Ribosomal frameshifting in response to hypomodified tRNAs in Xenopus oocytes

We used Xenopus oocytes as an intracellular system to study ribosomal frameshifting. Microinjection of oocytes with a construct encoding the naturally occurring UUU or AAC codon at the frameshift site demonstrated that the level of frameshifting was similar or lower than found normally in retroviral frameshifting in mammalian cells, suggesting that oocytes are a reliable system to study this event. Phenylalanine (Phe) or asparagine (Asn) tRNAs with and without the highly modified wyebutoxine (Y) or queuosine (Q) base, respectively, were microinjected to assess their ability to promote frameshifting. inhibited the level of frameshifting, while promoted frameshifting providing evidence that the hypomodified form does not act only to enhance frameshifting, but is an essential requirement. Both and were used indiscriminately in frameshifting, whether the frameshift site contained the wild-type AAC, or the mutant AAU codon, suggesting that Q base modification status does not influence this process.     

Applicability of new spin trap agent, 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide, in biological system

Electron spin resonance using spin-trapping is a useful technique for detecting direct reactive oxygen species, such as superoxide (). However, the widely used spin trap 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) has several fundamental limitations in terms of half-life and stability. Recently, the new spin trap 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) was developed by us. We evaluated the biological applicability of DPhPMPO to analyze in both cell-free and cellular systems. DPhPMPO had a larger rate constant for and formed more stable spin adducts for than DMPO in the xanthine/xanthine oxidase (X/XO) system. In the phorbol myristate acetate-activated neutrophil system, the detection potential of DPhPMPO for was significantly higher than that of DMPO (k_DMPO = 13.95 M⁻¹ s⁻¹, k_DPhPMPO = 42.4 M⁻¹ s⁻¹). These results indicated that DPhPMPO is a potentially good candidate for trapping in a biological system.     

Mixed-valence porphyrin π-cation radical derivatives: Electrochemical investigations

The electrochemistry of [Cu(OEP)] and [Ni(OEP)] are compared with the mixed-valence π-cations and . These electrochemical studies, carried out with cyclic voltammetry and hydrodynamic voltammetry, show that the mixed valence π-cations have distinct electrochemical properties, although the differences between the [M(OEP)]^+/0 and processes are subtle.     

Crystal structures and magnetic properties of Ni-bisdithiolene complexes with decamethylmetallocenium cations

Three new compounds of formula (1), (2), and (3) have been synthesised, and structurally and magnetically characterised (dmit²⁻ = 1,3-dithiol-2-thione-4,5-dithiolato; dmid²⁻ = 1,3-dithiol-2-one-4,5-dithiolato). Their structural features and magnetic behaviours are compared with those of and . The result of this is that the interactions between the Ni(dmit)₂ units in 1 are of ferromagnetic-type, as suggested previously for . The change from acetonitrile to acetone when going from to 2 results in stronger ferromagnetic intermolecular interactions. This better cooperativity is due to a significant increase of the number of contacts between the various moieties within the . On the contrary, the inclusion of larger solvents such as benzonitrile in this type of complexes results in a totally different structural arrangement, which leads to an antiferromagnetic behaviour for 3.     

NAD(P)H- and superoxide-dependent nitric oxide degradation by rat liver mitochondria

Mitochondria consume nitric oxide (NO) mainly through reaction with superoxide anion (). Here, we analyzed the sources for NO degradation by isolated rat liver mitochondria. Electron leakage from complex III and reverse electron transport to complex I accounted for -dependent NO degradation by mitochondria in the presence of a protonmotive force. Mitochondria incubated with NAD(P)H also presented intense generation and NO degradation rates that were insensitive to respiratory inhibitors and abolished after proteinase treatment. These results suggest that an outer membrane-localized NAD(P)H oxidase activity, in addition to the electron leakage from the respiratory chain, promotes -dependent NO degradation in rat liver mitochondria.     

Inhibition by purine nucleotides of the release of reactive oxygen species from muscle mitochondria: indication for a function of uncoupling proteins as superoxide anion transporters

Release of reactive oxygen species (ROS), measured as the sum of hydrogen peroxide (H₂O₂) and superoxide anion radical (), from respiring rat heart and skeletal muscle mitochondria was significantly decreased by millimolar concentrations of GTP or GDP. Attempts to differentiate between the two forms of ROS showed that the release of rather than that of H₂O₂ was affected. Meanwhile, intramitochondrial ROS accumulation, measured by inactivation of aconitase, increased. These results suggest that guanine nucleotides inhibit the release of from mitochondria. As these nucleotides are known inhibitors of uncoupling proteins (UCPs), it is proposed that UCPs may function as carriers of , thus enabling its removal from the matrix compartment.     

Curcumin dramatically enhances retinoic acid-induced superoxide generating activity via accumulation of p47-phox and p67-phox proteins in U937 cells

The membrane bound cytochrome b558 composed of large gp91-phox and small p22-phox subunits, and cytosolic proteins p40-, p47- and p67-phox are important components of superoxide ()-generating system in phagocytes and B lymphocytes. A lack of this system in phagocytes is known to cause serious life-threatening infections. Here, we describe that curcumin, a polyphenol responsible for the yellow color of curry spice turmeric, dramatically activates the -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and curcumin, the -generating activity increased more than 4-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and curcumin slightly enhanced gene expressions of the five components compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and curcumin caused remarkable accumulation of protein levels of p47-phox (to 7-fold) and p67-phox (to 4-fold) compared with those of the RA-treatment alone. These results suggested that curcumin dramatically enhances RA-induced -generating activity via accumulation of cytosolic p47-phox and p67-phox proteins in U937 cells. Therefore, it should have the potential as an effective modifier in therapy of leukemia and/or as an immunopotentiator.