Functional expression of murine LRP1 requires correction of Lrp1 cDNA sequences

Here, we describe the reconstruction of a functional 14 kbp full-length murine Lrp1 cDNA from overlapping partial cDNAs, which were described before [Biochim. Biophys. Acta 1173 (1993) 71]. The reconstructed full-length cDNA needed sequence correction (by mutagenesis) due to nucleotide errors present in the underlying partial cDNAs. These mistakes compromised the proteolytical maturation of the LRP precursor (4545 aa) into its alpha- and beta-subunits. To identify these mistakes initially, detailed sequence analyses and comparison of genomic and cDNA sequences of different murine strains proved to be necessary to obtain correct wild-type sequences. Comparison of Lrp1 cDNA sequences of CBA mice with Lrp1 genomic exon sequences of 129P3/J mice (like in man 89 exons) revealed only 24 nucleotide differences within about 14.8 kbp. Only 1 out of 23 nucleotide differences in the protein coding region affected an amino acid residue: Thr versus Ala at amino acid residue position 2642 in 129P3/J and CBA, respectively. After correction by mutagenesis, both a 129P3/J and a CBA-based version of a full-length wild-type Lrp1 cDNA were functionally expressed in an LRP-deficient mutant CHO cell line. Transient expression showed the expected maturation of the LRP precursor into its two subunits. Furthermore, stable transfection restored the sensitivity to exposure to Pseudomonas aeruginosa toxin A (PEA). Since LRP is the unique receptor for this toxin, this indicates that the toxin could enter the cells after binding to and endocytosis by its genuine receptor. This murine LRP expression system will be instrumental in future experimental dissection of this multifunctional receptor.     

Differential response of a(2)-macroglobulin-deficient mice in models of lethal TNF-induced inflammation

Tumor necrosis factor (TNF) is an essential mediator in the pathogenesis of Gram-negative septic shock. Injection of TNF into normal mice leads to systemic, lethal inflammation, which is indistinguishable from lipopolysaccharide (LPS)-induced lethal inflammation. alpha(2)-macroglobulin (A2M) is a major positive acute phase protein with broad-spectrum protease-inhibitory activity. Mouse A2M-deficient (MAM-/-) mice were significantly protected against lethal systemic inflammation induced by TNF. The protection is not due to faster clearance of the injected TNF. The induction of tolerance to TNF-induced lethality by repetitive administration of small doses of human TNF for five consecutive days was equally efficient in both mutant mice compared to wild-type mice. In D-(+)-galactosamine (GalN)-sensitized mice, TNF induces lethal inflammatory hepatitis. MAM(-/-) mice are equally sensitive to the lethal combination of TNF/GalN. Furthermore, interleukin-1-induced desensitization to TNF/GalN was not impaired in MAM(-/-) mice. We conclude that MAM plays a mediating role in TNF-induced lethal shock and that MAM deficiency does not reduce changes in efficiency of tolerance and desensitization to TNF and TNF/GalN-induced lethality, respectively.     

Disentangling and ranking the influences of multiple environmental factors on plant and soil-dwelling arthropod assemblages in a river Rhine floodplain area

Floodplains of large rivers are among the most dynamic and diverse, yet most threatened ecosystems on earth. For a solid underpinning of river conservation and rehabilitation measures, it is critical to unravel the influences of the multiple stressors affecting floodplain ecosystems. Using canonical correspondence analysis with variance partitioning, we disentangled and ranked the influences of three floodplain ecosystem stressors (land use, flooding and soil contamination) on terrestrial plant and soil-dwelling arthropod assemblages in a floodplain area along the river Rhine in The Netherlands. We included five biotic assemblages: plant species (73 taxa), ground beetle species (57 taxa), ground beetle genera (29 taxa), beetle families (32 taxa) and arthropod groups at taxonomic levels from family to class (10 taxa). Plant and arthropod assemblages were primarily related to land use, which explained 19–30% of the variation in taxonomic composition. For plant species composition, flooding characteristics were nearly as important as land use. Soil metal contamination constituted a subordinate explanatory factor for the plant assemblages only (3% of variation explained). We conclude that the taxonomic composition of terrestrial plant and arthropod assemblages in our study area is related to land use and flooding rather than soil metal contamination.     

