Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptoms

<p>Abstract</p> <p>Background</p> <p><it>Verticillium longisporum </it>is one of the most important pathogens of <it>Brassicaceae </it>that remains strictly in the xylem during most stages of its development. It has been suggested that disease symptoms are associated with clogging of xylem vessels. The aim of our study was to investigate extracellular defence reactions induced by <it>V. longisporum </it>in the xylem sap and leaf apoplast of <it>Brassica napus </it>var. <it>napus </it>in relation to the development of disease symptoms, photosynthesis and nutrient status.</p> <p>Results</p> <p><it>V. longisporum </it>(strain VL43) did not overcome the hypocotyl barrier until 3 weeks after infection although the plants showed massive stunting of the stem and mild leaf chlorosis. During this initial infection phase photosynthetic carbon assimilation, transpiration rate and nutrient elements in leaves were not affected in VL43-infected compared to non-infected plants. Proteome analysis of the leaf apoplast revealed 170 spots after 2-D-protein separation, of which 12 were significantly enhanced in response to VL43-infection. LS-MS/MS analysis and data base searches revealed matches of VL43-responsive proteins to an endochitinase, a peroxidase, a PR-4 protein and a β-1,3-glucanase. In xylem sap three up-regulated proteins were found of which two were identified as PR-4 and β-1,3-glucanase. Xylem sap of infected plants inhibited the growth of <it>V. longisporum</it>.</p> <p>Conclusion</p> <p><it>V. longisporum </it>infection did not result in drought stress or nutrient limitations. Stunting and mild chlorosis were, therefore, not consequences of insufficient water and nutrient supply due to VL43-caused xylem obstruction. A distinct array of extracellular PR-proteins was activated that might have limited <it>Verticillium </it>spreading above the hypocotyl. In silico analysis suggested that ethylene was involved in up-regulating VL43-responsive proteins.</p

Investigations with Drugs and Pesticides Revealed New Species- and Substrate-Dependent Inhibition by Elacridar and Imazalil in <i>Human</i> and <i>Mouse</i> Organic Cation Transporter OCT2

Multiple drugs are used to treat various indications as well as pesticides that are ingested unintentionally and enter the bloodstream. The residence time or bioavailability of these substances in circulation depends on several mechanisms, such as drug–drug interaction (DDI), drug–pesticide interaction, metabolizing enzymes and the hepatic and renal transport systems, involved in the elimination of the compounds from the body. One of these transporters is the Organic Cation Transporter 2 (OCT2) member of the solute carrier (SLC22) transporter family. OCT2 is highly expressed in the proximal tubule epithelial cells in human and mouse kidney, where it mediates the uptake of endogenous organic cations as well as numerous drugs and xenobiotics, and contributes to the first step of renal clearance. In this study, we examined OCT2 on two subjects: First, the transferability of data from mouse to human, since mice are initially examined in the development of new drugs to assess the renal excretion of organic cations. Second, to what extent the choice of substrate affects the properties of an inhibitor. For this purpose, the functional properties of hOCT2 and mOct2 were validated under the same experimental conditions with the known substrates metformin and 1-Methyl-4-phenylpyridinium iodide (MPP). While hOCT2 and mOct2 showed very low affinities for metformin with Km values of 3.9 mM and 3.5 mM, the affinity of hOCT2 and mOct2 for MPP (62 and 40 µM) was 64- and 89-fold higher, respectively. For our positive control inhibitor decynium22, we determined the following IC50 values for hOCT2 and mOct2: 2.2 and 2.6 µM for metformin uptake, and 16 and 6.9 µM for MPP uptake. A correlation analysis of the inhibitory effects of 13 drugs and 9 pesticides on hOCT2- and mOct2-mediated transport of metformin showed a correlation coefficient R2 of 0.88, indicating good interspecies correlation. Nevertheless, the bioenhancer elacridar and the fungicide imazalil showed species-dependent inhibitory potentials. Concentration-dependent inhibition of hOCT2- and mOct2-mediated metformin uptake by elacridar showed IC50 values of 20 µM and 1.9 µM and by imazalil 4.7 µM and 0.58 µM, respectively. In conclusion, although our data show comparable species-independent interactions for most compounds, there can be large species–specific differences in the interactions of individual compounds, which should be considered when extrapolating data from mice to humans. Furthermore, a comparison of the inhibitory potential of elacridar and imazalil on metformin uptake with that on MPP uptake reveals substrate-dependent differences in hOCT2 and mOct2 for both inhibitors. Therefore, it might be useful to test two different substrates in inhibition studies

Functional and Pharmacological Comparison of Human and Mouse Na+/Taurocholate Cotransporting Polypeptide (NTCP)

The Na+/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar Km values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC50 values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC50 for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM). In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans

BMC Plant Biology BioMed Central

Research article Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptom

Proteins significantly affected in the apoplast of <i>Verticillilum longisporum</i> VL43-infected compared with that of mock-infected <i>Arabidopsis thaliana</i> leaves.

<p>*Factor: protein abundance in samples of VL-treated plants/protein abundance in mock-inoculated plants,</p><p>**92% homology to PRX At437520.</p><p>Only those spots were analyzed that were reproducibly observed in two independent experiments. In each experiment three biological replicates were analyzed. For further details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-t004" target="_blank">Table 4</a>. Spot identification numbers refer to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-g001" target="_blank">Figure 1</a>. Peptides are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone.0031435.s003" target="_blank">Table S1</a>.</p

Influence of <i>Verticillium longisporum</i> VL43 infection on protein content of whole leaf extracts, in apoplastic washing fluid and on the number of protein spots after 2-D electrophoresis.

<p>Plants were analyzed 25 days post infection. Protein spots were determined by Proteomweaver software in silver stained gels (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031435#pone-0031435-g001" target="_blank">Fig. 1</a>). Data indicate means (N ± SE).</p

<i>Verticillium longisporum</i> VL43 induced disease symptoms in <i>Arabidopsis thaliana</i>.

<p>Measurements were taken 24 or 25 days post inoculation on the indicated number (n) of plants. Data indicate means (± SE). Values of <i>p</i>≤0.05 indicate significant differences between mock-inoculated and <i>Verticillium longisporum</i> (VL)-infected plants.</p

Typical 2-D gels showing <i>Arabidopsis thaliana</i> apoplastic leaf proteins after mock (A) or <i>Verticillium longisporum</i>

<p><b>(VL43) infection (B).</b> Proteins were extracted 25 dpi and 80 µg were loaded for separation. Gels were stained with silver nitrate. Numbers in the gels indicate protein spots analyzed by ESI-LC/MS from preparative Coomassie-stained gels.</p