Nuclear Localization Signals Enhance Germline Transmission of a Transgene in Zebrafish

We report that cytoplasmic injection into zebrafish eggs of 104 copies of plasmid DNA complexed to nuclear localization signal (NLS) peptides, as compared to 106 copies of naked DNA, increased nuclear uptake of transgene DNA early during embryo development and enhanced transgene integration frequency into the germline of founders. Monitoring the dynamics of nuclear uptake of DNA-NLS complexes by fluorescence in situ hybridization (FISH) of interphase nuclei indicates that NLS enhances both the proportion of nuclei importing DNA during early embryo development, and the amount of DNA imported by individual nuclei. The use of NLS increases the proportion of germline transgenic founders from 14 to 43% (P < 0.01) as assessed by polymerase chain reaction analysis of F1s. From germline transgenic DNA-NLS-injected founders, 47% transgenic F1s are obtained in wild-type crosses, as opposed to 6% from naked DNA-injec...     

In vivo simulation of human pharmacokinetics in the rabbit

The evaluation of drugs in vivo is often based on experimental models using small animals such as mice, rats and rabbits. However, these models could be improved to correspond more closely to the human situation if the pharmacokinetics of the drugs tested in animals were similar to that observed in humans. The use of a computer-controlled pump allowing an adequate flow of tobramycin and amikacin to be infused into rabbits enabled us to simulate the human pharmacokinetics of these antibiotics in vivo in this study. The function defining the rate of infusion required to perform the simulation of an intravenous bolus was first determined generally and symbolically for linear pharmacokinetic models independently from the number of compartments involved. The practical simulation of a decreasing monoexponential serum profile with a half-life of 2 h (one-compartment model for the human pharmacokinetics of aminoglycosides) was then studied for tobramycin and amikacin on the basis of a two-compartment model in the animal. The kinetics obtained had an apparent elimination half-life of 1.97 and 1.86 h, respectively. Linearity of the semilogarithmic regressions of the profiles obtained was quite sound. Finally, an a posteriori analysis of the pharmacokinetic model and its parameters is proposed on the basis of the results obtained after simulation.     

Effects of testosterone on Reelin expression in the brain of male European starlings

Reelin, a large glycoprotein defective in reeler mice, is assumed to determine the final location of migrating neurons in the developing brain. We studied the expression of Reelin in the brain of adult male European starlings that had been treated or not with exogenous testosterone. Reelin-immunoreactive cells and fibers were widely distributed in the forebrain including areas in and around the song control nucleus, HVC. No labeling was detected in other song control nuclei with the exception of nucleus uvaeformis, which was delineated by a dense cluster of Reelin-immunoreactive perikarya. Reelin is thus expressed in areas incorporating new neurons in adulthood, such as HVC. Reelin expression was sharply decreased by testosterone in HVC, nucleus uvaeformis and dorsal thalamus but not in other brain regions. These results are consistent with the idea that seasonal changes in Reelin expression modulate the incorporation of neurons within HVC. The presence of Reelin in other brain areas that do not incorporate new neurons in adulthood indicates, however, that this protein must play other unrelated roles in the adult brain. Additional studies should now be carried out to determine the specific role played by this protein in the seasonal plasticity of the songbird brain.     

Angiogenic synergism,vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2

The establishment of functional and stable vascular networks is essential for angiogenic therapy. Here we report that a combination of two angiogenic factors, platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2, synergistically induces vascular networks, which remain stable for more than a year even after depletion of angiogenic factors. In both rat and rabbit ischemic hind limb models, PDGF-BB and FGF-2 together markedly stimulated collateral arteriogenesis after ligation of the femoral artery, with a significant increase in vascularization and improvement in paw blood flow. A possible mechanism of angiogenic synergism between PDGF-BB and FGF-2 involves upregulation of the expression of PDGF receptor (PDGFR)-alpha and PDGFR-beta by FGF-2 in newly formed blood vessels. Our data show that a specific combination of angiogenic factors establishes functional and stable vascular networks, and provides guidance for the ongoing clinical trials of angiogenic factors for the treatment of ischemic diseases.     

