Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B.

<p>(A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.</p

Kaposi's sarcoma-associated herpesvirus infection promotes differentiation and polarization of monocytes into tumor-associated macrophages

<p>Tumor associated macrophages (TAMs) promote angiogenesis, tumor invasion and metastasis, and suppression of anti-tumor immunity. These myeloid cells originate from monocytes, which differentiate into TAMs upon exposure to the local tumor microenvironment. We previously reported that Kaposi's sarcoma-associated herpes virus (KSHV) infection of endothelial cells induces the cytokine angiopoietin-2 (Ang-2) to promote migration of monocytes into tumors. Here we report that KSHV infection of endothelial cells induces additional cytokines including interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-13 (IL-13) that drive monocytes to differentiate and polarize into TAMs. The KSHV-induced TAMs not only express TAM-specific markers such as CD-163 and legumain (LGMN) but also display a gene expression profile with characteristic features of viral infection. More importantly, KSHV-induced TAMs enhance tumor growth in nude mice. These results are consistent with the strong presence of TAMs in Kaposi's sarcoma (KS) tumors. Therefore, KSHV infection of endothelial cells generates a local microenvironment that not only promotes the recruitment of monocytes but also induces their differentiation and polarization into TAMs. These findings reveal a new mechanism of KSHV contribution to KS tumor development.</p

Co-fermentaion the phytase expressing <i>P</i>. <i>pastoris</i> and Endo H-P expressing <i>P</i>. <i>pastoris</i> with various initial ratio of cells innoculated in BMMY medium.

<p>M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 phytase expressed in <i>P</i>. <i>pastoris</i>; Lane 2–8 EndoH-P-expressing <i>P</i>. <i>pastoris</i> co-fermentated with phytase-expressing <i>P</i>. <i>pastoris</i> with inoculation ratio of 1:10, 1:25, 1:50, 1:100, 1:200, 1:500 and 1:1000 (Endo H-P: phytase), respectively.</p

Co-fermentaion of EndoH-P with the recombiant DNase I and phytase.

<p>M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 phytase expressed in <i>P</i>. <i>pastoris</i>; Lane 2 phytase co-induced with Endo H-P in <i>P</i>. <i>pastoris</i>; Lane 3 DNase I expressed in <i>P</i>. <i>pastoris</i>; Lane 4 DNase I co-induced with Endo H-P in <i>P</i>. <i>pastoris</i>; Lane 5 Endo H-P expressed in <i>P</i>. <i>pastoris</i>.</p

Deglycosylation of recombinant proteins expressed in <i>P</i>. <i>pastoris</i>.

<p>(A). SDS-PAGE of the recombinant proteins expressed in <i>P</i>. <i>pastoris</i> with or without digestion of Endo H-P with post-fermentation method. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 positive control(Horseradish Peroxidase) from the Glycoprotein Staining Kit;Lane 2 negative control(Soybean Trypsin Inhibitor) from Glycoprotein Staining Kit;Lane 3 mannosidase expressed in <i>P</i>. <i>pastoris</i>;Lane 4 deglycosylated mannosidase; Lane 5 phytase expressed in <i>P</i>. <i>pastoris</i>; Lane 6 deglycosylated phytase;Lane 7 <i>Dpn</i>I expressed in <i>P</i>. <i>pastoris</i>;Lane 8 deglycosylated <i>Dpn</i>I. (B). Glycoprotein staining of the recombinant proteins expressed in <i>P</i>. <i>pastoris</i> with or without Endo H-P digestion. All samples were loaded in the identical order with A.</p

Analysis of the enzymatic activity of deglycosylated DNase I and endo-1, 4-β-mannosidase obtained from co- and post-fermentation with Endo H-P.

<p>(A). M DNA molecular weight markers (the size of each band was indicated on the left); Lane 1 pHBM905A plasmid (about 300 ng); Lane 2 pHBM905A treated with 1 μl supernatant of DNase I; Lane 3–4 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with post-fermentation treatment; Lane 5 pHBM905A treated with 1 U commercial DNase I; Lane 6–7 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with co-fermentation treatment; Lane 8 pHBM905A treated with fermentation supernatant from <i>P</i>. <i>pastoris</i> bearing pHM905A plasmid (the negative control). (B). A hole of about 2mm was made with a hole puncher on a MD plate supplemented with 1% konjac powder and 0.05% trypan blue. The samples were added into the wells and the plate was incubated at 37°C, overnight. Sample 1: 1.5 μl supernatant of <i>P</i>. <i>pastoris</i> expressing glycosylated endo-1, 4-β-mannosidase; Sample 2: 2 μl supernatant of <i>P</i>. <i>pastoris</i> expressing glycosylated mannanase; Sample 3: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with post-fermentation treatment; Sample 4: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with co-fermentation treatment; Sample 5: 2 μl supernatant of EndoH-P.</p

The growth curves of phytase expressing <i>P</i>. <i>pastoris</i> with or without co-fermentation with Endo H-P expressing <i>P</i>. <i>pastoris</i>.

<p>(A).The growth curve of phytase- and Endo H-P-expressing <i>P</i>. <i>pastoris</i> during the vegetative growth phase. (B). The growth curve of phytase- and Endo H-P- expressing <i>P</i>. <i>pastoris</i> during the induction phase. The names of the strains were indicated at the bottom of the panel.</p

The hydrolytic activity of Endo H-P to proteins from mammals and baker's yeast.

<p>(A). The hydrolytic activity of Endo H-P to EPO expressed in CHO cells. Lane 1 EPO without treatment; Lane 2 EPO treated with commercial Endo H; Lane 3 EPO treated with purified Endo H-P; Lane 4 EPO treated with commercial PNGase F. (B). The hydrolytic activity of Endo H-P to CPY from baker's yeast. Lane 1 CPY without treatment; Lane 2 CPY treated with commercial Endo H; Lane 3 CPY treated with purified Endo H-P.</p

The alignments of <i>Endo H-P</i> with wild-type <i>Endo H</i>.

<p>The nucleotide sequence of <i>Endo</i> H-<i>P</i> was compared with that of wild-type <i>Endo</i> H, the result indicated that 220 nucleotides were altered in <i>Endo H-P</i>.</p

SDS-PAGE analysis of Endo H-P secreted in the cell culture supernatant of the shake flasks.

<p>M protein molecular weight marker (the size of each band was indicated on the left); Lane 1 cell culture supernatant (15 μl) of the strain bearing pHBM905A plasmid (negative control); Lane 2–7 cell culture supernatants (15 μl) collected from 1 to 6 days, respectively.</p