In the brain of Alzheimer's disease (AD) patients, neurotoxic amyloid peptides accumulate and are deposited as senile plaques. A major therapeutic strategy aims to decrease production of amyloid peptides by inhibition of gamma-secretase. Presenilins are polytopic transmembrane proteins that are essential for gamma-secretase activity during development and in amyloid production. By loxP/Cre-recombinase-mediated deletion, we generated mice with postnatal, neuron-specific presenilin-1 (PS1) deficiency, denoted PS1(n-/-), that were viable and fertile, with normal brain morphology. In adult PS1(n-/-) mice, levels of endogenous brain amyloid peptides were strongly decreased, concomitant with accumulation of amyloid precursor protein (APP) C-terminal fragments. In the cross of APP[V717I]xPS1 (n-/-) double transgenic mice, the neuronal absence of PS1 effectively prevented amyloid pathology, even in mice that were 18 months old. This contrasted sharply with APP[V717I] single transgenic mice that all develop amyloid pathology at the age of 10-12 months. In APP[V717I]xPS1 (n-/-) mice, long-term potentiation (LTP) was practically rescued at the end of the 2 hr observation period, again contrasting sharply with the strongly impaired LTP in APP[V717I] mice. The findings demonstrate the critical involvement of amyloid peptides in defective LTP in APP transgenic mice. Although these data open perspectives for therapy of AD by gamma-secretase inhibition, the neuronal absence of PS1 failed to rescue the cognitive defect, assessed by the object recognition test, of the parent APP[V717I] transgenic mice. This points to potentially detrimental effects of accumulating APP C99 fragments and demands further study of the consequences of inhibition of gamma-secretase activity. In addition, our data highlight the complex functional relation of APP and PS1 to cognition and neuronal plasticity in adult and aging brain.
Transgenic banana ( Musa acuminata ‘Gros Michel’) integrating either of two rice chitinase genes was generated and its resistance to Black Leaf Streak disease caused by the fungus Mycosphaerella fijiensis was tested using a leaf disk bioassay. PCR screening indicated the presence of the hpt selectable marker gene in more than 90 % of the lines tested, whereas more than three quarters of the lines contained the linked rice chitinase gene resulting in a co-transformation frequency of at least 71.4 %. Further, a unique stable integration of the transgenes in each line revealed some false negative PCR results and the expected co-transformation frequency of 100 %. The transgene insert number per line ranged from 1 to 5 and single transgene insert lines (25 % of all) were identified. Considerable delay in disease development (up to 63 days post-incoculation) over a monitoring period of 108 days occurred in nine lines with extracellularly targeted chitinase out of 17 transgenic lines tested and their necrotic leaf area decreased by 73–94 % compared to the untransformed susceptible control line. Finally, correlation between symptom development and rice chitinase expression was confirmed in two lines by Western analysis. The potential of rice chitinase genes to enhance resistance against M. fijiensis in banana was demonstrated as well as the usefulness of the leaf disk bioassay for early disease screening in transgenic banana lines. Electronic supplementary material The online version of this article (doi:10.1007/s11248-012-9631-1) contains supplementary material, which is available to authorized users.
Two metal‐lux fusions were constructed in Alcaligenes eutrophus by transposon Tn4431 mutagenesis. The resulting strains, named AE1239 (pMOL90::Tn4431) and AE1433 (pMOL30::Tn4431, pMOL28), were respectively emitting light in the presence of copper ions and in the presence of zinc, cadmium, cobalt, and lead ions. Both strains were freeze dried and used as microbial bioluminescent sensors for the evaluation of incinerator fly‐ashes (IFA) and soils contaminated by heavy metals. Two detection protocols (with or without direct contact between the bacterial sensors and the IFA) were used to test different batches of IFA that were either not treated or treated in order to be used as recycled material. The bacterial light production measured with a luminometer in the presence of increasing concentrations of IFA was compared with the IFA metal concentration measured by inductively coupled plasma emission spectrometer. The same methodology was used to compare different soils samples from a sandy soil area severely contaminated by heavy metals. Both biosensors could successfully assess the bioavailability of heavy metals. This bioassay appeared to be very simple and rapid since no pretreatment of the samples was required. Those biosensors can either be used to predict the potential risk of the use of heavy metal‐recycled materials in the environment or serve as a rapid screening tool for the efficacy of remediation practices used on contaminated soil. © 1996 by John Wiley & Sons, Inc.
Two metal-lux fusions were constructed in Alcaligenes eutrophus by transposon Tn4431 mutagenesis. The resulting strains, named AE1239 (pMOLSO::Tn4437) and AE1433 (pMOLSO::Tn4431, pMOL28), were respectively emitting light in the presence of copper ions and in the presence of zinc, cadmium, cobalt, and lead ions. Both strains were freeze dried and used as microbial bioluminescent sensors for the evaluation of incinerator fly-ashes (IFA) and soils contaminated by heavy metals. Two detection protocols (with or without direct contact between the bacterial sensors and the IFA) were used to test different batches of IFA that were either not treated or treated in order to be used as recycled material. The bacterial light production measured with a luminometer in the presence of increasing concentrations of IFA was compared with the IFA metal concentration measured by inductively coupled plasma emission spectrometer. The same methodology was used to compare different soils samples from a sandy soil area severely contaminated by heavy metals. Both biosensors could successfully assess the bioavailability of heavy metals. This bioassay appeared to be very simple and rapid since no pretreatment of the samples was required. Those biosensors can either be used to predict the potential risk of the use of heavy metal-recycled materials in the environment or serve as a rapid screening tool for the efficacy of remediation practices used on contaminated soil 0
Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression.
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