Assay of the glucuronoxylan methyltransferase activity of recombinant PtrGXM proteins.

<p>Maltose binding protein (MBP)-tagged PtrGXM proteins expressed in <i>E. coli</i> were purified with amylose resin, and used for assay of their methyltransferase activity. (A) SDS-polyacylamide gel electrophoresis detection of purified recombinant PtrGXM proteins (5 µg per lane). Shown are MBP (42.5 kD), and MBP-tagged PtrGXM1 (71.4 kD), PtrGXM2 (71.3 kD), PtrGXM3 (72.7 kD) and PtrGXM4 (72.4 kD). Molecular weight markers are indicated at right. (B) Recombinant PtrGXM proteins exhibit a methyltransferase activity that was able to transfer the radiolabeled methyl group from the <i>S</i>-adenosylmethionine donor onto the GlcA-substituted Xyl₄ acceptor but not GlcA, UDP-GlcA or Xyl₄. Error bars denote the se of three independent assays.</p

Kinetic properties of the PtrGXM methyltransferase activities.

<p>Recombinant PtrGXM proteins were assayed for the methyltransferase activity in the presence of various concentrations of the (GlcA)Xyl₄ acceptor. The results were analyzed by Lineweaver-Burk plots to determine the <i>K</i>_m and <i>V</i>_max values.</p

Detection of glucuronoxylan methyltransferase activity in <i>Populus</i> stem microsomes.

<p>Microsomes isolated from developing stems of <i>Populus trichocarpa</i> were incubated with the methyl donor, ¹⁴C-radiolabeled <i>S</i>-adenosylmethionine, and the GlcA-substituted Xyl₄ acceptor. The methyltransferase activity (CPM) was measured by the transfer of the radiolabeled methyl group onto the acceptor. Error bars in (A), (B) and (C) denote the se of two biological replicates. (A) <i>Populus</i> stem microsomes exhibit a methyltransferase activity toward the GlcA-substituted Xyl₄ acceptor. Microsomes (200 µg protein) were used for each reaction unless otherwise indicated. (B) Time course of the methyltransferase activity exhibited by the microsomes. (C) The methyltransferase activity increases with increasing amount of microsomes. (D) MALDI analysis of the reaction products catalyzed by the methyltransferase activity in <i>Populus</i> microsomes. Microsomes were incubated with the GlcA-substituted Xyl₄ acceptor in the absence (top panel) or presence (lower panel) of <i>S</i>-adenosylmethionine (SAM). The ions [M+Na]⁺ at <i>m/z</i> 745 and 759 correspond to the GlcA-substituted Xyl₄ acceptor [(GlcA)Xyl₄] and the methylated acceptor with an increase of 14 D [(MeGlcA)Xyl₄], respectively. The ions at <i>m/z</i> 767 and 781 are attributed to the doubly sodiated species [M+2Na]⁺ of (GlcA)Xyl₄ and (MeGlcA)Xyl₄, respectively. The identity of the ion at <i>m/z</i> 787 is not known.</p

Relative integrated values of GlcA and MeGlcA side chains in xylans from the wild type, <i>gxm1/2/3</i> and PtrGXM-complemented <i>gxm1/2/3</i> plants.

a<p>Ratio of MeGlcA relative to the total GlcA and MeGlcA side chains was calculated by dividing the integrated value of MeGlcA by that of the total GlcA and MeGlcA side chains based on the NMR data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087370#pone-0087370-g007" target="_blank">Fig. 7</a>.</p

¹H-NMR spectra of xylan from the <i>gxm1/2/3</i> mutant overexpressing PtrGXMs.

<p>Xylooligosaccharides generated by xylanase digestion of xylan were subjected to ¹H-NMR analysis. Resonances are labeled with the position of the assigned proton and the identity of the residue containing that proton. The resonances of H1 of α-d-GalA, H1 of α-l-Rha, H1 of 3-linked β-d-Xyl, H4 of α-d-GalA, and H2 of α-l-Rha are from the xylan reducing end tetrasaccharide sequence. G and M refer to the GlcA- and Me-α-GlcA-substituted xylosyl residues, respectively. Note the partial restoration of the resonances of Me-α-GlcA (red arrows) in the <i>gxm1/2/3</i> mutant overexpressing PtrGXMs.</p

MALDI spectra of the reaction products catalyzed by PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4 recombinant proteins.

