Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential. Prostate cancer is a major health problem for men in Western societies. Each year, approximately 230,000 American males are diagnosed with prostate cancer and nearly 30,000 die from this disease in vivo, efficiently delivers genes to both dividing and non-dividing cells and rarely causes any significant disease itself Bio-therapy provides an attractive opportunity to target androgen-independent prostate cancers. Unlike traditional chemotherapy, it can be designed and customized to specifically target cancers according to our understanding of the disease at a molecular level. The adenoviral vector has been used as a transfer vehicle to introduce genes into cancer cells because it is more efficient than non-viral gene transfer methods To overcome the limitations of traditional gene therapy with an replication deficient adenoviral vector, a Cancer-Targeting Gene-Viro-Therapy (CTGVT) was constructed by inserting an anti-tumor gene into a double-targeted oncolytic viral(OV) vector. It is actually an OV-gene, and has much better antitumor effect than that of either gene therapy alone or virotherapy alone. The oncolytic virus itself has anti-tumor power and selectively replicates several hundred-fold in tumor cells and thus, the therapeutic genes should also be selectively replicated several hundred-fold in tumor cells DD3 promoter and the E1B-55K gene was deleted. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical biomarker for prostate cancer PTEN is a well-known tumor suppressor gene that encodes a protein phosphatase PTEN with a CMV promoter was inserted into Ad.DD3.D55 to form Ad.DD3.D55-PTEN, which has a prostate cancer replication specificity and antitumor gene specificity. Ad.DD3.D55-PTEN efficaciously induces apoptosis in prostate cancer cells, eliminating prostate cancer xenografts with higher antitumor efficiency.In this study, we first generated a double-regulated adenovirus, Ad.DD3.D55, in which the E1A gene is under the control of DD3 promoter with WPRE was much higher in LNCaP and 22RV1 than in other cells, including the prostate cancer cell lines DU-145,PC3 and CL1. Although there were differences in the levels of expression in these prostate cell lines, these results indicate that WPRE could mediate enhancement of gene expression and maintain the relatively restricted activity of the DD3 promoter in prostate cell lines.The minimal DD3 promoter activity in the absence of WPRE was measured by luciferase-reporter expression, which showed the minimal DD3 promoter typically exhibited much more activity in the prostate cancer cell lines than in other cell lines. The pGL3-control, which contains the SV40 promoter and enhancer, was used as a positive control (activity = 1000). As shown in DD3 promoter and the E1B-55K gene has been deleted. The cytopathic effect of Ad.DD3.D55 was evaluated in normal and tumor cells by crystal violet staining, briefly Ad.DD3.D55 , which i staining. In prosThen we measured expression levels of E1A in the different cells infected with Ad.DD3.D55 (with WPRE) or Ad.DD3.D55(without WPRE) , respectPTEN expression cassette was introduced into the DD3 controlling double regulated-adenovirus Ad.DD3.D55 to generate Ad.DD3.D55-PTEN  and BEL-7404(human liver cancer cell line). The results showed that growth of infected prostate cancer cells were much inhibited with different degree, while the highest efficiency of inhibition by Ad.DD3.D55-PTEN was observed in LNCaP cells and CL1 cells . These dPTEN, but was also highly restricted to prostate cancer cells.The cytotoxicity of Ad.DD3.D55-PTEN was also analyzed by staining with crystal violet as shown in in vitro. As shown in We stained cells with Hoechst33258 to further analyze the apoptosis-inducing capacity of Ad.DD3.D55-PTEN The mechanism of Ad.DD3.D55-PTEN-induced apoptosis was studied. As shown in in vivo. As shown in The results presented above show that the anti-tumor effect of the CTGVT agent Ad.DD3.D55-PTEN is much better than either the oncolytic virus Ad.DD3.D55 or Ad.PTEN alone. Of the prostate cancer cell lines tested, Ad.DD3.D55-PTEN killed LNCaP and CL-1 cells with the highest efficiency. We selected the cell line CL1, an androgen-independent subclone of LNCaP, to evaluate the oncolytic potential of Ad.DD3.D55-PTEN because LNCaP cells have poor tumor-forming capability in vivo further illustrate the superiority of Ad.DD3.D55-PTEN with its viral DNA quantity up to 100 times greater than that of Ad-PTEN within 72 h and lasting for 2 weeks -mediated signal transduction, suppressing the PI3K/AKT pathway by reducing the level of PI3′K in the cell We observed varying sensitivities of the different prostate cancer cell lines to the cytotoxicity of Ad.DD3.D55-PTEN. The level of reporter expression driven by the DD3 promoter and WPRE in LNCaP cells was only 21.79% of that in 22RV1 cells , suggestin vivo after the period , Ad.DD3.D55-PTEN, by controlling the expression of the E1A gene with a minimal DD3 promoter and WPRE. Ad.DD3.D55-PTEN had excellent anti-tumor efficacy in both androgen-dependent and -independent prostate cancer cell lines. This report also provides a new strategy for constructing a CTGVT-PCa with tumor types specificity that have few tumor-specific promoters available.ad libitum6 22RV1 or CL1 cells in 150 µl DMEM were subcutaneously injected into the right flank of each mouse. When tumors reached 70–150 mm3 in size, mice were randomly assigned to one of four groups (six mice per group). Adenovirus (2×109 PFUs per mouse) or PBS was injected into the tumors every other day, with a total of three injections. The tumor volume (mm3) was measured with a vernier caliper every four days and calculated as (length×width2)/2. To study viral kinetics in vivo, tumors injected with Ad-PTEN or Ad.DD3.D55-PTEN were collected at 3, 6, 9, 12 and 15 days after the last injection and quickly frozen in liquid nitrogen. The total tumor DNA was extracted using the Genomic DNA MiniPreps Kit . Viral DNA was measured by real-time PCR using primers that anneal to the adenovirus E3 region. The oligonucleotide primers used for amplification are listed in Male BALB/c nude mice (4-week-old) were maintained and used in a light and temperature controlled room in an AAALAC-accredited facility, and given water and lab chow 2.The human cell lines used in this study were LNCaP, DU145, 22RV1,PC3 (prostate cancer), T-24 (bladder cancer), BEL-7404 (liver cancer), HeLa , BEAS-2B , HFL-I (embryonic lung fibroblast), L-02 (liver cell) and WISH (amnion cell). All cell lines were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences . CL1 cells (prostate cancer) were grown as described by Tso et al. DD3 promoter (AF279290 nt309–522) was cloned as described previously XbaI and NheI sites in the plasmid pGL3-DD3 to construct the plasmid pGL3-DD3-WPRE. The luciferase reporter gene assay was performed as previously described The minimal Different adenoviruses were generated by homologous recombination using Effectene Transfection Reagent  with the plasmid pBHGE3 and the plasmids pAd.DD3.D55 or pAd.DD3.D55-PTEN. Wild-type adenovirus (Ad.WT) was previously preserved in our laboratory The different cells were harvested at various times after infection with the indicated viruses at an MOI of 10. The total protein was extracted in cell lysis buffer . Protein concentrations were measured with the Enhanced BCA Protein Assay Kit . The protein samples were electrophoretically separated on an 8–12% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membranes were blocked for 30 min with 5% nonfat milk, sequentially incubated with primary and secondary antibodies and finally developed with ECL Western blot detection reagents  according to the manufacturer's instructions. Primary antibodies for