The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species.     

Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.     

A yeast-based model of alpha-synucleinopathy identifies compounds with therapeutic potential

We have developed a yeast-based model recapitulating neurotoxicity of alpha-synuclein fibrilization. This model recognized metal ions, known risk factors of alpha-synucleinopathy, as stimulators of alpha-synuclein aggregation and cytotoxicity. Elimination of Yca1 caspase activity augmented both cytotoxicity and inclusion body formation, suggesting the involvement of apoptotic pathway components in toxic alpha-synuclein amyloidogenesis. Deletion of hydrophobic amino acids at positions 66-74 in alpha-synuclein reduced its cytotoxicity but, remarkably, did not lower the levels of insoluble alpha-synuclein, indicating that noxious alpha-synuclein species are different from insoluble aggregates. A compound screen aimed at finding molecules with therapeutic potential identified flavonoids with strong activity to restrain alpha-synuclein toxicity. Subsequent structure-activity analysis elucidated that these acted by virtue of anti-oxidant and metal-chelating activities. In conclusion, this yeast-cell model as presented allows not only fundamental studies related to mechanisms of alpha-synuclein-instigated cellular degeneration, but is also a valid high-throughput identification tool for novel neuroprotective agents.     

Moorland pools as refugia for endangered species characteristic of raised bog gradients

In intact raised bog landscapes transitions from ombrotrophic into minerotrophic conditions occur. These gradients are lost from many bogs due to peat harvesting and drainage, and are difficult to restore. To determine which endangered species are characteristic of pristine raised bog gradients and their current status in degraded bogs, plants and macroinvertebrates were surveyed in Estonian intact raised bogs and Dutch degraded bog remnants. Dutch national distribution data were used to determine whether communities with these species occurred outside bog habitats. Water chemistry data were used to describe associated environmental conditions. Intact bog gradients were the preferred habitat for six plant species and fifteen macroinvertebrate species, all of which are endangered. In degraded bogs these species were scarce or not recorded. In intact bogs these species lived at sites where runoff from the bog massif came into contact with regional ground water resulting in a gradient in pH, alkalinity, Ca, Fe and ionic ratio. Analysis of Dutch national distribution data revealed aggregations of these endangered species in moorland pools. These pools contained water chemistry gradients similar to those found in pristine bogs, which occurred at sites were groundwater seepage and stream water came in contact. In the past, stream water has been used to increase pH and trophic status of moorland pools facilitating fisheries. Today, this practice offers a conservation strategy for the protection of endangered species for which no short-term alternatives are available.     

Do restoration measures rehabilitate fauna diversity in raised bogs? A comparative study on aquatic macroinvertebrates

To assess whether raised bog restorationmeasures contribute to the conservation andrestoration of the fauna diversity,macroinvertebrate species assemblages werecompared between water bodies created byrewetting measures and water bodies whichhave not been subject to restorationmeasures, but are remnants offormer peat cuttings and trenches used forbuckwheat culture in the past.The restoration sites were inhabited bycharacteristic raised bog species and rarespecies, but their numbers were higher atthe remnant sites not affected byrestoration management. A considerablenumber of characteristic and rare faunaspecies were only found at the remnantsites. The remnant sites includedconsiderably more variation inmacroinvertebrate species assemblages andhad a higher cumulative species richness.The number of characteristicmacroinvertebrate species was not clearlyrelated to the presence of a characteristicraised bog vegetation. In restoration sitesnumbers of rare and characteristic speciesper site tended to increase with the timeelapsed after rewetting. However,restoration measures will not automaticallyresult in restoration of a more or lesscomplete macroinvertebrate speciesspectrum, as restoration measures have sofar resulted in habitats for only a limitednumber of the characteristic species.When planning restoration measures, it isrecommended to protect the populations ofrare and characteristic species present inthe area, as these populations may becomethe sources for colonization of rewettedsites. Safeguarding habitat diversityduring the restoration process andrestoration of different elements of thehabitat diversity of complete raised bogsystems will result in the characteristicfauna diversity being conserved andrestored more successfully.