Automated target preparation for microarray-based gene expression analysis

DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables (1) the reduction of experimentally caused result variability, (2) the separation of (important) biological variability from (undesired) experimental variation, and (3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.     

Regional metabolite levels of the normal posterior fossa studied by proton chemical shift imaging

MR spectroscopy of the posterior fossa is pitted with numerous technical difficulties. It is, however, of great clinical interest in the study of the degenerative diseases and tumors of this area. We have developed a method to perform 2D CSI of this area, by using a sagittal slice and a careful positioning of outer volume saturation. We performed this acquisition in 30 healthy volunteers to determine the normal metabolic ratios in five voxels of this area (mesencephalon. pons. medulla oblongata, vermis, cerebellar white matter). The main technical difficulty was magnetic field inhomogeneity in the lower brainstem generated by dental alloys. However, 88% of the voxels were of sufficient quality to be analyzed. The statistically significant regional variations were a higher NAA/Cr ratio in the pons than in the medulla oblongata, higher Cho/Cr in the pons than in the mesencephalon and higher Cho/ Cr in the cerebellar white matter than in the vermis. We conclude that 2D CSI of the brainstem, although technically delicate can be performed in most patients.     

Poly(vinyl chloroformate) and derivatives: 3. Chemical modification of poly(vinyl chloroformate)

The chemical modification of poly(vinyl chloroformate) with compounds containing labile hydrogen atoms like amines, alcohols and phenols as well as with potassium cyanide has been investigated. Convenient conditions have been found in order to obtain soluble modified polymers with good substitution yields.     

Design, synthesis, and biological evaluation of the first podophyllotoxin analogues as potential vascular-disrupting agents

We designed and synthesized two novel series of azapodophyllotoxin analogues as potential antivascular agents. A linker was inserted between the trimethoxyphenyl ring E and the tetracyclic ABCD moiety of the 4-aza-1,2-didehydropodophyllotoxins. In the first series, the linker enables free rotation between the two moieties; in the second series, conformational restriction of the E nucleus was considered. We have identified several new compounds with inhibitory activity toward tubulin polymerization similar to that of CA-4 and colchicine, while displaying low cytotoxic activity against normal and/or cancer cells. An aminologue and a methylenic analogue were shown to disrupt endothelial cell cords on Matrigel at subtoxic concentrations, and an original assay of drug washout allowed us to demonstrate the rapid reversibility of this effect. These two new analogues are promising leads for the development of vascular-disrupting agents in the podophyllotoxin series.     

B-ring-modified isocombretastatin A-4 analogues endowed with interesting anticancer activities

A novel class of isocombretastatin A-4 (isoCA-4) analogues with modifications at the 3'-position of the B-ring by replacement with C-linked substituents was studied. Exploration of the structure-activity relationships of theses analogues led to the identification of several compounds that exhibit excellent antiproliferative activities in the nanomolar concentration range against H1299, MDA-MB231, HCT116, and K562 cancer cell lines; they also inhibit tubulin polymerization with potency similar to that of isoCA-4. 1,1-Diarylethylenes 8 and 17, respectively with (E)-propen-3-ol and propyn-3-ol substituents at the 3'-position of the B-ring, proved to be the most active in this series. Both compounds led to the arrest of various cancer cell lines at the G(2) /M phase of the cell cycle and strongly induced apoptosis. Docking of compounds 8 and 17 in the colchicine binding site indicated that their C3' substituents guide the positioning of the B-ring in a manner different from that observed for isoCA-4.     

Parameters affecting the synthesis of geranyl butyrate by esterase 30,000 from Mucor miehei

The factors affecting the synthesis of geranyl butyrate by esterase 30,000 of Mucor miehei were studied in a solvent-free system. The effects of substrate molar ratio, temperature, agitation speed, and initial addition of water were investigated. The equimolar ratio was most interesting for ester production in batch. There were no diffusion limitations, and the reaction could be realized at low agitation. The catalytic activity of the enzyme was irreversibly deactivated at 60°C, and the initial addition of water decreased the rate of conversion after 75 h of reaction. The enzyme activity increased with increased linear chainlength of the acid and was also affected by the alcohol structure. Esterase 30,000 gave the highest conversion of butyric acid with hexanol and terpenic alcohols (citronellol, nerol) and the lowest with the secondary alcohol (2-hexanol). Finally, five other industrial enzymatic preparations were investigated for their ability to synthesize geranyl butyrate and to hydrolyze olive oil. We observed, for the lipase from Rhizopus javanicua, that there is no relationship between hydrolytic and synthetic activities; this example shows that the hydrolytic lipase activity data cannot predict the capability of lipases in esterification reactions.     

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.     

Design of a composite ethidium-netropsin-anilinoacridine molecule for DNA recognition

Control of gene expression is a cherished goal of cancer chemotherapy. Small ligand molecules able to bind tightly to DNA in a well-defined configuration are being actively searched for. With this goal in mind, we have designed and synthesized the trifunctional molecule R-132, which combines a bispyrrole skeleton for minor groove DNA recognition and two different chromophores, anilinoacridine and ethidium. The affinity and mode of binding of R-132 to DNA were studied by a combination of complementary biochemical and biophysical techniques, which included absorption and fluorescence spectroscopy and circular and linear dichroism. A surface plasmon resonance biosensor analysis was also performed to quantify the kinetic parameters of the drug-DNA interaction process. Altogether, the results demonstrate that the three moieties of the hybrid molecule are engaged in the interaction process, thus validating the rational design strategy. At the biological level, R-132 stabilizes topoisomerase-II-DNA covalent complexes and displays potent cytotoxic activities, which are attributable to its DNA-binding properties. R-132 easily enters and accumulates in cell nuclei, as evidenced by confocal microscopy. R-132 therefore provides a novel lead compound for the design of gene-targeted anticancer agents.