E1A, E1B-55K, procaspase-3, poly(ADP-ribose) polymerase (PARP), Akt(Pan)(C67E7) and phospho-Akt(Ser473)(D9E) and all of the secondary antibodies were obtained from Santa Cruz Biotechnology . PTEN, cleaved-PARP and procaspase-8 antibodies were purchased from Cell Signaling Inc. . The procaspase-9 antibody was purchased from Thermo Fisher Scientific Inc. . The XIAP antibody was purchased from R&D Systems Inc.  and the tubulin antibody was manufactured by Sigma-Aldrich .2O) and documented by photography.Cells were seeded in 24-well plates and infected with Ad-PTEN, Ad.DD3.D55, Ad.DD3.D55-PTEN or wild type adenovirus at various MOIs. Five days after infection, cells were exposed to 2% crystal violet dissolved in 20% methanol for 5 min. The plates were then washed with distilled water  was added to each well after various times and the absorbance was measured in dual wavelength mode (595 nm and 655 nm). The percentage of cell viability was calculated as follows: (mean A595/A655 of infected cells)/(mean A595/A655 of uninfected cells)×100.Cells grown on glass coverslips were treated with viruses at an MOI of 10 for 48 h and stained with 1 µg/ml Hoechst 33258 .2O2 and 0.4% Triton X-100 to eliminate endogenous peroxidase activity. The sections were then blocked with the blocking serum and incubated with the corresponding antibody. The subsequent procedures were performed using the ABC Staining System  according to the manufacturer's instructions.Frozen tumor sections were incubated in 1% HThe apoptotic cells in tumor tissue sections were observed by using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining with a TACSTM 2 TdT-DAB In Situ Apoptosis Detection Kit  according to the manufacturer's instructions. The sections were counterstained with hematoxylin. All sections were visualized with an Olympus BX51 microscope .t-test.The differences between the groups were assessed using the Student's


Background:N-Glycosylation affects the biosynthesis of the endo-β1,4-glucanase KORRIGAN1 which is crucial for cell wall synthesis in plants.Results: Underglycosylated KORRIGAN1 displays reduced enzymatic activity. This is not observed upon changes to the N-glycan composition.Conclusion: Modified N-glycan processing exerts no direct effect on KORRIGAN1 function in planta.Significance: The relevance of several N-glycosylation sites for KORRIGAN1 activity is revealed. N-glycosylation and N-glycan processing frequently cause alterations in plant cell wall architecture, including changes in the structure of cellulose, which is the most abundant plant polysaccharide. KORRIGAN1 (KOR1) is a glycoprotein enzyme with an essential function during cellulose biosynthesis in Arabidopsis thaliana. KOR1 is a membrane-anchored endo-β1,4-glucanase and contains eight potential N-glycosylation sites in its extracellular domain. Here, we expressed A. thaliana KOR1 as a soluble, enzymatically active protein in insect cells and analyzed its N-glycosylation state. Structural analysis revealed that all eight potential N-glycosylation sites are utilized. Individual elimination of evolutionarily conserved N-glycosylation sites did not abolish proper KOR1 folding, but mutations of Asn-216, Asn-324, Asn-345, and Asn-567 resulted in considerably lower enzymatic activity. In contrast, production of wild-type KOR1 in the presence of the class I α-mannosidase inhibitor kifunensine, which abolished the conversion of KOR1 N-glycans into complex structures, did not affect the activity of the enzyme. To address N-glycosylation site occupancy and N-glycan composition of KOR1 under more natural conditions, we expressed a chimeric KOR1-Fc-GFP fusion protein in leaves of Nicotiana benthamiana. Although Asn-108 and Asn-133 carried oligomannosidic N-linked oligosaccharides, the six other glycosylation sites were modified with complex N-glycans. Interestingly, the partially functional KOR1 G429R mutant encoded by the A. thaliana rsw2-1 allele displayed only oligomannosidic structures when expressed in N. benthamiana, indicating its retention in the endoplasmic reticulum. In summary, our data indicate that utilization of several N-glycosylation sites is important for KOR1 activity, whereas the structure of the attached N-glycans is not critical.Defects in  N-Glycans covalently attached to asparagine residues have a vast number of diverse functions including support of protein folding, quality control processes, protein targeting, and modulation of protein-protein interactions  is a possible candidate glycoprotein that could be directly affected by changes in N-glycosylation. The precise molecular function of KOR1 and its orthologs from other species during synthesis of cellulose is still unclear  using the primers KOR1–6F (TATAGCGGCCGCCTTGATCGTTAAAACTGTGCCGC) and KOR1–7R (TATAGAATTCAAGGTTTCCATGGTGCTGGTG) and cloned into NotI- and EcoRI-digested baculovirus transfer vector pVTBacHis-1  and purification of the recombinant proteins were performed as described previously (m MES (pH 6.0), 250 mm NaCl, 30 mm CaCl2 by means of diafiltration.The KOR1 open reading frame lacking the N-terminal cytoplasmic region and transmembrane domain was amplified from BacHis-1 . In the eviously . At the N-glycosylation sites of KOR1 were introduced using the QuikChange site-directed mutagenesis kit (Stratagene) and Phusion® high-fidelity DNA polymerase. Mutagenesis was performed following the instructions of the manufacturer. The pVTBacHis-KOR1 construct was used as templates for site-directed mutagenesis, and the respective single N-glycosylation mutants were generated using the following primers: N216Q-F, CTTTCTCAAGACTTTCCAAAGTACTGCTGATTCC and N216Q-R, GGAATCAGCAGTACTTTGGAAAGTCTTGAGAAAG; N324Q-F, TCTAGCAAGTTCTATCAGTCAAGTATGTATTGG and N324Q-R, CCAATACATACTTGACTGATAGAACTTGCTAGA; N345Q-F, TATTATGCTACCGGACAAGTAACGTATCTCAAT and N345Q-R, ATTGAGATACGTTACTTGTCCGGTAGCATAATA; N408Q-F, GACTATGCTGGTCTGTTGGTGGAAGGTCCTTAG and N408Q-R, CTAAGGACCTTCCACCAACAGACCAGCATAGTC; N425Q-F, GCCTATTTTCAACAAATTTCAAAGAACCAATGGAGGTTTAA and N425Q-R, TTAAACCTCCATTGGTTCTTTGAAATTTGTTGAAAATAGGC; and N567Q-F, TGTCCGTATGAACTACCAATACACTGAACCGACTC and N567Q-R, GAGTCGGTTCAGTGTATTGGTAGTTCATACGGACA. The single mutant N324Q was used as template to generate the double mutant N324Q/N345Q, and this double mutant was subsequently used as template to obtain the triple mutant N216Q/N324Q/N345Q. To confirm the introduced mutations, all constructs were subjected to DNA sequencing using the following primers: for N216Q and N216Q/N324Q/N345Q primer, KOR1–3F (TTTGGTGGGAGGTTATTATGATGC); for N324Q, N345Q, and N324Q/N345Q primer, KOR1–14F (TGGACTATAAAAGGCCCGTGACTA); and for N408Q, N425Q, and N567Q, primer KOR1–12F (CAAGAAACCAAACCCAAACACG).Mutations in several A. thaliana ecotype Col-0 was used as a wild-type control. The rsw2-1 seeds  as described previously . Subsequently, KOR1 was excised and ligated into the XbaI/BamHI digested binary expression vector p29-Fc-GFP. p29 is derived from p27  and contFT lines  was doneFT lines . Infiltreviously . To geneR change , 15 was N-glycans of purified recombinant KOR1, proteins were separated by SDS-PAGE (10%) under reducing conditions and detected by Coomassie Brilliant Blue staining. The corresponding band was excised from the gel, destained, carbamidomethylated, in-gel digested with trypsin/chymotrypsin, and analyzed by LC-ESI-MS as described recently , 250 mm NaCl, and 30 mm CaCl2 were incubated for 60 min at 30 °C with 90 μl of 0.1% (carboxymethyl)cellulose 4M (Megazyme) resuspended in the same buffer. The reaction was stopped by the addition of 400 μl of ice-cold 50 mm sodium borate (pH 10.0). 