Anaerobic digestion of Ulva sp. 1. Relationship between Ulva composition and methanisation

Ulva often represents the main component of mass algal growths, and its composition and degradability make it a relatively good methanisation substrate. In ‘green tides’ Ulva sp. from Brittany, the low content oflignin-type components (polyphloroglucinols: 1.3% dry weight), and the large hemicellulosic fraction (9% dry weight) favour the substrate's accessibility to enzymes. Anaerobic degradation with a batch orcompletely stirred system is technically possible. However, the methane yield reached only 0.20 m³ kg⁻¹ volatile solids and the epuration rate 50% volatile solids in experiments in batch or completely stirred reactors. More generally, methanisation comes up against various practical obstacles: seasonal growth of Ulva, low density of alga in suspension for loading the digester, high S concentration leading to the production of a biogas with a high H2S content, and, finally, the existence of a refractory or slowly degradable part, which requires a compromise between productivity and biological yield. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of a hydrogen peroxide signalling pathway in the control of light-dependent germination in Arabidopsis

Germination is controlled by external factors, such as temperature, water, light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and demonstrated that ROS in addition to their role as cell wall loosening factor are essential signalling molecules in this process. Indeed, we showed that ROS are released prior to endosperm rupture, that their production is required for germination, and that class III peroxidases, as ROS level regulators, colocalized with ROS production. Among ROS, H₂O₂ modifies, during germination early steps, the expression of genes encoding for enzymes regulating ROS levels. This pointing out a regulatory feedback loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels. We followed the early visible steps of germination (testa and endosperm rupture) in Arabidopsis seeds treated by specific ROS scavengers and as the light quality perception is necessary for a regular germination, we examined the germination in presence of exogenous H₂O₂ in different light qualities. H₂O₂ either promoted germination or repressed germination depending on the light wavelengths, showing that H₂O₂ acts as a signal molecule regulating germination in a light-dependent manner. Using photoreceptors null-mutants and GA-deficient mutants, we showed that H₂O₂-dependent promotion of germination relies on phytochrome signalling, but not on cryptochrome signalling, and that ROS signalling requires GA signalling.     

Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased Susceptibility to Pentamidine and Melarsoprol

The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.     

Two msbB genes encoding maximal acylation of lipid A are required for invasive Shigella flexneri to mediate inflammatory rupture and destruction of the intestinal epithelium

Shigella flexneri is a Gram-negative pathogen that invades and causes inflammatory destruction of the human colonic epithelium, thus leading to bloody diarrhea and dysentery. A type III secretion system that delivers effector proteins into target eukaryotic cells is largely responsible for cell and tissue invasion. However, the respective role of this invasive phenotype and of lipid A, the endotoxin of the Shigella LPS, in eliciting the inflammatory cascade that leads to rupture and destruction of the epithelial barrier, was unknown. We investigated whether genetic detoxification of lipid A would cause significant alteration in pathogenicity. We showed that S. flexneri has two functional msbB genes, one carried by the chromosome (msbB1) and the other by the virulence plasmid (msbB2), the products of which act in complement to produce full acyl-oxy-acylation of the myristate at the 3' position of the lipid A glucosamine disaccharide. A mutant in which both the msbB1 and msbB2 genes have been inactivated was impaired in its capacity to cause TNF-alpha production by human monocytes and to cause rupture and inflammatory destruction of the epithelial barrier in the rabbit ligated intestinal loop model of shigellosis, indicating that lipid A plays a significant role in aggravating inflammation that eventually destroys the intestinal barrier. In addition, neutralization of TNF-alpha during invasion by the wild-type strain strongly impaired its ability to cause rupture and inflammatory destruction of the epithelial lining, thus indicating that TNF-alpha is a major effector of epithelial destruction by Shigella.     

CO2 permeability of the rat erythrocyte membrane and its inhibition

We have studied the CO₂ permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs ¹⁸O-labelled CO₂. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO₃⁻ permeability. For normal rat erythrocytes, we find at 37 °C a CO₂ permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10⁻³cm/s. We studied whether the rat erythrocyte membrane possesses protein CO₂ channels similar to the human red cell membrane by applying the potential CO₂ channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO₂ permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO₂ channels that are responsible for at least 50% of its CO₂ permeability.