Effects of phosphorus-mobilizing bacteria on tomato growth and soil microbial activity

Aims

The aim of our study was to clarify whether inoculating a soil with Pseudomonas sp. RU47 (RU47) bacteria would stimulate the enzymatic cleavage of organic P compounds in the rhizosphere and bulk soil, promoting plant growth. Adding either viable or heat treated RU47 cells made it possible to separate direct from indirect effects of the inoculum on P cycling in soil and plants.

Methods

We performed a rhizobox experiment in the greenhouse with tomato plants (Solanum lycopersicum) under low P soil conditions. Three inoculation treatments were conducted, using unselectively grown soil bacteria (bacterial mix), heat treated (HT-RU47) and viable RU47 (RU47) cells, and one not inoculated, optimally P-fertilized treatment. We verified plant growth, nutrient availability, enzyme activities and microbial community structure in soil.

Results

A plant growth promotion effect with improved P uptake was observed in both RU47 treatments. Inoculations of RU47 cells increased microbial phosphatase activity (PA) in the rhizosphere.

Conclusions

Plant growth promotion by RU47 cells is primarily associated with increased microbial PA in soil, while promotion of indigenous Pseudomonads as well as phytohormonal effects appear to be the dominant mechanisms when adding HT-RU47 cells. Thus, using RU47 offers a promising approach for more efficient P fertilization in agriculture.

     

Toward establishing structure–activity relationships for oxygenated coumarins as differentiation inducers of promonocytic leukemic cells

The presumption that some coumarins might be lead compounds in the search for new differentiation agents against leukemia is based on the fact that natural coumarins, 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin (C-2) and 5-methoxy-6,7-methylenedioxycoumarin (C-1) inhibit proliferation and induce differentiation in U-937 cells [Riveiro, M. E.; Shayo, C.; Monczor, F.; Fernandez, N.; Baldi, A.; De Kimpe, N.; Rossi, J.; Debenedetti, S.; Davio, C. Cancer Lett. 2004, 210, 179–188]. These promising findings prompted us to investigate the anti-leukemia activity of a broader range of related polyoxygenated coumarins. Twenty related natural or synthetically prepared coumarins, including a range of 5-substituted ayapin derivatives which have become easy accessible via newly developed synthesis methods, were evaluated, where treatments with 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-3) and 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-2) were able to inhibit the cell growth and induce the differentiation of U-937 cells after 48 h treatment. These results provide insight into the correlation between some structural properties of polyoxygenated coumarins and their in vitro leukemic differentiation activity.     

Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function,Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.     

The self‐sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5‐hydroxylation of diclofenac

P450 monooxygenases are able to catalyze the highly regio‐ and stereoselective oxidations of many organic molecules. However, the scale‐up of such bio‐oxidations remains challenging due to the often‐low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti‐inflammatory drug that is persistent in the environment along with the 4'‐ and 5‐hydroxy metabolites. Here we have used the self‐sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5‐hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5‐hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram‐scale production of human drug metabolites through recombinant whole cell biocatalysis.     

Presenilin-dependent intramembrane proteolysis of CD44 leads to the liberation of its intracellular domain and the secretion of an Abeta-like peptide

Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.     

A tandem Kunitz protease inhibitor (KPI106)–serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula

Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza‐specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two‐hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate.     

Cytoprotective function of sAppalpha in human keratinocytes

sAPPalpha, the soluble form of the beta-amyloid precursor protein, has been shown to act as a potent epidermal growth factor by stimulating keratinocyte proliferation and migration. In this report we provide evidence for a cytoprotective role of sAPPalpha. As a model we used HaCaT cells and normal human keratinocytes (NHK) cultured in the absence of fetal calf serum and bovine pituitary extract. Under these conditions keratinocytes began to undergo apoptosis at increasing rates after 96 h of culture. Surprisingly, keratinocytes were protected from apoptosis by the addition of 50 nM recombinant sAPPalpha. Subsequent experiments were performed to elucidate the regulatory basis of the cytoprotective role of sAPPalpha. We found that recombinant sAPPalpha facilitated the substrate adhesion of keratinocytes in the first 30 minutes after seeding. The basis for this adhesion-promoting function was shown by the ability of recombinant sAPPalpha to continuously coat the culture dish thereby promoting the ability to bind keratinocytes. A second mechanism explaining the cytoprotective role was found in the significant inhibition of apoptosis by recombinant sAPPalpha. In HaCaT cells moderate UV-B irradiation was sufficient to induce apoptosis. In contrast, induction of apoptosis in NHK required additionally the depletion of endogenous sAPPalpha suggesting that sAPPalpha mediates protection against UV-B irradiation. Staurosporine-induced apoptosis rates were significantly reduced by about 59% after addition of recombinant sAPPalpha. These results show that sAPPalpha exerts a pronounced cytoprotective effect and that this effect is mediated by facilitated cell adhesion and by the antiapoptotic function of sAPPalpha.     

Mate Choice for Non-siblings in Wild House Mice: Evidence from a Choice Test and a Reproductive Test

Two experiments were designed to test whether wild house mice discriminate between olfactory cues from different kin and, if so, whether given preferences would relate to actual reproductive decisions. Experimental animals were mice born to the offspring of wild-caught house mice. Litter-mates stayed together until 60 d of age and were then housed individually. In a choice test, animals were placed in the middle of an arena with 4 openings which led to small cages containing bedding material from opposite-sex animals of known kinship (full-sib, cousin, unrelated) or clean material. Test animals (11 oestrous females, 11 males tested with oestrous females' bedding, 8 males tested with material from non-oestrous females) preferred conspecific to control bedding. Males tested with oestrous females' bedding significantly preferred unrelated to full-sib odours. In a second experiment, 34 males were each mated simultaneously to 3 females (sister, cousin, unrelated) and these groups were then housed together for 5, 10, and 15 d. Females were checked for litters during the next 20 d. Reproductive rate increased significantly in the 15 d cohabitation group, and significantly more cousin and unrelated females than sisters gave birth to a litter.