Target fishing reveals PfPYK-1 and PfRab6 as potential targets of an antiplasmodial 4-anilino-2-trichloromethylquinazoline hit compound.

We present investigations about the mechanism of action of a previously reported 4-anilino-2-trichloromethylquinazoline antiplasmodial hit-compound (Hit A), which did not share a common mechanism of action with established commercial antimalarials and presented a stage-specific effect on the erythrocytic cycle of P. falciparum at 8 < t < 16 h. The target of Hit A was searched by immobilising the molecule on a solid support via a linker and performing affinity chromatography on a plasmodial lysate. Several anchoring positions of the linker (6,7 and 3') and PEG-type linkers were assessed, to obtain a linked-hit molecule displaying in vitro antiplasmodial activity similar to that of unmodified Hit A. This allowed us to identify the PfPYK-1 kinase and the PfRab6 GTP-ase as potential targets of Hit A.

Structuration and deformation of colloidal hydrogels.

The aim of the present paper is to determine the optimum conditions for the formation of homogeneous colloidal silica hydrogels by aggregation and drying processes, avoiding mechanical instabilities at the surface. Aggregation is controlled by adding monovalent salt to the silica nano-particle suspension while the drying of the sol is also modulated by changing the evaporation rate. A phase diagram reveals two regions in the parameter plane, ionic strength versus evaporation rate: a region where the drop undergoes an isotropic shrinkage and forms the required homogeneous gel and a region where mechanical instabilities appear due to the formation of a solid skin at the gel surface. The frontier between these two regions can be determined by equating the following two characteristic times: the gelation time and the time for skin formation. Permeability measurements of the final gel provide an estimate of the drying stress which is compared to the yield stress of the material. In accordance with the determined phase diagram, our study shows that instabilities appear when the drying stress is larger than the yield stress.

First (e,e) coincidence measurements on solvated sodium benzoate in water using a magnetic bottle time-of-flight spectrometer.

Understanding the mechanisms of X-ray radiation damage in biological systems is of prime interest in medicine (radioprotection, X-ray therapy…). Study of low-energy rays, such as soft-X rays and light ions, points to attribute their lethal effect to clusters of energy deposition by low-energy electrons. The first step, at the atomic or molecular level, is often the ionization of inner-shell electrons followed by Auger decay in an aqueous environment. We have developed an experimental set-up to perform electron coincidence spectroscopy on molecules in a water micro-jet. We present here the first results obtained on sodium benzoate solutions, irradiated at the oxygen and carbon K-edges.

Modulation of the expression and activity of cathepsin S in reconstructed human skin by neohesperidin dihydrochalcone.

Dysregulation of cathepsin S (Cat S), a cysteine protease involved in extracellular-matrix and basement membrane (BM) degradation, is a concomitant feature of several inflammatory skin diseases. Therefore, Cat S has been suggested as a potential therapeutic target. Flavonoids, which were identified as regulatory molecules of various proteolytic enzymes, exert beneficial effects on skin epidermis. Herein, thirteen flavonoid compounds were screened in vitro and in silico and neohesperidin dihydrochalcone (NHDC) was identified as a potent, competitive, and selective inhibitor (Ki=8±1 µM) of Cat S. Furthermore, Cat S-dependent hydrolysis of nidogen-1, a keystone protein of BM architecture, as well elastin, collagens I and IV was impaired by NHDC, while both expression and activity of Cat S were significantly reduced in NHDC-treated human keratinocytes. Moreover, a reconstructed human skin model showed a significant decrease of both mRNA and protein levels of Cat S after NHDC treatment. Conversely, the expression of nidogen-1 was significantly increased. NHDC raised IL-10 expression, an anti-inflammatory cytokine, and mediated STAT3 signaling pathway, which in turn dampened Cat S expression. Our findings support that NHDC may represent a valuable scaffold for structural improvement and development of Cat S inhibitors to preserve the matrix integrity and favor skin homeostasis during inflammatory events.

In situ and time-resolved infrared detection of the reactivity induced by electrons in polymer films.