500 μl of freshly prepared 2,2′-bicinchoninic acid solution  and reagent B ) was added, and the samples were incubated for 15 min at 95 °C. Samples were cooled on ice and then centrifuged for 3 min prior to measurement of their absorbance at 562 nm. Samples containing heat-inactivated enzyme were used as controls. The amount of reducing ends generated was then deduced using a glucose standard curve (0–100 nmol). All assays were done in duplicates. Enzymatic deglycosylation of KOR1 under non-reducing conditions prior to activity assays was performed without (control) or with endoglycosidase H (Endo H)  or peptide N-glycosidase F (PNGase F)  in 50 mm MES (pH 6.0), 250 mm NaCl, 30 mm CaCl2 for 60 min at 30 °C. Activity assays were then performed as outlined above. To analyze enzyme stability, rKOR1 proteins were incubated for 30 to 240 min at 30 °C prior to activity tests. Statistical analyses were performed using Student's t test, with p < 0.05 considered significant.Solutions (10 μl) of purified recombinant KOR1 (rKOR1) produced in insect cells (0.35–4 μg) in 50 mA. thaliana KOR1 contains eight potential N-glycosylation sites . To analyze the role of N-glycosylation for KOR1 enzymatic activity we expressed an A. thaliana KOR1 variant lacking the N-terminal 90 amino acids (which include the cytoplasmic tail and the single transmembrane domain) in S. frugiperda Sf21 cells using a baculovirus expression system. Immunoblot analysis of cell extracts and culture supernatants revealed that recombinant wild-type KOR1 (rKOR1 WT) was successfully expressed in Sf21 cells and secreted into the culture medium. The secreted His6-tagged rKOR1 protein was purified by means of nickel-chelate affinity chromatography and subjected to enzymatic deglycosylation with Endo H, which cleaves oligomannosidic N-glycans, and PNGase F, which cleaves oligomannosidic and complex N-glycans lacking core α1,3-fucose , the predominant structures present on the other seven N-glycosylation sites were Golgi-processed paucimannosidic N-glycans (Man3GlcNAc2 and Man3FucGlcNAc2). Only minor peaks with masses that match to oligomannosidic N-glycans were found , although the rKOR1 content of the respective cell extracts was similar (data not shown). This suggests that absence of the N-glycan attached to Asn-567 interferes with proper folding of the enzyme in the endoplasmic reticulum of the insect cells in a subtle manner. In contrast, the amount of secreted rKOR1 N324Q/N345Q was strongly reduced as compared with wild-type and rKOR1 N345Q (B and C), indicating that folding of the double mutant was heavily impaired. LC-ESI-MS analysis of purified rKOR1 forms confirmed that all single-mutant proteins lacked only the N-glycan usually attached to the mutated asparagine residue (data not shown).To examine the impact of individual  of KOR1 . In addionserved –20. To f mutants A, althouR1 N345Q , B and CN-glycosylation sites are not critical for maximal enzymatic activity. Mutations at glycosylation sites Asn-216, Asn-324, and Asn-345 reduced KOR1 enzymatic activity by 15–30%. Removal of the oligomannosidic N-glycan on glycosylation site Asn-567 resulted in the most pronounced difference with ∼50% of wild-type activity (We chose to assay enzymatic activity of rKOR1 WT and the mutant variants using carboxymethylcellulose ((carboxymethyl)cellulose 4M) as substrate and a reducing sugar assay to monitor the released glucose. In previous studies, (carboxymethyl)cellulose 4M was proven to be the best substrate, when KOR1 orthologs were analyzed with different known cellulose derivatives , 20. rKOactivity .N-glycans, rKOR1 WT and mutated forms were incubated at 30 °C over a time range from 30 to 240 min prior to activity assays. rKOR1 WT and all single mutants except for N425Q retained at least 90% of their enzymatic activity after preincubation for 120 min (data not shown). In contrast, the activity of rKOR1 N425Q was found to be reduced to 69% (120 min) or 44% (240 min). The sensitivity of the activity assay was tested using varying amounts of purified proteins. At least 350 ng of recombinant protein per assay were needed to obtain reliable results. The hydrolysis rate of (carboxymethyl)cellulose 4M did increase in a linear manner up to 4 μg of enzyme protein used per assay.To ensure that the effect on KOR1 activity is not a matter of protein instability due to the loss of individual B. napus and P. tremula x tremuloides KOR1 orthologs caused an almost complete loss of enzymatic activity . In accordance with data for rKOR1 WT, the shift in mobility was more pronounced after treatment with PNGase F. The slight shift of rKOR1 N567Q in the Endo H-digested sample results very likely from removal of minor amounts of oligomannosidic N-glycans present on the other seven N-glycosylation sites.Previously, it was shown that enzymatic deglycosylation of the heterologously produced activity , 20. To observed A. In accB), whereas Endo H treatment of rKOR1 resulted in a loss of 20–30% compared with the initial enzymatic activity (B). Interestingly, the specific activity of the single mutants N216Q, N324Q, N345Q, N408Q, and N425Q after deglycosylation with PNGase F was at least as high as that of deglycosylated rKOR1 WT. The activities of glycosylation mutants treated with Endo H were comparable with that of Endo H-digested rKOR1 WT, being reduced by 20–30% as compared with the undigested forms.Upon deglycosylation with PNGase F, rKOR1 displayed ∼50% residual enzymatic activity B, whereaactivity B. IntereN-glycosylation profile for rKOR1 activity, we expressed rKOR1 WT in insect cells in the presence of kifunensine (rKOR1 KIF), a class I α-mannosidase inhibitor that prevents processing of oligomannosidic glycans in the endoplasmic reticulum. As a consequence of kifunensine treatment, all eight N-glycans on rKOR1 should contain Man5–9GlcNAc2 oligosaccharides instead of the paucimannosidic structures detected on the wild-type enzyme. Immunoblot analysis and subsequent affinity purification showed that rKOR1 KIF was present in the culture supernatant in amounts comparable with untreated rKOR1 WT (A). The reduced mobility on SDS-PAGE indicated that rKOR1 KIF indeed contains largely unprocessed oligomannosidic N-glycans (A). LC-ESI-MS analysis of glycopeptides from rKOR1 KIF confirmed the presence of predominantly oligomannosidic N-glycans (Man7GlcNAc2 to Man9GlcNAc2) . Notably, the enzymatic activity of rKOR1 KIF was indistinguishable from rKOR1 WT . Collectively, these data suggest that N-glycosylation is important for KOR1 activity, but a distinct N-glycan structure is not required for its function.To analyze the significance of the observed rKOR1 WT A. The re-glycans A. LC-ESIrKOR1 WT , indicatN. benthamiana leaf epidermal cells. To facilitate purification and subcellular localization, the Fc domain of the human IgG1 heavy chain and GFP were fused to the C-terminal end of KOR1. To distinguish between complex and oligomannosidic N-glycans, KOR1-Fc-GFP was expressed in N. benthamiana wild-type and ΔXTFT lines  as predominant glycoform  was generated, which lacks all N-glycosylation sites in the potential catalytic cleft of KOR1. All detectable glycopeptides derived from the mutant variants displayed a similar glycosylation profile as observed for wild-type KOR1 (A and B). These findings demonstrate that the N-glycans of the wild-type enzyme and the single, double, and triple mutants tested are all properly processed in the plant Golgi. However, enzymatic analysis of wild-type and mutant KOR1-Fc-GFP could not be performed due to insufficient amounts of purified protein.To determine whether individual ype KOR1 , A and BN. benthamiana showed the presence of oligomannosidic and complex N-glycans. To investigate the glycosylation of endogenous A. thaliana KOR1, proteins extracted from A. thaliana Col-0 seedlings as well as from the fut11 fut12 mutant  . Consequently, to investigate the nature of the N-glycans on KOR1 G429R, we expressed a KOR1 G429R-Fc-GFP variant