Potent inhibitors of anthrax lethal factor from green tea

The anthrax lethal factor (LF) has a major role in the development of anthrax. LF is delivered by the protective antigen (PA) inside the cell, where it exerts its metalloprotease activity on the N-terminus of MAPK-kinases. PA+LF are cytotoxic to macrophages in culture and kill the Fischer 344 rat when injected intravenously. We describe here the properties of some polyphenols contained in green tea as powerful inhibitors of LF metalloproteolytic activity, and how the main catechin of green tea, (-)epigallocatechin-3-gallate, prevents the LF-induced death of macrophages and Fischer 344 rats.     

Neurosteroid modulation of the presynaptic NMDA receptors regulating hippocampal noradrenaline release in normal rats and those exposed prenatally to diazepam

Prenatal exposure to diazepam (DZ), a positive allosteric modulator of the gamma-aminobutyric acid(A) (GABA(A)) receptor complex, exerts profound effects that become more evident during puberty and in many cases are sex-specific, suggesting that such exposure interferes with the activity of steroid hormones. Apart from their well known effects on the genome, the reduced metabolites of many steroid hormones also interact directly with membrane receptors, including those for N-methyl-D-aspartate (NMDA). In this study, we compared the effects of several neurosteroids on NMDA receptors from normal rats and those exposed in utero to DZ (1.25mg/kg per day) from the 14th through the 20th day of gestation.In superfused rat hippocampal synaptosomes, activation of the NMDA receptor stimulates the basal release of [3H]noradrenaline ([3H]NA), which was used in our study as an index of receptor function. [3H]NA release was evoked in a concentration-dependent manner by NMDA (100 microM) plus glycine (GLY). The maximal increase (68.23+/-3.86%) with respect to basal release was achieved with a GLY concentration of 10 microM, and the EC(50) for GLY was 0.1 microM. Release stimulated by 100 microM NMDA + 0.1 microM GLY was not modified by any of the neurosteroids tested, with the exception of pregnenolone sulfate (PREG-S), which produced a 78.57+/-3.94% reduction in release at the maximal concentration used (0.3 microM). In synaptosomes from animals exposed in utero to DZ, the inhibitory effect of PREG-S was reduced by 46.55+/-2.33%.Given the important roles played by NMDA receptors in physiological and pathological processes within the central nervous system (CNS), characterization of NMDA receptor modulation is an important objective. The fact that this modulation can be altered by exposure in utero to DZ indicates that the behavioral abnormalities observed in exposed animals might be partially attributed to an altered sensitivity of NMDA receptors to the modulatory effects of neurosteroids.     

Cellular responses to H(2)O(2) and bleomycin-induced oxidative stress in L6C5 rat myoblasts

In muscle cells, reactive oxygen species (ROS) are continually generated. It is believed that these molecules have a well-established role as physiological modulators of skeletal muscle functions, ranging from development to metabolism and from blood flow to contractile functions. Moreover, ROS may contribute to the development of muscle fatigue, inflammation, and degeneration, and may be implicated in many muscle diseases. The aim of the present study was to verify the role of short or prolonged exposure to oxidative stress, generated by different concentrations of H(2)O(2), on growth, chromosomal aberrations, and apoptosis induced in cultured L6C5 rat muscle cells used as model for myoblasts. Our results indicate that, in L6C5 cells, reactive oxygen intermediates (ROI) can activate distinct cell pathways leading to cell growth induction and development of resistant phenotype, or to chromosomal aberrations, cell cycle arrest, or cell death. The positive vs. negative effects of H(2)O(2)-altered redox potential in myoblasts are strictly related to the intensity of oxidative stress, likely depending on the types and number of cellular targets involved. Among these, DNA molecules appear to be very sensitive to breakage by H(2)O(2), although DNA damage is not directly responsible for ROI-induced apoptosis in L6C5 rat myoblasts.     

C2-symmetric inhibitors of Plasmodium falciparum plasmepsin II: synthesis and theoretical predictions

A series of C(2)-symmetric compounds with a mannitol-based scaffold has been investigated, both theoretically and experimentally, as Plm II inhibitors. Four different stereoisomers with either benzyloxy or allyloxy P1/P1' side chains were studied. Computational ranking of the binding affinities of the eight compounds was carried out using the linear interaction energy (LIE) method relying on a complex previously determined by crystallography. Within both series of isomers the theoretical binding energies were in agreement with the enzymatic measurements, illustrating the power of the LIE method for the prediction of ligand affinities prior to synthesis. The structural models of the enzyme-inhibitor complexes obtained from the MD simulations provided a basis for interpretation of further structure-activity relationships. Hence, the affinity of a structurally similar ligand, but with a different P2/P2' substituent was examined using the same procedure. The predicted improvement in binding constant agreed well with experimental results.     

alpha1-Adrenoceptor antagonists. 5. Pyridazinone-arylpiperazines. Probing the influence on affinity and selectivity of both ortho-alkoxy groups at the arylpiperazine moiety and cyclic substituents at the pyridazinone nucleus

Our previous work on pyridazinone-arylpiperazine derivatives suggested some structural features that a compound should have to show high affinity and good selectivity for alpha(1) adrenoceptors (AR) with respect to alpha(2)-AR. Accordingly, two classes of new alkoxyphenylpiperazinylheptylpyridazinones were designed and synthesized to evaluate the effect of the alkoxy substituent on affinity and selectivity. As expected, affinity increased with larger alkoxy groups. Affinity values are all comparable with that of the reference compound (prazosin), with the exception of compound 1c found 4.5-fold more active than prazosin.     

