULTS: PEI-mediated transfection in the presence of bafilomycin A1 resulted in a 7-74-fold decrease in reporter gene expression depending on the cell line used. In contrast, the efficiency of the monocationic lipid, DOTAP, was not importantly altered in the presence of the drug. Furthermore, the present data show that PEI cannot destabilize erythrocyte membranes, even at acidic pH, and that PEI, complexed or not to DNA, can increase the transfection efficiency of the cationic polymer, polylysine, when added at the same time to the cells. CONCLUSIONS: The transfection efficiency of PEIs partially relies on their ability to capture the protons which are transferred into the endosomes during their acidification. In addition, PEI is able to deliver significant amounts of DNA into cells and the DNA complexes involved in the expression of the transgene escape within 4 h from the endosomes.

Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R.

Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr(52-96)) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr(52-96) was 10- to 1,000-fold more active. Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.

Carcase characteristics of a heavy Japanese quail line under introgression with the roux gene.

1. The roux plumage sex-linked recessive gene may be used for early sexing of Japanese quail in crossbreeding production systems with wild-type and roux lines. However, associated effects of the gene on carcase and meat composition need to be assessed. 2. Quail carcases from pure Line K males and females (100% K), a heavy meat line which was used as the recipient line for the introgression of the roux gene, and from same-age roux or wild-type females from the second generation of introgression (75% K) were dissected. The effects of sex, line and plumage colour on carcase components and on protein and lipid contents of various tissues were estimated. 3. Expected sex differences in carcase weights were obtained, with marginally higher values for females. However, weights of parts and carcase yield (ratio of empty carcase weight without head, neck and feet over live body weight) were similar for both sexes in pure Line K which had a 68% carcase yield. Lipid contents in leg meat were higher in males (3.1%) than in females (2.7%). 4. The roux gene did not seem to have any major impact on carcase parts or composition. However, in roux birds, leg skin was marginally higher in lipids and pectoralis major lower in proteins than in wild-type ones.

[Detection and purification of a liver microsomal enzyme inducing the release of a glycopeptide inhibitor of hepatocyte proliferation from human alpha 2 macroglobulin]. / Détection et purification d'une enzyme microsomiale hépatique induisant la libération du glycopeptide inhibiteur de la prolifération des hépatocytes à partir de l'alpha 2 macroglobuline humaine.

The interaction rat liver microsomes/human alpha 2 macroglobulin releases in vivo an inhibitory peptide of the hepatocyte proliferation. Treatment of Triton X 100 on adult rat liver microsomes enables the solubilisation of a proteolytic enzyme. Its partial purification was obtained by Ultrogel AcA 44 filtration followed by a DEAE Sephacel chromatography. This enzyme shows a proteolytic activity in presence of calcium on synthetic substrates including Phe, Tyr and Trp. A whole enzyme inhibition is got after treatment by DFP or benzamidine. In presence of highly purified human alpha 2 macroglobulin this enzyme releases a glycopeptide of low molecular weight, which inhibits the hepatocyte proliferation during the G1-S transition in baby rat.