Detection of Oxidation Products of 5-Methyl-2′-Deoxycytidine in Arabidopsis DNA

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-29-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5- HmdC, 5-formyl-29-deoxycytidine (5-FodC), 5-carboxyl-29-deoxycytidine (5-CadC), 5-hydroxymethyl-29-deoxyuridine (5- HmdU), and the (59S) diastereomer of 8,59-cyclo-29-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 106 nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tetfamily dioxygenase enzymes in Arabidopsis.

Cloning and Identification of a Complete cDNA Coding for a Bactericidal and Antitumoral Acidic Phospholipase A(2) from Bothrops jararacussu Venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bioactive Metabolites Produced by Penicillium sp.1 and sp.2, Two Endophytes Associated with Alibertia macrophylla (Rubiaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteinase-activated receptor-2 (PAR2) agonist causes periodontitis in rats

Proteinase-activated receptor-2 (PAR2) is a G-protein-coupled receptor that mediates cellular responses to extracellular proteinases. Since PAR2 is expressed by oral epithelial cells, osteoblasts, and gingival fibroblasts, where its activation releases interleukin-8, we hypothesized that PAR2 activation may participate in periodontal disease in vivo. We investigated the role of PAR2 activation in periodontal disease in rats. Radiographic and enzymatic (myeloperoxidase) analysis revealed that topical application of PAR2 agonist causes periodontitis but also exacerbates existing periodontitis, leading to significant alveolar bone loss and gingival granulocyte infiltration. Inhibition of matrix metalloproteinase (MMP) and cyclo-oxygenase (COX) decreased PAR2 agonist-induced periodontitis. More specifically, the overexpression of COX-1, COX-2, MMP-2, and MMP-9 in gingival tissues suggests that they are involved in PAR 2-induced periodontitis. In conclusion, PAR2 agonist causes periodontitis in rats through a mechanism involving prostaglandin release and MMP activation. Inhibition of PAR2 may represent a novel approach to modulate host response in periodontitis.

Effects of arachidonic acid upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells.

1. The effects of arachidonic acid upon the volume-sensitive Cl- current present in cultured osteoblastic cells (ROS 17/2.8) was studied using the whole-cell patch-clamp technique. 2. Arachidonate produced two distinct phases of inhibition, a rapid phase occurring within 10-15 s of application preceding a slower phase that occurred 2 min after onset of arachidonate superfusion. Accompanying the slower inhibitory phase was an acceleration of the time-dependent inactivation exhibited by the current at strongly depolarized potentials (> + 50 mV). The half-maximal inhibitory concentrations (IC50) were 177 +/- 31 and 10 +/- 4 microM for the two phases respectively. 3. Arachidonate was still effective in the presence of inhibitors of cyclo-oxygenase (indomethacin, 10 microM), lipoxygenase (nordihydroguaretic acid, 10-100 microM) and cytochrome P450 (SKF525A, 100 microM; ethoxyresorufin, 10 microM; metyrapone, 500 microM; piperonyl butoxide, 500 microM; cimetidine, 1 mM). The effects of arachidonate could not be produced by another cis unsaturated fatty acid, oleic acid. 4. Measurements of cell volume showed that arachidonate effectively inhibited the regulatory volume decrease elicited by ROS 17/2.8 cells in response to a reduction in extracellular osmolarity. 5. It is concluded that the volume-sensitive Cl- conductance in ROS 17/2.8 cells is directly modulated by arachidonate and may represent a physiological mechanism by which volume regulation can be controlled in these cells.

A combinatorial approach to the chemical synthesis and biological evaluation of 3,4,5-trisubstiituted furan-2(5H)-ones

Many important natural products contain the furan-2(5H)-one structure. The structure of this molecule lends itself to manipulation using combinatorial techniques due to the presence of more than one site for the attachment of different suhstituents. By developing different reaction schemes at the three sites available for attachment on the furan-2(5H)-one scaffold, combinatorial chemistry techniques can be employed to assemble libraries of novel furan 2(5H)-ones. These libraries can then be entered into various biological screening programmes. This approach will enable a vast diversity or compounds to be examined, in the hope or finding new biologically active Iead structures. The work in this thesis has investigated the potential that combinatorial chemistry has in the quest for new biologically active lead structures based on the furan-2(5H)-one structure. Different reactions were investigated with respect to their suitability for inclusion in a library. Once sets of reactions at the various sites had been established, the viability of these reactions in the assembly of combinatorial libraries was investigated. Purification methods were developed, and the purified products entered into suitable biological screening tests. Results from some of these tests were optimised using structure activity relationships, and the resulting products re-screened. The screening tests performed were for anticancer and antimicrobial activity, cholecystokinin (CCK-B) antagonism and anti-inflammatory activity (in the quest for novel cyclo-oxygenase (COX-2) selective non-steroidal anti-inflammatory drugs). It has been shown that many reactions undergone by the furan-2(5H)-one structure are suitable for the assembly of a combinatorial library. Investigation into the assembly of different libraries has been carried out with initial screening results included. From this work, further investigation into combinatorial library assembly and structure activity relationships of screened reaction products can be undertaken.

(SP,SP)-(–)-(E)-1,2-Bis(methyl­phenyl­phosphino­yl)ethene

The title compound, C(16)H(18)O(2)P(2), possesses two stereogenic P atoms and shows a distorted s-cis conformation of each O=P-C=C moiety. This has been suggested on the basis of the stereochemical result of 1,3-dipolar cyclo-additions with nitro-nes and is now confirmed by the present crystal structure analysis. There are two crystallographically independent molecules in the asymmetric unit.