Heavy metal interference with growth hormone signalling in trout hepatoma cells RTH-149

We have studied the effects of heavy metals (Hg²⁺, Cu²⁺, Cd²⁺) on growth hormone (GH) activation of tyrosine kinase and Ca²⁺ signaling in the trout (Oncorhynchus mykiss) hepatoma cell line RTH-149. Molecular cloning techniques using primer designed on Oncorhynchus spp. growth hormone receptor (GHR) genes allowed to isolate a highly homologous cDNA fragment from RTH-149 mRNA. Thereafter, cells were analysed by Western blotting or, alternatively, with Ca²⁺ imaging using fura-2/AM. Exposure of cells to ovine GH alone produced a stimulation of the JAK2/STAT5 pathway and intracellular free Ca²⁺ variations similar to what has been observed in mammalian models. Cell pre-exposure to Cu²⁺, Hg²⁺ or Cd²⁺ affected cell response to GH by enhancing (Cu²⁺) or inhibiting (Cd²⁺) the phosphorylation of JAK2 and STAT5. Heavy metals induced the activation of the MAP kinase p38, and pre-exposure to Hg²⁺ or Cu²⁺ followed by GH enhanced the effect of metal alone. Image analysis of fura2-loaded cells indicated that pre-treatment with Hg²⁺ prior to GH produced a considerable increase of the [Ca²⁺]_i variation produced by either element, while using Cu²⁺ or Cd²⁺ the result was similar but much weaker. Data suggest that heavy metals interfere with GH as follows: Hg²⁺ is nearly ineffective on JAK/STAT and strongly synergistic on Ca²⁺ signaling; Cu²⁺ is activatory on JAK/STAT and slightly activatory on Ca²⁺; Cd²⁺ is strongly inhibitory on JAK/STAT and slightly activatory on Ca²⁺; heavy metals could partially activate STAT via p38 independently from GH interaction.Published online: March 2005     

Thermodynamic and kinetic characterization of a beta-hairpin peptide in solution: an extended phase space sampling by molecular dynamics simulations in explicit water

The folding of the amyloidogenic H1 peptide MKHMAGAAAAGAVV taken from the syrian hamster prion protein is explored in explicit aqueous solution at 300 K using long time scale all-atom molecular dynamics simulations for a total simulation time of 1.1 mus. The system, initially modeled as an alpha-helix, preferentially adopts a beta-hairpin structure and several unfolding/refolding events are observed, yielding a very short average beta-hairpin folding time of approximately 200 ns. The long time scale accessed by our simulations and the reversibility of the folding allow to properly explore the configurational space of the peptide in solution. The free energy profile, as a function of the principal components (essential eigenvectors) of motion, describing the main conformational transitions, shows the characteristic features of a funneled landscape, with a downhill surface toward the beta-hairpin folded basin. However, the analysis of the peptide thermodynamic stability, reveals that the beta-hairpin in solution is rather unstable. These results are in good agreement with several experimental evidences, according to which the isolated H1 peptide adopts very rapidly in water beta-sheet structure, leading to amyloid fibril precipitates [Nguyen et al., Biochemistry 1995;34:4186-4192; Inouye et al., J Struct Biol 1998;122:247-255]. Moreover, in this article we also characterize the diffusion behavior in conformational space, investigating its relations with folding/unfolding conditions.     

Design, synthesis, physical and chemical characterisation, and biological interactions of lectin-targeted latex nanoparticles bearing Gd–DTPA chelates: an exploration of magnetic resonance molecular imaging (MRMI)

The physical and chemical parameters involved in the design and synthesis of biospecifically targeted nanoparticulate contrast media for magnetic resonance molecular imaging (MRMI) were explored in this pilot investigation. Latex nanoparticles 100, 400 and 900 nm in diameter were doubly derivatised, first with tomato lectin and then with gadolinium^III-diethylenetriamine pentaacetic acid (Gd-chelates) to target them to epithelial and endothelial glycocalyceal N-glycans and to generate contrast enhancement in magnetic resonance imaging (MRI). After intravenous injection into mice, human placental cotyledons or human Vena saphena magna, contrasty images of the vascular structures were obtained in 1.5 T MRI with spatial resolution 0.1 mm in the imaging plane and 0.6 mm in the z axis, persisting >60 min and resistant to washing out by buffer rinses. Ultrastructural analysis of the nanoparticles revealed the targeting groups at the nanoparticle surfaces and the distribution of the Gd-chelates within the nanoparticles and enabled counts for use in determining relaxivity. The relaxivity values revealed were extremely high, accounting for the strong MR signals observed. Occasionally, nanoparticles larger than 100 nm were seen in close spatial association with disrupted regions of cell membrane or of collagen fibrils in the extracellular matrix. The data suggest that 100-nm nanoparticles generate adequate contrast for MRMI and cause least disruption to endothelial cell surfaces.