The real time and in situ analysis of chemical reactions such as polymerization reactions, polymer degradation, and oxidation of polymers is of utmost importance. Surprisingly, only few experimental tools allowing this are available. To bridge this gap, we have developed a new experimental setup coupling a 60 keV electron gun with an infrared spectrometer operating in the mid-IR region (800-4000 cm-1) and associated with the rapid scan mode. The measurements are performed using the infrared reflection absorption spectroscopy approach. We worked here with 0.5-25 ms pulses for which the dose per pulse ranges from 4 to 200 kGy. Combining pulse electrons with rapid scan analysis enables performing experiments with a time resolution of 80 ms (and above). Therefore, this new platform can, for instance, give direct insights into reaction mechanisms at stake during the degradation of polymers. First results on a bisphenol A polycarbonate irradiated under vacuum enabling the validation of this new setup and also the recording of time-resolved spectra are presented. Reaction mechanisms are then proposed.

The effect of myoglobin crowding on the dynamics of water: an infrared study.

Solutions containing 8 and 32 wt% myoglobin are studied by means of infrared spectroscopy, as a function of temperature (290 K and lower temperatures), in the mid- and far-infrared spectral range. Moreover, ultrafast time-resolved infrared measurements are performed at ambient temperature in the O-D stretching region. The results evidence that the vibrational properties of water remain the same in these myoglobin solutions (anharmonicity, vibrational relaxation lifetime…) and in neat water. However, the collective properties of the water molecules are significantly affected by the presence of the protein: the orientational time increases, the solid-liquid transition is affected in the most concentrated solution and the dynamical transition of the protein is observed, from the point of view of water, even in the least concentrated solution, proving that the water and myoglobin dynamics are coupled.

A trapped water network in nanoporous material: the role of interfaces.

The infrared spectra of water confined in well controlled pore glasses were recorded as a function of the pore size ranging from 8 to 320 nm and in the 30-4000 cm(-1) spectral range using the ATR technique. The experiments prove that even in the large pores, the water network is significantly perturbed. The energy of the connectivity (or hindered translation) band (around 150 cm(-1)) is found to increase when the pore size decreases, indicating that confinement increases the H-bonding between neighbouring water molecules. Moreover, a drastic decrease of the FWHM of the connectivity band was observed upon confinement. This can be related to some ordering induced by the rigid walls of the pores. Furthermore, the partial filling of pores causes a significant modification to the water network, resembling heating of the trapped liquid and suggesting a role played by the water/air interface.

Radiolysis of water in nanoporous gold.

Nanoporous gold was used as a high specific surface material to describe the water radiolysis in the vicinity of metal surfaces. Porous gold monoliths were prepared by electrolytic dissolution of silver in Au-Ag alloys and characterized by small angle neutron scattering. The hydroxyl radical production under gamma irradiation was measured by benzoate scavenging in water confined in 50 nm porous gold or in silica glasses of similar pore size, and in bulk water. Whereas a silica interface induces minimal modifications to the HO˙ radical production with respect to bulk water, HO˙ production near gold is enhanced more than seven times on the short time scale, and almost completely suppressed by reaction with the metal on the long time scale.

Preparative purification of antiamyloidogenic stilbenoids from Vitis vinifera (Chardonnay) stems by centrifugal partition chromatography.

Five stilbenoids, E-resveratrol, E-piceatannol, (+) E-(epsilon)-viniferin, (+)-ampelopsin A and vitisin C were isolated from methyl tert-butyl ether (MtBE) stem extract of Vitis vinifera (Chardonnay cv). Their purification on a preparative scale was obtained by centrifugal partition chromatography (CPC) using quaternary Arizona solvent systems composed of n-heptane/ethyl acetate/methanol/water. We tested 23 Arizona solvent systems to partition the extract and found that systems K and M (Hept/EtOAc/MeOH/water, 1:2:1:2 and 5:6:5:6, respectively; v/v) were the best to separate the stilbenes mentioned above. This support-free liquid-liquid chromatographic procedure made it possible to isolate ampelopsin A from V. vinifera for the first time. The antiamyloidogenic activity of the isolated stilbenes was evaluated versus beta-amyloid fibrils. E-resveratrol and (+)-ampelopsin A were found to be the most active compounds with 63 and 46% inhibition at 10microM, respectively. These findings suggest that E-resveratrol and (+)-ampelopsin A may function as attractive new candidates for protecting against brain cell dysfunction in vivo in AD by inhibiting the aggregation of Abeta.