in N. benthamiana, purified the protein, and analyzed its glycopeptides by LC-ESI-MS. Although the obtained amounts of purified KOR1 G429R-Fc-GFP were considerably lower than for KOR1-Fc-GFP, we could identify four of the eight glycopeptides. The identified glycopeptides displayed a predominant peak corresponding to the mass of Man9GlcNAc2, with additional peaks corresponding to Man8GlcNAc2 and Glc1Man9GlcNAc2 . The presence of oligomannosidic structures and glucose-containing oligosaccharides instead of complex N-glycans indicates that KOR1 G429R-Fc-GFP is retained by quality control processes in the ER and hence does not reach the Golgi. Collectively, these data strongly suggest that the severe phenotype of cgl1 rsw2-1 is not caused by an N-glycan processing defect that directly affects KOR1 glycosylation.The phenotype of the 1 G429R) . The deshe Golgi . The amon status A. ConseqN-Glycosylation is a major co- and post-translational modification in plants and the majority of all proteins destined for different compartments of the endomembrane pathway are glycosylated. The N-glycan biosynthesis and processing pathways of A. thaliana are already quite well understood  could not be efficiently expressed in insect cells, whereas the full-length KOR1-Fc-GFP variant with the same mutations was produced in comparable amounts as wild-type KOR1 in N. benthamiana. The presence of complex N-glycans demonstrates further that this mutant variant is processed in the plant Golgi, suggesting normal transport through the endomembrane system. In future studies, it remains to be shown whether the different N-glycan mutants can complement kor1 mutants and whether N-glycosylation of KOR1 is required for its function in plants.Notably, the effects of the different A. thaliana seedlings using affinity purification with different antibodies against KOR1. However, despite numerous efforts, it was not possible to enrich sufficient amounts of endogenous KOR1 to perform glycosylation site analysis by mass spectrometry. Consequently, we transiently expressed a KOR1 fusion protein in leaves of N. benthamiana for analysis of its N-glycan composition when produced in planta. In accordance with data from endoglycosidase treatment and immunoblotting of endogenous KOR1 from A. thaliana seedlings, we detected oligomannosidic as well as processed complex N-glycans. Notably, we observed minor differences between N-glycans present on recombinant KOR1 produced in insect cells and KOR1 expressed in N. benthamiana leaf epidermal cells. These differences are found on the first two and the last N-glycosylation sites and are very likely caused by the presence/absence of different protein domains. In insect cells, we expressed a soluble KOR1 variant lacking the N-terminal cytoplasmic region and the single transmembrane domain to obtain a secreted form of the protein that can be readily purified from cell supernatants and used for in vitro activity assays. These constructs were designed to allow a direct comparison with previous studies relying on recombinant enzymes produced in P. pastoris  in the variant produced in insect cells, which lacks the 90 most N-terminal amino acid residues.We attempted to purify native KOR1 from pastoris , 20. In N-glycan maturation as well as the different fate of the double N-glycosylation site mutant. Plants contain, for example, two calnexin and three calreticulin proteins, and calreticulin 3 seems specific for plants and has been found to play an important role in ER quality control processes of heavily glycosylated leucine-rich repeat receptor kinases  in planta. The presence of oligomannosidic N-glycans on KOR1-G429R-Fc-GFP (which is indicative of ER retention) further corroborates this finding. The observed additive phenotype of cgl1 rsw2-1 and mns3 rsw2-1 could be caused by a combination of the KOR1 defect in rsw2-1 and the N-glycan processing defect involving one or several other glycoproteins that could (but not necessarily have to) act in the same cellular pathway (cellulose synthesis). Alternatively, the less severe phenotype of rsw2-1 compared with other kor1 mutants (N-glycan processing in mns3 and cgl1 could still directly affect the subcellular localization or function of KOR1 G429R.The finding that the perature , 23 led function , 24. Blos mutant –53. The  mutants , 17 indirsw2-1 with other N-glycosylation and N-glycan processing mutants (N-glycans in cellulose biosynthesis.Further studies such as the combination of  mutants , 54, 55


Granulocyte colony-stimulating factor (G-CSF), a hematopoietic cytokine, was recently used to treat patients of acute myocardial infarction with beneficial effect. However, controversy exists as some patients developed re-stenosis and worsened condition post G-CSF delivery. This study presents a new disease model to study G-CSF induced cardiac thrombosis and delineate its possible mechanism. We used iron loading to mimic condition of chronic cardiac dysfunction and apply G-CSF to mice to test our hypothesis.Eleven out of fifteen iron and G-CSF treated mice (I+G) showed thrombi formation in the left ventricular chamber with impaired cardiac function. Histological analysis revealed endothelial fibrosis, increased macrophage infiltration and tissue factor expression in the I+G mice hearts. Simvastatin treatment to I+G mice attenuated their cardiac apoptosis, iron deposition, and abrogated thrombus formation by attenuating systemic inflammation and leukocytosis, which was likely due to the activation of pAKT activation. However, thrombosis in I+G mice could not be suppressed by platelet receptor inhibitor, tirofiban.Our disease model demonstrated that G-CSF induces cardiac thrombosis through an inflammation-thrombosis interaction and this can be attenuated via statin therapy. Present study provides a mechanism and potential therapy for G-CSF induced cardiac thrombosis. Granulocyte colony-stimulating factor (G-CSF), a hematopoietic cytokine, induces mobilization of the hematopoietic stem cells from the bone marrow into the peripheral blood circulation. In traditional bone marrow transplantation, G-CSF is given to healthy donors for allogenic hematopoietic cell collection ,2. Recenin vivo disease model to study G-CSF induced cardiac thrombosis in mice is presented. We assumed that patients with atherosclerosis, diabetes, chronic heart failure, or other diseases with chronic inflammation or vasculopathy may be at higher risk for thrombosis after G-CSF treatment. Since chronic iron loading increases vascular oxidative stress and accelerate atherosclerosis [In the present study, a new clerosis -17; we pclerosis ,19. UsinIn order to test whether G-CSF can mobilize autologous stem cells, we divided male C57BL/6 mice (bw 25-30 gm) into four groups (n = 5/group) and injected them with 50, 100, 200 μg/kg bw G-CSF or saline daily for 5 days respectively. Blood serum was then harvested for flow analysis.in vivo cardiac echocardiography at the end of the second and fourth week. Similar protocols of iron loading and G-CSF supplement to mice were previously described [Male C57BL/6 mice (body weight (bw): 25-30 gm) were divided into four experimental groups (n = 15-18/group). (1) Iron loading and G-CSF supplement (I+G group): 10 mg/25 gm bw/day iron dextran (Sigma-Aldrich Co. U.S.A.), was injected five times/week intraperitoneally (ip) for 4 weeks, and 100 μg/kg bw recombinant human G-CSF , was administered five times/week subcutaneously during the second week. (2) G group: Dextrose (0.1 ml of 10%) instead of iron dextran was injected five times/week for 4 weeks. G-CSF was administered as in I+G group. (3) I group: 0.1 ml saline (instead of G-CSF) was administered subcutaneously five times/week during the second week and iron dextran was injected as I+G group. (4) Control or C group: Only 10% dextrose and saline solutions were administered as in I+G group Figure . Mice unescribed .st, 3rd, and 5th/week) in addition to four weeks of I+G treatment. Mice were divided into the following four groups (n = 10/group), I+G group, I+G plus simvastatin group (I+G+St), iron only group (I), and control or C. Protocols for iron loading and G-CSF supplement were the same as before. A third set of male C57BL/6 mice were injected with tirofiban  using Alzet minipumps  for the first two weeks in addition to four weeks of I+G treatment. Mice were divided into the following three groups (n = 10/group), I+G group, I+G plus tirofiban group, and control group. Complete blood counts and leukocyte classification were checked with the CELL-DYN® 3700  and serum C-reactive protein , ICAM-1 and MCP-1 level were determined with the Quantikine® ELISA  using an ELISA plate reader at 450 nm with a correction at 570 nm.The second set of male C57BL/6 mice were injected (ip) with 10 mg/kg bw simvastatin  for first two weeks . The anterior chest was shaved and laid in a left decubitous position with application of gel on the chest wall for better scanhead-skin contact. The echocardiography system  was equipped with 2D, M-mode, and pulse wave Doppler imaging. Heart rate, left-ventricle (LV) dimension in both systolic and diastolic stages, the LV fractional shortening/ejection fraction and mitral valvular inflow with diastolic E and A waves in Doppler flow mapping were measured.Mice were perfused through the LV with 4% paraformaldehyde in 0.1 M PBS. The paraffin-embedded cardiac cross sections (5 μm) were stained with Hematoxylin & Eosin, Masson's trichrome and iron-specific-Prussian blue. Trichrome-stained sections were used to detect a cumulative index of myocardial damage, including fibrosis and inflammation. The cardiac coronary artery and liver paraffin section were stained with Hematoxylin & Eosin.2O2 and blocking 5% fetal bovine serum. The sections were then incubated with first antibody with rabbit anti-tissue factor , mouse anti-8-OHdG , mouse anti-HNEJ-2 , mouse anti-CD45  and mouse anti-CD34 . Thereafter treated with a 1:200 dilution of biotinylated anti-mouse and anti-rabbit IgG antibody , followed by horseradish peroxidase (HRP)-conjugated streptavidin-biotin complex (Vectastain Elite ABC kit standard) for 1 hour at room temperature and then used 3,3-diaminobenzidine (DAB) as a chromogen , and counterstained with Contrast GREEN Solution  for microscopic studies.Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M PBS and post fixed with the same fixative overnight at 4°C. Coronal heart were paraffin-embedded and tissue sections were cut into 5 μm thickness. After blocking deparaffinized sections and then treated with epitope retrieval buffer  in 95~100°C for 30 min, and then quenched with 30% HFor immunofluorescent staining, sections were first rehydrated and epitope retrieval buffer  in 95~100°C for 30 min. Sections were then washed and blocked with 5% fetal bovine serum for 1 hr. Sections were then double-stained with antibodies against TF  and CD13 (1:100) overnight at 4°C. Different Fluorescein  and Rhodamine  secondary antibodies (Jackson ImmunoResearch Lab. Inc.) were used to obtain fluorescent colors. The stained sections were counterstained with DAPI to visualize nuclei by ProLong antifade (Invitrogen) mounting reagent.Flow cytometry analysis was performed with FACSCalibur and CellQuest Pro software  using directly conjugated mAbs against the following markers: CD11b-PE and Ly-6G-FITC or CD45-PE and CD117-PE (c-kit) (BD biosciences) with corresponding isotype matched controls. Blood samples were washed with PBS buffer and red blood cells were removed by RBC lysis buffer. Briefly, mAbs and cells were incubated for 30 minutes at 4°C and unbound reagents were removed by washing. Cells were then resuspended in fixing buffer  for flow analysis.Assays were performed using Applied Biosystems PRISM 7700 sequence detection system with cDNAs derived from mice treated with or without G-CSF following iron injection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. Thermal cycler conditions were as follows: hold for 2 min at 50°C and 10 min at 95°C, followed by two-step PCR for 35 cycles of 95°C for 15 s, then 60°C for 1 min. Forward and reverse primers and a fluorescence-labeled probe were as follows: ICAM-1 sense, 5'- CGC AAG TCC AAT TCA CAC TGA -3', and antisense, 5'- ATT TCA GAG TCT GCT GAG AC -3); MCP-1 sense, 5'- CAG CCA GAT GCA GTT AAC GC -3', and antisense, 5'- GCC TAC TCA TTG GGA TCA TCT TG -3'); tissue factor sense, 5'- AAG GAT GTG ACC TGG GCC TAT GAA -3', and antisense, 5'- ACT GCT GAA TTA CTG GCT GTC CGA T-3'); TNF-α sense, 5'- TAC TGA ACT TCG GGG TGA TTG GTC C -3', and antisense, 5'- GGT TCT CTT CAA GGG ACA AGG CTG -3') and GAPDH sense, 5'-GGA GCC AAA CGG GTC ATC ATC TC-3', and antisense, 5'-GAG GGG CCA TCC ACA GTC TTC T-3'). The relative expression ratio of each transcript  in comparison to GAPDH was calculated as described.Myocardium protein extracts were prepared by using a protein extraction kit (NE-PER), and total protein concentrations was determined by BCA™ protein assay reagent. Western Blot chemiluminescence reagents were obtained from PIERCE . Proteins were separated by polyacrylamide gel electrophoresis and transferred to PVDF membranes for Western blot analysis. Blots were incubated with either anti-p-AKT (1:1000), anti-AKT (1:1000), anti-eNOS (1:1000) , anti-MPO (1:500)  and anti-β-actin (1:2000) antibodies in non-fat dry milk in wash buffer overnight at 4°C. Blots were then incubated with peroxidase conjugated anti-rabbit  or anti-goat  for 1 hour at room temperature. Proteins were visualized by enhanced chemiluminescence, immunoblot signals were quantitated using a Fujifilm Medical Systems U.S.A., Inc.t-test. Statistical analysis was performed with one-way ANOVA by Tukey test for multiple comparisons. The differences were considered significant at a value of P < 0.05.Statistical analysis was done by SPSS for Windows (version 12.0). All data are described as means ± standard deviation (S.D.). The two groups were compared using the Student's th week showed that heart functions in the I+G group was abnormal with decrement in fractional shortening and mild chamber dilation in the left ventricle (LV) without affecting the heart rate  and CD45(+) cells from G-CSF injected mice to confirm that G-CSF can mobilize stem cells and leukocytes in a dosage dependent manner in our mice model before analyzing any phenotype Figure . Echocarte Table . In addik Figure , Table 1The mural thrombi found in I+G mice were mainly located in the apex region of the LV Figure , but alsCellular compositions of the all groups were examined by immunohistochemistry. Tissue factor was upregulated within the myocardium where it may be mediated by the infiltrating cells in both I and I+G groups, with more prominent in the latter group Figure . ConfocaIn order to elucidate the role of G-CSF in our I+G model, we compared the heart tissue from both I group and I+G group for oxidative stress, leukocyte infiltration and inflammatory profile between them. As expected, I+G hearts had higher levels of 4-HNE and 8-OHdG (both are index of oxidative stress), and increased expression of CD45 (leukocyte marker) Figures  and 4B. th week and compared. Incidence of thrombi formation were 0/10 in the control group, 7/10 in the I+G, and 2/10 in the I+G+St groups (p < 0.05 versus I+G group), respectively. Concomitant TUNEL assay and iron staining showed a significant decrease in apoptotic cardiomyoctes  and the control group was collected at the end of 4 Figures  and 5C a Figures  and 5D ith week recheck, leukocytosis was aggravated in the I+G mice, but attenuated in the I+G+St mice  and endothelial nitric oxide synthase (eNOS) increased in the hearts of the G plus statin and I+G+St groups, as compared to other groups Figure . These rin vivo can be attenuated by simvastatin therapy, but not by tirofiban treatment. Our results confirmed that G-CSF can induce in vivo cardiac thrombosis through inflammation-thrombosis interaction.Results of the present study demonstrate that G-CSF supplement on iron loading hearts can recruit neutrophils/monocytes and up-regulate tissue factors, ICAM-1, TNF-alpha, and MCP-1 thus further activating inflammatory processes in the endo-myocardium and induce cardiac thrombosis. Chronic iron loading can increase cardiac oxidative stress. Whereas G-CSF treatment activates serial events of inflammation-thrombosis circuitry and that leads to intra-cardiac thrombus formation. This inflammation-associated cardiac thrombosis in vivo mouse model could be a novel avenue for investigating inflammation and thrombosis interactions in the cardiac endothelium, compared to previous studies that focused mainly on the vascular endothelium [Iron overload is known to accelerate arterial thrombosis through increased vascular oxidative stress and impaired vascular reactivity ,21 and iothelium ,28.Iron loading has multiple effects on all body tissues, including cardiac myocytes and macrophages. For example, in a similar iron overload model (with chronic iron treatment for 12 weeks) showed increased cardiac interstitial fibrosis in addition to inflammatory infiltration . Iron-ovOur results showing that G-CSF can promote inflammatory profiles and cardiac thrombosis that leads to cardiac dysfunction, are in contrast to previous reports showing G-CSF therapy to be beneficial in acute myocardial infarction ,4,31,32 HMG-CoA reductase inhibitors, or statins, are known to improve cardiac dysfunction through their anti-inflammatory and anti-oxidative action. Statins also affect endothelial function through the production of nitric oxide ,19. Presin vivo disease model to elucidate the mechanism of post G-CSF cardiac thrombosis, which could have major clinical implication.Recently meta-analysis results from 10 clinical trials for stem cell mobilization by G-CSF therapy for myocardial recovery after AMI showed neither improvement of LV function or the reduction in infarct size in patients with AMI after reperfusion . In ordeThe authors declare that they have no competing interests.WSL and CFC designed the experiments and analyzed the data. WSL and HL performed the in vivo study. HL performed the in vitro study. TK analyzed the cardiac pathology. WTKC and TK help to coordinate this study. CFC wrote the manuscript. All authors have read and approved the final manuscript.Histology of I+G mice and blood parameters of I+G mice with tirofiban treatment. A figure demonstrating histology of other organs in I+G mice and a table listing blood parameters of I+G mice with or without tirofiban therapy.Click here for file


Both unmethylated and methylated B-DNA were found to be more flexible than Z-DNA. However, methylation significantly destabilized the BII, relative to the BI, state through the Gp5mC steps. In addition, methylation decreased the free energy difference between B- and Z-DNA. Comparisons of α/γ backbone torsional angles showed that torsional states changed marginally upon methylation for B-DNA, and Z-DNA. Methylation-induced conformational changes and lower energy differences may contribute to the transition to Z-DNA by methylated, over unmethylated, B-DNA and may be a contributing factor to biological function.Methylation of cytosine at the 5-carbon position (5mC) is observed in both prokaryotes and eukaryotes. In humans, DNA methylation at CpG sites plays an important role in gene regulation and has been implicated in development, gene silencing, and cancer. In addition, the CpG dinucleotide is a known hot spot for pathologic mutations genome-wide. CpG tracts may adopt left-handed Z-DNA conformations, which have also been implicated in gene regulation and genomic instability. Methylation facilitates this B-Z transition but the underlying mechanism remains unclear. Herein, four structural models of the dinucleotide d(GC) DNA methylation is one of the main epigenetic modifications contributing to gene regulation and a considerable amount of scientific effort has been devoted to understanding the mechanisms, roles, and effects of methylation in healthy and diseased states DNA has a flexible backbone structure characterized by fluctuations in the torsional angles α, γ, ε, ζ, and sugar pucker (δ) See . The α, anti to the syn conformation of the alternating purines and has been shown to involve base extrusion at both B-Z junctions Most repetitive DNA sequences can assume different structures apart from canonical B-DNA, including cruciforms, triplexes, hairpins, quadruplexes and Z-DNA Molecular dynamics (MD) simulations have been used extensively to study DNA structure and dynamics 5 repeat sequence were constructed in ideal B-, methylated B-, Z-, and methylated Z-DNA forms. Two 50 nanoseconds MD simulations in explicit solvent under physiological conditions were performed for each, reaching a 0.4 microsecond aggregate simulation time. An analysis of the trajectories was then performed to examine the effect of methylation on the BI/BII stability in B-DNA and the sampling of non-canonical α/γ backbone torsional states (g+/t (gauche+/trans), g−/t (gauche−/trans), and g+/g− (gauche+/gauche−)) during the B- and Z-DNA simulations. The results indicate that methylation lowered the free energy difference between B- and Z-DNA. This suggests a lower energetic barrier, which may help explain the more facile change to Z-DNA by the methylated, rather than unmethylated, B-DNA.In an attempt to bridge this knowledge gap, structural models of a d(GC•GC)In this study, the effects of cytosine methylation on DNA dynamics are examined, including flexibility, base pair step parameters, base pair geometry and backbone torsion angles  in both The average structures of the heavy atoms of non-terminal bases obtained from the trajectories are used to calculate root mean square deviations (RMSD) . The resThe sequence averaged conformational parameters from the eight independent simulations for base pairs , base paBase pair step parameters are within one standard deviation of the experimentally observed values  are 1.6 kcal/mol in B-DNA and 2.08 kcal/mol in 5mCB-DNA. Interestingly, when the free energy profiles for the CpG versus GpC steps are separated, an increase in ΔGBI–BII from 1.02 kcal/mol (B-DNA) to 1.98 kcal/mol (5mCB-DNA) is revealed for the GpC steps, whereas no effect is observed for the CpG steps. (ΔGBI–BII = 0.16 between B-DNA and 5mCB-DNA) (mCB-DNA) . Also, tmCB-DNA)  are alsoparmbsc0 parameter set The effects of methylation on the BI/BII states suggest that methylation caused significant backbone torsional rearrangements. In solution, free B-DNA is mostly found in the canonical (g−/g+) α/γ states; in protein-DNA complexes, on the other hand, DNA exhibits a higher percentage (∼15%) of non-canonical states trans conformations in the cytosine backbone (GpC steps) after methylation, thereby switching to g+ Our results confirm that unmethylated B-DNA almost exclusively samples canonical α/γ states and methylation only marginally increases sampling of non-canonical states, particularly for the Gp5mCp steps. By contrast, unmethylated and methylated Z-DNA sample non-canonical conformations extensively (∼30–50% of the time) . It has MM/PBSA parm99 force field in the form of extensive α/γ transitions Parmbsc0 force field MD simulations have been extensively employed to study nucleic acids dynamics The overlapping conformational states by concerted phosphate backbone torsional angle switches observed in both methylated B- and unmethylated/methylated