RecA-Mediated,Targeted Mutagenesis in Zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into 1-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in 1 or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems. Current address (Ying Yang): First Hospital of Beijing University, Beijing 100034, P.R. China. Current address (Christopher D. Kaufman): Max Delbrück Center for Molecular Medicine, Robert Rössle Strasse 10, D-13092 Berlin, Germany. Current address (Perry B. Hackett): Discovery Genomics, Inc. 614 McKinley Pl. NE Minneapolis, MN 55413     

Crop management and agronomic context of the Farm Scale Evaluations of genetically modified herbicide-tolerant crops

The Farm Scale Evaluations of genetically modified herbicide-tolerant crops (GMHT) were conducted in the UK from 2000 to 2002 on beet (sugar and fodder), spring oilseed rape and forage maize. The management of the crops studied is described and compared with current conventional commercial practice. The distribution of field sites adequately represented the areas currently growing these crops, and the sample contained sites operated at a range of management intensities, including low intensity. Herbicide inputs were audited, and the active ingredients used and the rates and the timings of applications compared well with current practice for both GMHT and conventional crops. Inputs on sugar beet were lower than, and inputs on spring oilseed rape and forage maize were consistent with, national averages. Regression analysis of herbicide-application strategies and weed emergence showed that inputs applied by farmers increased with weed densities in beet and forage maize. GMHT crops generally received only one herbicide active ingredient per crop, later and fewer herbicide sprays and less active ingredient (for beet and maize) than the conventional treatments. The audit of inputs found no evidence of bias.     

Protein production in Escherichia coli for structural studies by X-ray crystallography

The arrival of genomic sequences to the database has provided a seemingly unlimited supply of targets for protein structure determination and the possibility of solving the structure of an entire proteome. Based on our experience with the proteomes of Pyrobaculum aerophilum and Mycobacterium tuberculosis, we have developed a simple strategy for the production of proteins for structural studies by X-ray crystallography. Our scheme demonstrates a strong protein target commitment and includes the expression of genes from these organisms in Escherichia coli. These proteins are expressed with affinity tags and purified for characterization and crystallization. We have identified protein solubility and crystallization as the two major bottlenecks in the process toward the determination of protein structures by X-ray diffraction. Strategies to overcome these bottlenecks are discussed.     

Tipping the apoptotic balance in Alzheimer's disease

The mechanisms underlying the selective neuronal death in Alzheimer's disease are largely unresolved. Nonetheless, it is apparent that the environment of the diseased brain is extremely rich in pro-apoptotic stimuli and that these lead to an activation of the apoptotic death cascade. However, there is surprisingly little evidence for the completion of the death pathway indicating that the apoptotic death program is terminated by a mechanism termed abortosis. This review discusses the concept of abortosis in relation to Alzheimer's disease.     

Amyotrophic lateral sclerosis: a novel hypothesis involving a gained 'loss of function' in the JNK/SAPK pathway

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that mainly affects motor neurons. Despite intensive research efforts inspired by the mile-stone discovery linking the Cu/Zn superoxide dismutase 1 (SOD1) gene to a subset of familial cases, the mechanisms underlying disease pathogenesis are still largely unknown. Nonetheless, the recent finding of a second gene associated with familial form of the disease, ALS2, is likely to be of great help in elucidating the key pathways involved in motor neuron degeneration. Here, we provide evidence that the JNK/SAPK pathway plays a critical neuroprotective role in susceptible motor neurons in ALS. The involvement of the JNK/SAPK pathway integrates our knowledge about these two known genetic factors into a single pathogenic pathway involved in both sporadic and familial ALS.     

Channels, pumps, and exchangers in the gill and kidney of freshwater fishes: their role in ionic and acid-base regulation

In freshwater fishes, the gill and kidney are intricately involved in ionic and acid-base regulation owing to the presence of numerous ion channels, pumps, or exchangers. This review summarizes recent developments in branchial and renal ion transport physiology and presents several models that integrate epithelial ion and acid-base movements in freshwater fishes. At the gill, three cell types are potentially involved in ionic uptake: pavement cells, mitochondria-rich (MR) PNA(+) cells, and MR PNA(-) cells. The transfer of acidic or basic equivalents between the fish and its environment is accomplished largely by the gill and is appropriately regulated to correct acid-base imbalances. The kidney, while less important than the gill in overall acid or base excretion, has an essential role in regulating systemic acid-base balance by controlling HCO(3) (-) reabsorption from the filtrate.     