Solid-state NMR characterization of a controlled-pore glass and of the effects of electron irradiation.

Controlled-pore glasses (CPGs) are silica-based materials which provide an adequate model system for a better understanding of the radiation chemistry of glasses, especially under nanoscopic confinement. This paper presents a characterization of a nanoporous CPG before and after electron irradiation using multinuclear solid-state magnetic resonance (NMR). 1H MAS NMR has been used for studying the surface proton sites and it is observed that the irradiation leads to a dehydration of the material. Accordingly, concerning the silicon sites near the surface, the observed variation of the Q4, Q3 and Q2 species from 1H-29Si CPMAS spectra shows an increase of the surface polymerization under irradiation, implying in majority a Q2 to Q3/Q4 conversion mechanism. Similarly, 1H-17 O CPMAS measurements exhibit an increase of Si-O-Si groups at the expenses of Si-OH groups. In addition, modifications of the environment of the residual boron atoms are also put in evidence from 11B MAS and MQMAS NMR These data show that MAS NMR methods provide sensitive tools for the characterization of these porous glasses and of the tiny modifications occurring under electron irradiation.

Localization and quantification of carbon-centered radicals on any amino acid of a protein.

A general strategy to localize and quantify carbon-centered radicals within proteins is described. The methodology was first exemplified on amino acids and then on a peptide. This method is applicable to any protein system regardless of size, and the site of hydrogen abstraction by *OH on all residues within proteins is easily and accurately detected.

Radiolysis of confined water: molecular hydrogen formation.

The formation of molecular hydrogen in the radiolysis of water confined in nanoscale pores of well-characterised porous silica glasses and mesoporous molecular sieves (MCM-41) is examined. The comparison of dihydrogen formation by irradiation of both materials, dry and hydrated, shows that a large part of the H2 comes from the surface of the material. The radiolytic yields, G(H2)=(3+/-0.5)x10(-7) mol J(-1), calculated using the total energy deposited in the material and the water, are only slightly affected by the degree of hydration of the material and by the pore size. These yields are also not modified by the presence of hydroxyl radical scavengers. This observation proves that the back reaction between H2 and HO(.) is inoperative in such confined environments. Furthermore, the large amount of H2 produced in the presence of different concentrated scavengers of the hydrated electron and its precursor suggests that these two species are far from being the only species responsible for the H2 formation. Our results show that the radiolytic phenomena that occur in water confined in nanoporous silica are dramatically different to those in bulk water, suggesting the need to investigate further the chemical reactivity in this type of environment.

[Antibacterial effect of two glycine betaine analogues against Escherichia coli]. / Activité antibactérienne de deux analogues de la glycine bétaïne vis-à-vis d'Escherichia coli.

Strains of enterobacteria that cause urinary tract infections are able to grow in urine with high tonicity. In such conditions bacterias adapt to osmotic forces by incorporation of osmoprotectant compounds including glycine betaine. Accumulation of toxic analogues in bacteria, using inducible betaine transporters, has been previously proposed for development of antibiotics. In this study we report antibacterial effect of two analogues against 82 strains of Escherichia coli isolated form urinary tract infections. Minimal inhibitory concentrations have been measured with and without osmotic stress. The betaine analogues have antibacterial effect against E. coli strains, but only in presence of an osmotic stress.

Solid phase synthesis of sulphonamides: novel ligands of 5-HT6 receptors.

Using solid phase synthesis techniques, we have rapidly obtained a series of eight aryl sulphonamides derived from putrescine. These conjugates with various aryl groups were evaluated for their affinity towards 5-HT6 receptors in man. This evaluation revealed the interest of two compounds which present the same activity level, in the submicromolar range, as two reference derivatives. The most potent will be considered as a new lead for further investigations.

Constituents of the stem bark of Ormosia wayana.

