Radical trapping properties of imidazolyl nitrones

The ability of ten imidazolyl nitrones to directly scavenge free radicals (R(*)) generated in polar ((*)OH, O(*)(2)(-), SO(*)(3)(-) cysteinyl, (*)CH(3)) or in apolar (CH(3)-(*)CH-CH(3)) media has been studied. When oxygen or sulfur-centered radicals are generated in polar media, EPR spectra are not or weakly observed with simple spectral features. Strong line intensities and more complicated spectra are observed with the isopropyl radical generated in an apolar medium. Intermediate results are obtained with (*)CH(3) generated in a polar medium. EPR demonstrates the ability of these nitrones to trap radicals to the nitrone C(alpha) atom (alpha radical adduct) and to the imidazol C(5) atom (5-radical adduct). Beside the nucleophilic addition of the radical to the C(alpha) atom, the EPR studies suggest a two-step mechanism for the overall reaction of R(*) attacking the imidazol core. The two steps seem to occur very fast with the (*)OH radical obtained in a polar medium and slower with the isopropyl radical prepared in benzene. In conclusion, imidazolyl nitrones present a high capacity to trap and stabilize carbon-centered radicals.     

Long-term gene expression in dividing and nondividing cells using SV40-derived vectors

Among the goals of gene therapy is long-term expression of delivered transgenes. Recombinant Tag-deleted SV40 vectors (rSV40s) are especially well suited for this purpose. rSV40s deliver transgene expression that endures for extended periods of time in tissue culture and in vivo, in both dividing and nondividing cells. These vectors are particularly effective in transducing some cell types that have been almost unapproachable using other gene delivery systems, such as quiescent hematopoietic progenitor cells and their differentiated derivatives. Other cellular targets include neurons, brain microglia, hepatocytes, dendritic cells, vascular endothelium, and others. Because rSV40s do not elicit neutralizing antibodies they are useful for in vivo gene delivery in settings where more than one administration may be desirable. The key characteristics of these vectors include their high production titers and therefore suitability for large cell pools, effectiveness in delivering intracellular proteins, and untranslated RNAs, maintenance of transgene expression at constant levels for extended times, suitability for constitutive or conditional promoters and for combinatorial gene delivery and ability to integrate into genomes of both dividing and nondividing cells.     

Comparative labelling of α1-acid glycoprotein by (3H)-borohydride or (125I)-iodide for use in a radioimmunoassay

The labelling of α₁-acid glycoprotein (AGP) with (³H)-sodium borohydride was compared to the labelling with (¹²⁵I)-sodium iodide by the chloramine T method in view to its use in a radioimmunoassay. The tritium labelling allowed to reach a high specific radioactivity similar to that obtained with iodide ((³H)-AGP: 29.8 mCi/mg; (¹²⁵I)-AGP: 30.5 mCi/mg). Each mole of sialic acid residue of AGP contains one atom of tritium. The stability of (³H)-AGP was better than that of (¹²⁵I)-AGP as indicated by its immunoreactivity as a function of time. Immunoreactivities and standard curves were similar for the two tracers but affinity of antiserum was higher for (¹²⁵I)-AGP than for (³H)-AGP. Tritium labelling by (³H)-borohydride will be very useful for glycoprotein antigens which cannot be labelled with (¹²⁵I)-iodide.     

Laser Raman investigation of intact single muscle fibers on the state of water in muscle tissue

Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nulilus). The spectra in the region of the O-H (or O-²H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of “structured” intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 ± 0.01 for H₂O and 1.05 ± 0.03 for ²H₂O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state at ?5°C, which corresponds to 1 g of water per of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.     

Involvement of FtsZ in coupling of nucleoid separation with septation

The cell-cycle parameters of an Escherichia coli strain expressing essential division gene ftsZ at one-fifth of its normal level, because of antisense regulation by DicF RNA, have been analysed. Inhibition of FtsZ expression affects neither the generation time nor the replication initiation mass, the C period, or the constriction period, but it does dramatically retard the initiation of constriction relative to replication termination. Separation of the nucleoids is equally postponed, indicating that division is not coupled to termination of replication, but to partitioning. The severe inhibition of nucleoid separation by DicF RNA, and its suppression by overproduction of FtsZ, suggest a role for FtsZ in the control of separation, and consequently in the coupling of separation and division. We suggest that the normal pattern of nucleoid separation previously found in cells deficient in ftsZ function was a consequence of the loss of a negative effect exerted by FtsZ on separation. In agreement with this view, we find that nucleoid separation is temporarily inhibited after arrest of FtsZ synthesis, but is later resumed as FtsZ is further diluted into the elongating filaments.     

Effects of the New Arginase Inhibitor N-Hydroxy-nor- -Arginine on NO Synthase Activity in Murine Macrophages

In stimulated murine macrophage, arginase and nitric oxide synthase (NOS) compete for their common substrate, -arginine. The objectives of this study were (i) to test the new α-amino acid N^ω-hydroxy-nor- -arginine (nor-NOHA) as a new selective arginase inhibitor and (ii) to elucidate the effects of arginase inhibition on -arginine utilization by an inducible NOS. Nor-NOHA is about 40-fold more potent than N^ω-hydroxy- -arginine (NOHA), an intermediate in the -arginine/NO pathway, to inhibit the hydrolysis of -arginine to -ornithine catalyzed by unstimulated murine macrophages (IC₅₀ values 12 ± 5 and 400 ± 50 μM, respectively). Stimulation of murine macrophages with interferon-γ and lipopolysaccharide (IFN-γ + LPS) results in clear expression of an inducible NOS (iNOS) and to an increase in arginase activity. Nor-NOHA is also a potent inhibitor of arginase in IFN-γ + LPS-stimulated macrophage (IC₅₀ value 10 ± 3 μM). In contrast to NOHA, nor-NOHA is neither a substrate nor an inhibitor for iNOS and it appears as a useful tool to study the interplays between arginase and NOS. Inhibition of arginase by nor-NOHA increases nitrite and -citrulline accumulation for incubation times higher than 12 h, under our conditions. Our results allow the determination of the kinetic parameters of the two competitive pathways and the proposal of a simple model which readily explains the differences observed between experiments. This model readily accounts for the observed effects and should be useful to predict the consequences of arginase inhibition in the presence of an active NOS on -arginine availability.     

Reactive oxygen species generation and antioxidant systems in plant mitochondria

In living cells, reactive oxygen species (ROS) play a key role in signaling but these compounds can also damage macromolecules. As in other compartments, the mitochondrial ROS concentrations need to be tightly controlled. Plant mitochondria contain several antioxidant systems that are not only able to scavenge ROS and limit their production but also to repair damages to macromolecules and possibly to serve as redox sensors. They comprise ascorbate- and glutathione-dependent pathways as well as systems based on thioredoxin (TRX)- and glutaredoxin (GRX)-like molecules. This review describes the various mitochondrial redox pathways for ROS control in plants with special emphasis on the poorly studied GRX and TRX systems and provides perspectives for future research in this area.     

PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes

Exosomes are small vesicles secreted by different immune cells and which display anti-tumoral properties. Stimulation of RBL-2H3 cells with ionomycin triggered phospholipase D2 (PLD2) translocation from plasma membrane to intracellular compartments and the release of exosomes. Although exosomes carry the two isoforms of PLD, PLD2 was enriched and specifically sorted on exosomes when overexpressed in cells. PLD activity present on exosomes was clearly increased following PLD2 overexpression. PLD2 activity in cells was correlated to the amount of exosome released, as measured by FACS. Therefore, the present work indicates that exosomes can vehicle signaling enzymes.