Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.     

Type 2 cytotoxic T lymphocytes modulate the activity of dendritic cells toward type 2 immune responses

Activated CD8+ T cells can differentiate into type 1 (Tc1) cells, producing mainly IFN-gamma, and type 2 (Tc2) cells, producing mostly IL-4, IL-5, and IL-10. Tc1 cells are potent CTL involved in the defense against intracellular pathogens and cancer cells. The role of Tc2 cells in the immune response is largely unknown, although their presence in chronic infections, cancer, and autoimmune diseases is associated with disease severity and progression. Here, we show that mouse Tc2 cells modify, through a cell-to-cell contact mechanism, the function of bone marrow-derived dendritic cells (DC). Indeed, Tc2-conditioned DC displayed a reduced expression of MHC class II and costimulatory molecules, produced IL-10 instead of IL-12, and favored the differentiation of both naive CD4+ and CD8+ T cells toward type 2 cells in the absence of added polarizing cytokines. The novel function for Tc2 cells suggests a type 2 loop in which Tc2 cells modify DC function and favor differentiation of naive T cells to type 2 cells. The type 2 loop may at least in part explain the unexpected high frequency of type 2 cells during a chronic exposure to the Ag.     

Effect of temperature on the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases

A study on the response of the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases to temperature variations has been carried out. The thermodynamic properties of the heat inactivation process and of the reaction with the substrates glutamate and α-ketoglutarate have been investigated. The heat inactivation of nuclear glutamate dehydrogenase proceeds at a faster rate than that of the mitochondrial enzyme in the temperature range 40–51 °C; the enthalpy of activation of the inactivation process is higher and the entropy is almost double, compared to the values of mitochondrial glutamate dehydrogenase. The effect of temperature on the maximal velocity shows that, with both glutamate and α-ketoglutarate, the enthalpy of activation with nuclear glutamate dehydrogenase is double and the decrease in entropy almost half of the values of the mitochondrial enzyme. The variation of the apparent K_m with temperature shows a decrease of the affinity of both enzymes for glutamate, with no major difference in the thermodynamic properties of the reaction. With α-ketoglutarate, on the other hand, the affinity of nuclear glutamate dehydrogenase decreased, whereas that of the mitochondrial enzyme increased with temperature. The process is therefore exothermic with the former enzyme, endothermic with the latter; furthermore, it occurs with a decrease in enthropy with nuclear glutamate dehydrogenase, but with a large increase with the mitochondrial enzyme. The studies on the effect of temperature on the activity were carried out in the range 20–44 °C.     

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.     

Pachybrachis sassii, a new species from the Mediterranean Giglio Island (Italy) (Coleoptera, Chrysomelidae, Cryptocephalinae)

Pachybrachis sassii, new species is described from Giglio Island, of the Tuscan Archipelago (Italy). The new species belongs to the nominotypical subgenus and is closely related to Pachybrachis salfii Burlini, 1957, from which it differs in the shape of the median lobe of the aedeagus and in the pattern of the yellow raised spots on the elytra and pronotum. Ecological observations are made. The neotype of Pachybrachis salfii from Colloreto, Monte Pollino (Italy) is designated.     

The intercalation to DNA of bipyridyl complexes of platinum(II) with thioureas

The non-covalent interaction of the complexes [Pt(bpy)(R,R'NCSNR',R')(2)]Cl(2) (bpy=2,2'-bipyridine; R=R'=R'=R'=H; R=Me, R'=R'=R'=H; R=n-Bu, R'=R'=R'=H; R=p-tolyl, R'=R'=R'=H; R=Et, R'=H, R'=Et, R'=H) with calf thymus DNA has been studied at pH 7 and 25 degrees C. The processes give rise to: (i) reversible bathochromic shifts and strong hypochromicity of the absorption bands of the complexes, (ii) induced circular dichroism and (iii) an increase both in the melting temperature and viscosity of the DNA comparable to that observed for other well known metallointercalators. The binding constants, K(B), have been determined spectrophotometrically using the McGhee von Hippel equation. Plot of logK(B) vs -log[Na(+)] for the complex with unsubstituted thiourea gives a straight line with a slope value close to that expected for a dicationic intercalator. The binding affinity of the various complexes for DNA is independent of the thiourea nature; this suggests that the intercalation occurs through stacking of the bpy moiety while the ancillary ligands lie outside the nucleobases far away from the sugar phosphate backbone. The data show also that the electronic effects of the ligand substituents are not transmitted to the intercalating unit.