The isolation of lupeol, beta-sitosterol acetate and three quinolizidinic alkaloids, (+)-lupanine, (-)-sparteine and (-)-3 beta-hydroxy-13 alpha-tigloyloxy-lupanine (1), from the stem bark of Ormosia wayana is reported.

Fractionation of low-molecular-mass heparin by centrifugal partition chromatography in the ion-exchange displacement mode.

Centrifugal partition chromatography in the ion-exchange displacement mode allowed a preparative and efficient fractionation of low-molecular-mass heparins from enoxaparin sodium. Amberlite LA2--a lipophilic liquid secondary amine--was chosen as a weak anion exchanger. The biphasic system methyl isobutyl ketone-water was selected. Protonated LA2 (10%, v/v) was added to the organic stationary phase. Hydroxide (Na+, OH-) was chosen as a displacer in the aqueous mobile phase. The observed pH and concentration profiles are typical of displacement chromatography, as supported by numerical simulation. The Dubois test for the analysis of sugar content and an analysis of sulfur content (and consequently sulfatation rate) were carried out to monitor the effectiveness of the procedure. Moreover, the fractions were analyzed by high-performance size-exclusion chromatography and the 1H NMR spectra confirmed the fractionation of the sample of enoxaparin sodium.

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N1-(n-octanesulfonyl)spermine and human colon cancer cells.

N(1)-(n-octanesulfonyl)spermine (N(1) OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N(1) OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N(1)OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N(1)OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N(1)OSSpm is an example, are suited to preferentially kill tumour cells.

Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii.

A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned. The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively. The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa. Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants. Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs. The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies. Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T. gondii. Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages. Southern blot analysis indicated that SOD occured as a single copy gene in T. gondii genome.

EPR and ligand field studies of iron superoxide dismutases and iron-substituted manganese superoxide dismutases: relationships between electronic structure of the active site and activity.

The problem of metal selectivity of iron/manganese superoxide dismutases (SODs) is addressed through the electronic structures of active sites using electron paramagnetic resonance and ligand field calculations. Studies of wild-type iron(III) SOD (FeSOD) from Escherichia coli and from Methanobacterium thermoautotrophicum and iron-substituted manganese(III) SOD (Fe(sub)MnSOD) from E. coli and from Serratia marcescens are reported. EPR spectroscopy of wild-type enzymes shows transitions within all three Kramers doublets identified by their g values. From the temperature dependence of the observed transitions, the zero-field splitting is found to be negative, D = -2 +/- 0.2 cm-1. The electronic structure is typical of a distorted trigonal bipyramid, all the EPR features being reproduced by ligand field analysis. This unique and necessary electronic structure characterizes wild-type enzymes whatever their classification from the amino acid sequence into iron or manganese types, as E. coli FeSOD or M. thermoautotrophicum FeSOD. In iron-substituted manganese SODs, reduced catalytic activity is found. We describe how inhomogeneity of all reported substituted MnSODs might explain the activity decrease. EPR spectra of substituted enzymes show several overlapping components. From simulation of these spectra, one component is identified which shares the same electronic structure of the wild-type FeSODs, with the proportion depending on pH. Ligand field calculations were performed to investigate distortions of the active site geometry which induce variation of the excitation energy of the lowest quartet state. The corresponding coupling between the ground state and the excited state is found to be maximum in the geometry of the native SODs. We conjecture that such coupling should be considered in the electron-transfer process and in the contribution of the typical electronic structure of FeSOD to the activity.

Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode.

Centrifugal partition chromatography (CPC) in the pH-zone refining mode allowed a preparative and efficient isolation of vindoline, vindolinine, catharanthine and vincaleukoblastine from a crude mixture of Catharanthus roseus alkaloids. The separation protocol was tested with a synthetic mixture of vindoline, catharanthine and vincaleukoblastine. The fraction content was analyzed and the results compared with theoretical chromatograms obtained by numerical simulation. The increase in injected sample mass results in an improvement of the purity of the isolated compounds. This observation, confirmed by theory, is of prime importance for the development of preparative pH-zone refining CPC as a preparative separation method.