Utilization of Energy Nutrients by Cerebellar Slices

We performed an ontogenetic study about the utilization of glycine, glutamine, -hydroxybutyrate and glycerol as energy nutrients by rat cerebellum slices. Production of CO₂ from glycerol and glutamine increased with the animals' age and glutamine was the most used nutrient for CO₂ production. In adult age, glutamine oxidation to CO₂ was 15 to 35 times higher than all other nutrients studied. CO₂ production from glycine decreased markedly with age and 10 day-old rats showed an oxidation 7.5 times higher than that of adult rats. At fetal age and at 10 postnatal days, glycine oxidation to CO₂ was only 2 times lower than glutamine oxidation to CO₂. Lipid synthesis from -hydroxybutyrate was highest in adult rats. We did not observe any difference in the utilization of -hydroxybutyrate between slices of cerebral cortex and cerebellum at the ages of 10 days and adult. The main nutrients used for lipid synthesis were glycerol and -hydroxybutyrate.     

External nickel inhibits epithelial sodium channel by binding to histidine residues within the extracellular domains of alpha and gamma subunits and reducing channel open probability

Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni(2+) on cloned mouse alpha-beta-gamma ENaC expressed in Xenopus oocytes. Ni(2+) reduced amiloride-sensitive Na(+) currents of the wild type mouse ENaC in a dose-dependent manner. The Ni(2+) block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni(2+) was accompanied by moderate inward rectification at concentrations higher than 0.1 mm. ENaC currents were also blocked by the histidine-reactive reagent diethyl pyrocarbonate. Pretreatment of the oocytes with the reagent reduced Ni(2+) inhibition of the remaining current. Mutations at alphaHis(282) and gammaHis(239) located within the extracellular loops significantly decreased Ni(2+) inhibition of ENaC currents. The mutation alphaH282D or double mutations alphaH282R/gammaH239R eliminated Ni(2+) block. All mutations at gammaHis(239) eliminated Ni(2+)-induced inward current rectification. Ni(2+) block was significantly enhanced by introduction of a histidine at alphaArg(280). Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni(2+) block. Although alphaH282C-beta-gamma channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), alpha-beta-gamma H239C channels were insensitive to MTSET. From patch clamp studies, Ni(2+) did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni(2+) reduces ENaC open probability by binding to a site consisting of alphaHis(282) and gammaHis(239) and that these histidine residues may participate in ENaC gating.     

NO-flurbiprofen maintains duodenal blood flow,enhances mucus secretion contributing to lower mucosal injury

This study investigates possible mechanisms behind the reduced gastrointestinal ulcerogenicity of nitric oxide (NO)-flurbiprofen compared with flurbiprofen. The duodenal mucosa of Inactin-anaesthetised rats was exteriorized for intravital microscopy. Blood flow was measured with laser-Doppler flowmetry (LDF), mucus thickness with micropipettes, ICAM-1 and P-selectin expression with dual-labeled antibody technique, and mucosal integrity by (51)Cr-EDTA permeability. Exposure of the duodenum to flurbiprofen (1.0 mg/ml) for 90 min significantly reduced LDF to 70 +/- 4%, whereas NO-flurbiprofen (1.3 mg/ml) had no significant effect. Mucus accumulation after 60-min exposure was 75 +/- 23 microm (control), -1 +/- 17 microm (flurbiprofen), and 104 +/- 35 microm (NO-flurbiprofen). Mucosal permeability to (51)Cr-EDTA was unchanged in the control and NO-flurbiprofen groups but increased significantly from 1.0 +/- 0.2 to 3.7 +/- 0.7 microl x min(-1) x g(-1) after 90-min exposure to flurbiprofen. Expression of ICAM-1 was significantly increased after oral flurbiprofen but not by NO-flurbiprofen. Positive effects of NO-flurbiprofen compared with flurbiprofen on mucus formation, blood flow, and adhesion molecule expression likely contribute to the reduced mucosal injury observed with NO-flurbiprofen.