Electrochemical Characteristics of Nitroheterocyclic Compounds of Biological Interest. VIII Stability of Nitro Radical Anions from Cyclic Voltammetric Studies

The stability of the one electron addition product of four biologically important nitroheterocyclic compounds has been examined electrochemically. Using cyclic voltammetry the tendency of the nitro radical anion to undergo disproportionation was studied by two methods of analysis. The first was based on determining the voltammetric time-constant required for half of the reduction product, RNO₂, to react further. The second concerned the minimum volume of dimethylformamide which had to be added to the aqueous electrolytic medium to give a specific cyclic voltammetric response. Both methods were found to compare well with the results obtained for RNO₂T stabilities using a theoretically derived procedure for a second order reaction following a charge-transfer step. The use of these alternative approaches for quantifying the reactivity of reduction products is discussed. The time-constant method in particular may be useful in studying complex reaction pathways.     

2,4-Diaminopyrimidine MK2 inhibitors. Part II: Structure-based inhibitor optimization

We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC₅₀ values as low as 19 nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFα production in peripheral human monocytes.     

In vitro propagation of <Emphasis Type="Italic">Pimelea spicata</Emphasis> R.Br (Thymelaeaceae), an endangered species of the Sydney region,Australia

Micropropagation was assessed as an ex situ conservation strategy for the endangered Australian plant Pimelea spicata (Thymelaeaceae). Although regeneration of this species was achieved, several physiological problems were observed and examined. Explants of P. spicata had a higher multiplication rate on MS medium, than on ½ MS, but there was a significantly higher percentage of necrotic shoot tips on the higher salt medium. Increasing calcium concentration and gas exchange exacerbated shoot-tip necrosis. A number of hyperhydrated shoots were produced in all treatments, the cause of which could not be determined, although less hyperhydicity was observed in the ½ MS treatment. Shoots, rooted in vitro on ½ MS in the absence of plant growth regulators, were successfully acclimatised to greenhouse conditions, while direct rooting of microshoots using IBA gel treatment proved unsuccessful. This is the first report of tissue culture propagation of this endangered species.     

The role of glucolipid in the biosynthesis of glycoprotein in Tetrahymena pyriformis

Cell-free preparations from Tetrahymena pyriformis catalyze the incorporation of glucose from UDP-glucose into a glucolipid having properties which are identical to those of other dolichyl phosphoryl sugar derivatives. Kinetic and other experiments have provided evidence that this glucolipid serves as glucose donor for two other types of glucosylated substances, one of which has been tentatively identified as an oligosaccharide lipid and the other a glycoprotein or glycoproteins. In addition, the partially purified glucolipid served as a glucosyl donor to these cell components, suggesting that in this protozoan, at least part of the glycoprotein is synthesized by reactions involving lipid-linked sugars in a manner analogous to that which has been observed in glycoprotein synthesis in mammalian cells.     

Carrying capacity in a heterogeneous environment with habitat connectivity

A large body of theory predicts that populations diffusing in heterogeneous environments reach higher total size than if non‐diffusing, and, paradoxically, higher size than in a corresponding homogeneous environment. However, this theory and its assumptions have not been rigorously tested. Here, we extended previous theory to include exploitable resources, proving qualitatively novel results, which we tested experimentally using spatially diffusing laboratory populations of yeast. Consistent with previous theory, we predicted and experimentally observed that spatial diffusion increased total equilibrium population abundance in heterogeneous environments, with the effect size depending on the relationship between r and K. Refuting previous theory, however, we discovered that homogeneously distributed resources support higher total carrying capacity than heterogeneously distributed resources, even with species diffusion. Our results provide rigorous experimental tests of new and old theory, demonstrating how the traditional notion of carrying capacity is ambiguous for populations diffusing in spatially heterogeneous environments.     

DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5′-CCA AAG₆ XA₈C CGG G-3′, where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4′-oxidized abasic sites. Biochemistry, 46, 3096–3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3′ to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.     

ATP synthase complex of Plasmodium falciparum: dimeric assembly in mitochondrial membranes and resistance to genetic disruption

The rotary nanomotor ATP synthase is a central player in the bioenergetics of most organisms. Yet the role of ATP synthase in malaria parasites has remained unclear, as blood stages of Plasmodium falciparum appear to derive ATP largely through glycolysis. Also, genes for essential subunits of the F(O) sector of the complex could not be detected in the parasite genomes. Here, we have used molecular genetic and immunological tools to investigate the localization, complex formation, and functional significance of predicted ATP synthase subunits in P. falciparum. We generated transgenic P. falciparum lines expressing seven epitope-tagged canonical ATP synthase subunits, revealing localization of all but one of the subunits to the mitochondrion. Blue native gel electrophoresis of P. falciparum mitochondrial membranes suggested the molecular mass of the ATP synthase complex to be greater than 1 million daltons. This size is consistent with the complex being assembled as a dimer in a manner similar to the complexes observed in other eukaryotic organisms. This observation also suggests the presence of previously unknown subunits in addition to the canonical subunits in P. falciparum ATP synthase complex. Our attempts to disrupt genes encoding β and γ subunits were unsuccessful, suggesting an essential role played by the ATP synthase complex in blood stages of P. falciparum. These studies suggest that, despite some unconventional features and its minimal contribution to ATP synthesis, P. falciparum ATP synthase is localized to the parasite mitochondrion, assembled as a large dimeric complex, and is likely essential for parasite survival.     

Scanning N-glycosylation mutagenesis of membrane proteins

N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc₃Man₉GlcNAc₂-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12–14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.     

Synthesis, in vitro, and in vivo antibacterial activity of nocathiacin I thiol-Michael adducts

The synthesis and antibacterial activity of a series of nocathiacin I derivatives (4-20) containing polar water solubilizing groups is described. Thiol-Michael adducts containing acidic polar groups have reduced antibacterial activity whereas those with basic polar groups have retained very good antibacterial activity.