<p>Purified recombinant PtrGXMs were incubated with the GlcA-substituted Xyl₄ [(GlcA)Xyl₄] acceptor and the <i>S</i>-adenosylmethionine methyl donor and the reaction products were purified and subjected to MALDI analysis. Note the appearance of a new ion peak at <i>m/z</i> 759 corresponding to (MeGlcA)Xyl₄ (red arrows) with an increase of 14 D relative to the (GlcA)Xyl₄ acceptor (<i>m/z</i> 745) in the products of reactions incubated with PtrGXMs but not with MBP. The ions at <i>m/z</i> 767 and 781 are attributed to the doubly sodiated species [M+2Na]⁺ of (GlcA)Xyl₄ and (MeGlcA)Xyl₄, respectively.</p

Phylogenetic and expression analyses of four <i>Populus GXM</i> genes.

<p>(A) Phylogenetic relationship of DUF579-containing proteins from <i>Populus</i> and Arabidopsis. The amino acid sequences of DUF579-containing proteins from <i>Populus</i> and Arabidopsis were aligned using ClustalW and their phylogenetic relationship was analyzed using the neighbor-joining method in MEGA5.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087370#pone.0087370-Tamura1" target="_blank">[40]</a>. Bootstrap values resulted from 1,000 replicates are shown at the nodes. (B) Quantitative PCR analysis of the expression of <i>PtrGXM</i> genes in developing leaves, petioles, stems without secondary growth (stem I), and stems with secondary growth (stem II) of <i>Populus</i>. The expression level of each gene in leaves was taken as 1. (C) Quantitative PCR analysis of the induction of expression of <i>PtrGXM</i> genes in the leaves of transgenic <i>Populus</i> plants overexpressing PtrWND2B. The control is transgenic <i>Populus</i> plants transformed with an empty vector. Error bars in (B) and (C) denote the se of three biological replicates.</p

In situ hybridization analysis of <i>PtrGXM1</i>, <i>PtrGXM2</i>, <i>PtrGXM3</i> and <i>PtrGXM4</i> mRNAs in <i>Populus</i> stems.

<p>Cross sections of stems were hybridized with digoxigenin-labeled antisense probes of <i>PtrGXM1</i> (A), <i>PtrGXM2</i> (B), <i>PtrGXM3</i> (C), and <i>PtrGXM4</i> (D) or the sense probe of <i>PtrGXM1</i> as the control (E). The hybridized sections were incubated with alkaline phosphatase-conjugated antibodies and the hybridization signals are shown as purple color. co, cortex; pf, phloem fiber; sx, secondary xylem. Bar in (A) = 160 µm for (A) to (E).</p

MALDI-TOF mass spectra of xylooligosaccharides generated by xylanase digestion of xylan from the <i>gxm1/2/3</i> mutant overexpressing PtrGXMs.

<p>The ions at <i>m/z</i> 745 and 759 are attributed to xylotetrasaccharides bearing a GlcA residue [(GlcA)Xyl₄] and a methylated GlcA residue [(MeGlcA)Xyl₄], respectively. The ion at <i>m/z</i> 761 corresponds to the xylan reducing end pentasaccharide, β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-α-l-Rha-(1→2)-α-d-GalA-(1→4)-d-Xyl (X-X-R-GA-X). Note the partial restoration of the ion signal at <i>m/z</i> 759 corresponding to (MeGlcA)Xyl₄ (red arrows) in the <i>gxm1/2/3</i> mutant overexpressing PtrGXMs.</p

Pharmacokinetics of paclitaxel/17-AAG-loaded micelles in nude mice bearing human ovarian tumor SKOV-3 xenografts.

<p>The dual drug-loaded micelles were i.v. administered at the doses of 20 mg/kg paclitaxel and 37.5 mg/kg 17-AAG. For the free drug-treated group, the mice received the same combined doses of free paclitaxel and 17-AAG dissolved in DMSO. Each data point was the average+SE, n = 4 mice per group. A, the micellar formulation resulted in over 10-fold increase in paclitaxel concentrations in plasma. The plasma concentration of paclitaxel following the free drug administration was below the detection limit at 4 h. B, the micellar formulation resulted in over 3-fold increase in 17-AAG concentrations in plasma. The plasma concentration of 17-AAG was below the detection limit at 4 h for both groups. C, the micellar formulation caused a 3.5-fold increase of paclitaxel (***, p = 0.0001) in the tumor without significant affecting the drug distribution to normal organs. D, the micellar formulation caused a 1.7-fold increase of 17-AAG (***, p = 0.0005) in the tumor without significant affecting the drug distribution to normal organs.</p