Theoretical calculations, synthesis and base pairing properties of oligonucleotides containing 8-amino-2'-deoxyadenosine

Theoretical calculations on double and triple helices containing 8-amino-2'-deoxyadenosine were made to analyze the possible differences in base pairing properties between 8-aminoadenine and adenine. These calculations indicate a strong preferential stabilization of the triplex over the duplex when adenine is replaced by 8-aminoadenine. In addition, a protected phosphoramidite derivative of 8-amino-2'-deoxyadenosine was prepared for the introduction of 8-aminoadenine into synthetic oligonucleotides using the phosphite-triester approach. DNA triple helical structures are normally observed at acidic pH. However, when oligonucleotides carrying 8-aminoadenine are used, very stable triple helical structures can be observed even at neutral pH. Biological applications of triple helices could benefit from the use of 8-aminoadenine derivatives.     

The epitheliome: agent-based modelling of the social behaviour of cells

We have developed a new computational modelling paradigm for predicting the emergent behaviour resulting from the interaction of cells in epithelial tissue. As proof-of-concept, an agent-based model, in which there is a one-to-one correspondence between biological cells and software agents, has been coupled to a simple physical model. Behaviour of the computational model is compared with the growth characteristics of epithelial cells in monolayer culture, using growth media with low and physiological calcium concentrations. Results show a qualitative fit between the growth characteristics produced by the simulation and the in vitro cell models.     

Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells

Placental growth factor (PlGF) competes with vascular endothelial growth factor (VEGF) for binding to VEGF receptor (VEGFR)-1 but does not bind VEGFR2. Experiments show that PlGF can augment the response to VEGF in pathological angiogenesis and in models of endothelial cell survival, migration, and proliferation. This synergy has been hypothesized to be due to a combination of the following: signaling by PlGF through VEGFR1 and displacement of VEGF from VEGFR1 to VEGFR2 by PlGF, causing increased signaling through VEGFR2. In this study, the relative contribution of PlGF-induced VEGF displacement to the synergy is quantified using a mathematical model of ligand-receptor binding to examine the effect on ligand-receptor complex formation of VEGF and PlGF acting together. Parameters specific to the VEGF-PlGF system are used based on existing data. The model is used to simulate in silico a specific in vitro experiment in which VEGF-PlGF synergy is observed. We show that, whereas a significant change in the formation of endothelial surface growth factor-VEGFR1 complexes is predicted in the presence of PlGF, the increase in the number of VEGFR2-containing signaling complexes is less significant; these results were shown to be robust to significant variation in the kinetic parameters of the model. Synergistic effects observed in that experiment thus appear unlikely to be due to VEGF displacement but to a shift from VEGF-VEGFR1 to PlGF-VEGFR1 complexes and an increase in total VEGFR1 complexes. These results suggest that VEGFR1 signaling can be functional in adult-derived endothelial cells.     

S-allylmercaptocysteine scavenges hydroxyl radical and singlet oxygen <Emphasis Type="Italic">in vitro</Emphasis> and attenuates gentamicin-induced oxidative and nitrosative stress and renal damage <Emphasis Type="Italic">in vivo</Emphasis>

Background  

Oxidative and nitrosative stress have been involved in gentamicin-induced nephrotoxicity. The purpose of this work was to study the effect of S-allylmercaptocysteine, a garlic derived compound, on gentamicin-induced oxidative and nitrosative stress and nephrotoxicity. In addition, the in vitro reactive oxygen species scavenging properties of S-allylmercaptocysteine were studied.     

Practical aspects of using the microbatch method in screening conditions for protein crystallization

The microbatch technique is a simple and efficient method for screening for protein crystallization conditions both by hand and using automated systems. Many of the problems associated with more commonly used methods such as vapour diffusion can be overcome using this method. Despite its promise microbatch has not been widely utilized as a viable screening procedure. This review aims to describe the method from a practical point of view with an emphasis on screening crystallization conditions, outlining current progress and discussion of the advantages compared to other methods.     

The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors

Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.     

The ecdysone inducible gene expression system: unexpected effects of muristerone A and ponasterone A on cytokine signaling in mammalian cells

Inducible gene expression systems in mammalian cells have been shown to be valuable processes to study the specific function of a protein in differentiation, proliferation or survival/apoptosis. Usually, these systems use as inducible reagents, compounds that are thought to be neutral and devoid of physiological or biologically undesirable effects in mammalian cells. We recently used the ecdysone inducible gene expression system in hematopoietic cells and found that the two inducer analogs of ecdysone, muristerone A and ponasterone A, altered the signaling pathways induced by IL-3 in the pro-B cell-line, Ba/F3. Indeed, we showed that these two analogs potentiate the IL-3-dependent activation of the PI 3-kinase/Akt pathway, which could ultimately interfere with the growth, and/or survival of these cells.     

Synthesis of a new 5'-O-triphosphate analog of 5-methyl 2'-O-deoxycytidine. Preliminary in vitro labelling for non-radioactive detection of DNA

We report the synthesis of the triphosphate of 5-methyl 4-N-[6-(p-bromobenzamido)hex-1-yl]-2'-O-deoxycytidine 3A. We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-[n-[6-(p-bromobenzamido) caproyl amino]alk-1-yl]-2'-deoxycytidine compounds, and confirmed their presence by 1H-NMR studies. In vitro DNA labeling with modified nucleotides is preliminarily evaluated.     

Slit antagonizes netrin-1 attractive effects during the migration of inferior olivary neurons

Inferior olivary neurons (ION) migrate circumferentially around the caudal rhombencephalon starting from the alar plate to locate ventrally close to the floor-plate, ipsilaterally to their site of proliferation. The floor-plate constitutes a source of diffusible factors. Among them, netrin-1 is implied in the survival and attraction of migrating ION in vivo and in vitro. We have looked for a possible involvement of slit-1/2 during ION migration. We report that: (1) slit-1 and slit-2 are coexpressed in the floor-plate of the rhombencephalon throughout ION development; (2) robo-2, a slit receptor, is expressed in migrating ION, in particular when they reach the vicinity of the floor-plate; (3) using in vitro assays in collagen matrix, netrin-1 exerts an attractive effect on ION leading processes and nuclei; (4) slit has a weak repulsive effect on ION axon outgrowth and no effect on migration by itself, but (5) when combined with netrin-1, it antagonizes part of or all of the effects of netrin-1 in a dose-dependent manner, inhibiting the attraction of axons and the migration of cell nuclei. Our results indicate that slit silences the attractive effects of netrin-1 and could participate in the correct ventral positioning of ION, stopping the migration when cell bodies reach the floor-plate.