Z-DNA  agree wiIn summary, MD simulations were used to further understand the role of cytosine methylation on both the canonical B-DNA and non-canonical left-handed Z-DNA structures. The results show that methylation lowers the free energy difference between B and Z-DNA resulting in the increased population of Z-DNA. We suggest that methylation-induced differences in the CpG and GpC steps' backbone dynamics may facilitate the initial step in the mechanism of B to Z transitions.5 repeats were built using the canonical B- and Z-DNA settings in w3DNA Four structural models of 10 base paired d(GC•GC)parm99parmbsc0 corrections parm99+ ions and solvated with approximately 5000 water molecules for the B-DNA models and 5450 water molecules for the Z-DNA models in truncated octahedral boxes. Additional Na+ and Cl− ions were added by randomly replacing water molecules, to bring the system to 150 mM salt concentration . The radial distribution functions of counterions around DNA for all simulations (data not shown) agree well with the previously published distributions 2 harmonic restraints on all solute atoms. The harmonic restraints were then reduced to 50, 10, 5, and 1 kcal/mol/Å2 in 20 ps intervals, followed by 380 ps of unrestrained equilibration at constant pressure and temperature. Two independent 50 ns-long production runs were performed for each system starting with different initial velocities. The aggregate simulation time was 400 nanoseconds. Trajectories were analyzed using the MM/PBSA and PTRAJ modules of AMBER 10, as well as Curves+ MD simulations were performed using the AMBER 10 simulation package MM+EPB+ESA−TS, where EMM is the molecular mechanics energy, EPB is Poisson Boltzmann energy, ESA is the nonpolar solvation free energy and TS is the entropic contribution. ESA was assumed to be proportional to the solvent accessible surface area (SA), i.e. ESA = γSA+b, with the coefficients set to default γ = 0.00542 kcal/Å2 mol and b = 0.92 kcal/mol. The AMBER nmode program was used to estimate the vibrational entropies after one thousand step energy minimization Conformational free energies were calculated by the MM/PBSA method using the perl scripts available within the AMBER 10 Figure S1Time evolution of root mean square deviation (RMSD) of the heavy atoms from their mean positions during the simulations. A. B-DNA B. 5mCB-DNA C. Z-DNA D. 5mCZ-DNA. Black and red lines represent the two independent runs for each system. The terminal bases are excluded.(PNG)Click here for additional data file.Figure S2Representative distributions of base pair parameters in B-DNA (black) and 5mCB-DNA (red) simulations. Vertical dashed lines indicate the mean ± one standard deviation of the crystallographically determined values see . Top row(PNG)Click here for additional data file.Figure S3Representative distributions of base pair step parameters in B-DNA (black) and 5mCB-DNA (red) simulations. Vertical dashed lines indicate the mean ± one standard deviation of the crystallographically determined values see . Top row(PNG)Click here for additional data file.Figure S4Representative distributions of base pair step parameters for GpC and CpG steps in B-DNA and 5mCB-DNA simulations. Black and green lines show the CpG steps, whereas red and blue lines show the GpC steps for the B-DNA and 5mCB-DNA simulations, respectively. Y-axes show the counts. X-axes are in Angstroms in the top row  and degrees in the bottom row .(PNG)Click here for additional data file.Figure S5Representative distributions of base pair parameters in Z-DNA (black) and 5mCZ-DNA (red) simulations. Y-axes show the densities. X-axes are in degrees in the top row  and Angstroms in the bottom row .(PNG)Click here for additional data file.Figure S6Representative distributions of base pair parameters for GpC and CpG steps in Z-DNA and 5mCZ-DNA simulations. Black and green lines show the CpG steps, whereas red and blue lines show the GpC steps for the Z-DNA and 5mCZ-DNA simulations, respectively. Y-axes show the densities. X-axes are in Angstroms in the top row  and degrees in the bottom row .(PNG)Click here for additional data file.Figure S7Representative distributions of base pair step parameters in Z-DNA (black) and 5mCZ-DNA (red) simulations. Note that the two bimodal distributions in slide and twist correspond to GpC vs. CpG steps . y-axes show the densities. x-axes are in Angstroms in the top row  and degrees in the bottom row .(PNG)Click here for additional data file.Figure S8Representative distributions of base pair step parameters for GpC and CpG steps in Z-DNA and 5mCZ-DNA simulations. Black and green lines show the CpG steps, whereas red and blue lines show the GpC steps for the Z-DNA and 5mCZ-DNA simulations, respectively. Y-axes show the densities. X-axes are in Angstroms in the top row  and degrees in the bottom row .(PNG)Click here for additional data file.Figure S9Representative distributions of sugar phosphate backbone torsional angles in B-DNA (black) and 5mCB-DNA (red) simulations. The x axes are in degrees.(PNG)Click here for additional data file.Figure S10Representative distributions of sugar pucker parameters in B-DNA (black) and 5mCB-DNA (red) simulations. The x axes are in degrees.(PNG)Click here for additional data file.Figure S11Representative distributions of sugar phosphate backbone torsional angles in Z-DNA (black) and 5mCZ-DNA (red) simulations. The x axes are in degrees.(PNG)Click here for additional data file.Figure S12Representative distributions of sugar pucker parameters in Z-DNA (black) and 5mCZ-DNA (red) simulations. The x axes are in degrees.(PNG)Click here for additional data file.Figure S13Time evolution of ε- ζ for individual bases in representative unmethylated B-DNA (blue) and methylated 5mCB-DNA (red) simulations for base pairs 2–4 showing the jumps between BI and BII states. Left columns are Watson strand, and right columns are Crick strand. A ε-ζ<0 indicates that the base is in the BI conformation.(PNG)Click here for additional data file.Figure S14Time evolution of ε- ζ for individual bases in representative B-DNA (blue) and 5mCB-DNA (red) simulations for base pairs 6–9 showing the jumps between BI and BII states. Left columns are Watson strand, and right columns are Crick strand. A ε-ζ<0 indicates that the base is in BI conformation.(PNG)Click here for additional data file.Figure S15Cumulative means of the fraction of BI conformations (top) and the fraction of canonical conformations (bottom) in B-DNA (black) and 5mCB-DNA (red) simulations. Solid and dashed lines indicate the two independent MD runs. Note that in one methylated B-DNA trajectory the 3′ terminal base pair breaks and reforms around 20–25 ns.(PNG)Click here for additional data file.Figure S16Scatter plots of α vs. γ for CpG and GpC steps of Z-DNA simulations. (A) Z-DNA GpC steps; (B) Z-DNA CpG steps; (C) 5mCZ-DNA Gp5mC steps; (D) 5mCZ-DNA 5mCpG steps. The plots are color-coded based on the density of points.(PNG)Click here for additional data file.Table S1Sequence-averaged conformational parameters I: Base pair parameters(DOC)Click here for additional data file.Table S2Sequence-averaged conformational parameters II: Base pair step parameters(DOCX)Click here for additional data file.Table S3Sequence-averaged conformational parameters III: Helical parameters(DOCX)Click here for additional data file.Table S4Sequence-averaged conformational parameters IV: Backbone torsions(DOCX)Click here for additional data file.Table S5Average geometric parameters from high resolution X-ray structures(DOCX)Click here for additional data file.Table S6Percent of sugar pucker values from the eight simulations.(DOCX)Click here for additional data file.Table S7Percent of α/γ conformational states for the 8 composite MD simulations.(DOCX)Click here for additional data file.


Human T-cell leukemia virus type I (HTLV-I) has efficiently adapted to its host and establishes a persistent infection characterized by low levels of viral gene expression and slow proliferation of HTLV-I infected cells over decades. We have previously found that HTLV-I p30 is a negative regulator of virus expression.In this study we show that p30 targets multiple cell cycle checkpoints resulting in a delayed entry into S phase. We found that p30 binds to cyclin E and CDK2 and prevents the formation of active cyclin E-CDK2 complexes. In turn, this decreases the phosphorylation levels of Rb and prevents the release of E2F and its transcriptional activation of genes required for G1/S transition. Our studies also show that HTLV-II p28 does not bind cyclin E and does not affect cell cycle progression.In contrast to HTLV-I, the HTLV-II-related retrovirus is not oncogenic in humans. Here we report that the HTLV-I p30 delays cell cycle progression while its homologue, HTLV-II p28, does not, providing evidence for important differences between these two related retrovirus proteins. Cell cycle progression from G1 to S phase is regulated by the sequential activation of two kinase-complexes, CDK4/6-cyclin D and Cyclin E-CDK2 .Unlike the cyclin D-dependent kinases, the activity of cyclin E-Cdk2 is intermittent and reaches a maximum at the G1- to S-phase transition -6. CycliHuman T-cell leukemia virus type I (HTLV-I) was originally isolated from a patient with cutaneous T-cell lymphoma . HTLV-I We previously demonstrated that p30 is a post-transcriptional repressor of HTLV-I replication . AdditioLentiviral particles expressing p30-myc or GFP were prepared by transfection of 293FT cells with HR-CMV-p30myc or GFP with pDLN and VSV-G, respectively, as previously reported . The genHela cells and 293FT were obtained from the ATCC (American Type Culture Collection). They were maintained in Dubelcco modified Eagle medium, DMEM, complemented with 10% of fetal bovine serum (Gibco) and 1% penicillin-streptomycin. Jurkat T cells were cultured in RPMI complemented with 10% of fetal bovine serum (Gibco) and 1% penicillin-streptomycin. Cells were maintained at 37°C in a humid incubator with 5% CO2 concentration. Polyfect transfection reagent (Qiagen) and Calcium phosphate complexes (Invitrogen) were used to transfect HeLa and 293FT cells, respectively. The luciferase reporter (pGL3) whose expression is driven by six E2F responsive elements (6xE2F-Luc)  was usedFor cell cycle synchronization and release, 10 h after transfection (GFP vectors), cells were treated overnight with Hydroxyurea (2 mM) to arrest cells in the G1 phase of the cell cycle. They were next washed and released from block for the time indicated in the figure legends. For the accurate counting of cells going through the cell cycle, in some experiments cells were treated with Nocodazole (40 ng/ml) after release from Hydroxyurea to block them in G2/M. For the cell cycle distribution analysis, cells were resuspended in media containing the Dye Cycle Violet (Excitation at 405 nm and Emission at 450 nm) (Invitrogen) and incubated for 30 min at 37°C before being analyzed by an LSRII flow cytometer using FACS DIVA 6.1 software.i-S epifluorescence microscope equipped with a device camera and the NIS elements software (Nikon). The images were collected by using the objectives 20× and 100×.GFP or p30-myc expressing viruses have been titered by immuno-fluorescence to reach 100% of cells transduced. For immunostaining, Hela cells seeded on coverslips were transduced for 48 Hours with the viral particles expressing p30-myc. They were then fixed in 3.7% Paraformaldehyde (PFA) for 15 min at RT, washed with PBS for 5 min followed by permeabilization with 0.5% Triton X-100 on ice for 5 min. They were next blocked for 1 h in PBS with 0.5% gelatin and 0.25% bovine serum albumin at room temperature. Cells were incubated overnight with anti-myc primary antibody , washed 3 times with PBS plus gelatin 0.2% and then incubated with anti Mouse Alexa-596 conjugated secondary antibody (Invitrogen). They were next washed and mounted by using DABCO mounting medium . Fluorescent images were captured by using a Nikon TT Experiments. The fold = 2-DDCT.The expression of E2F, cyclinE and p21 was quantified by real time PCR using the Sybr Green method (Applied Biosystems). Briefly, the total mRNA of cells transfected or not transfected with the p30 expression vector was extracted by Trizol, DNase-I treated and reverse-transcribed using the High capacity RNA to cDNA kit (Applied Bioscience) as recommended by the manufacturer. Real time PCR (40 cycles) was performed in a total of 25 ul containing RT master mix, water, primers at 0.5 uM each and cDNA as recommended by the manufacturer using Applied Bioscience Step One plus real time PCR Software. The GAPDH gene was used as an internal amplification control. GAPDH primers: Forward 5'-gaaggtgaaggtcggagtc-3' and Reverse 5'-gaagatggtgatgggatttc-3'. Real time primers: cyclinE forward: tcagggtatcagtggtgcga, cyclinE reverse: caaatccaagctgtctctgtg, p21 forward: tgcgttcacaggtgtttctg, p21 reverse: gccattagcgcatcacagt, E2F forward: accctgacctgctgctctt and E2F reverse: tctcggccaggtactgatg. The results are determined by the Relative Standard Curve and comparative CCell extracts were prepared using NP-40 lysis buffer ), 150 mM NaCl, and a set of proteases (inhibitors). The immunoprecipitation was performed overnight and the proteins were separated by SDS page, transferred to PVDF membrane and western blotted using the standard protocol of WB: Blocking with TBS/Milk 5%, primary antibody overnight incubation and secondary antibody 2 h incubation (in TBS Milk 2%), washed 3 times in TBST 0.05% and signals were revealed with super signal chemiluminescent substract (Thermo Scientific). Primary antibodies used in this study: Rabbit polyclonal anti phospho(Thr821/826)-Rb (Santa Cruz), Goat polyclonal anti Actin sc-1615 (Santa Cruz), Rabbit polyclonal anti cyclinE (Santa Cruz C-19 clone), Mouse monoclonal anti-myc tag (9E10) and Mouse monoclonal anti-HA (3F10), Mouse monoclonal anti Flag (Roche), goat polyclonal anti Rb sc-50-G (Santa Cruz), Rabbit polyclonal anti E2F-1 sc-193 (Santa Cruz), Mouse monoclonal anti PCNA  and Rabbit polyclonal anti p21waf sc-397 (Santa Cruz).Using genome-wide microarray analyses, we and others have previously shown that HTLV-I regulatory protein p30 modulates a number of genes involved in the cell cycle. In addition, p30 has been shown to promote the accumulation of cells in the G2-M phase of the cell cycle to facilitate early viral spread and increase T-cell survival following infection . Since oWe next wanted to study the effect of p30 on the expression of endogenous G1/S regulators. Since transfection efficiencies are not sufficient for such studies, we cloned p30 with a carboxy-terminal myc tag into a lentiviral vector. High titer pseudotype virus particles were prepared by transfection of 293FT cells with HR-CMV-p30myc, pDLN and VSV-G. A GFP expressing virus was also prepared in the same conditions, titered by immuno-fluorescence and used as control. Expression of p30 was readily detected by western blot using an anti-myc antibody forty-eight hours after infection of HeLa cells Figure , and, inE2F and cyclin E mRNA in the presence of p30  4 and 6 control early G1, progression through G1/S is controlled by the cyclin E-CDK2 complexes. In the absence of Cyclin E-CDK2 activity, E2F remains bound to Rb and cannot activate the transcription of genes involved in S phase entry. Previous studies have shown that Tax directly alters the ratio of Rb-bound and Rb-unbound E2F resulting in activation of E2F-dependent transcription and S-phase entry . Activitcyclin E . To idenWhile HTLV-I is the etiological agent of ATL and TSP/HAM, infection with HTLV-II is not oncogenic in humans. We next investigated the effect of the HTLV-I p30 homologue, HTLV-II p28 , on cyclin vivo. In fact, while HTLV-I expression can be detected in TSP/HAM patients, the virus is mainly silent in ATL patients [tax/rex mRNA prevents its nuclear export, thereby repressing virus expression [Because HTLV-I is highly immunogenic and presents a low variability, reducing expression of viral proteins is essential to virus maintenance patients  and expapatients . We havepression . This mapression . These fpression .In the present study, we demonstrate that the HTLV-I accessory protein p30, but not its HTLV-II homologue p28, delays entry of cells into S phase and cell cycle progression. While proliferation of infected cells is required at times, uncontrolled proliferation of infected cells may lead to their activation and expression of viral genes. Therefore, it seems logical that the virus evolved proteins to balance proliferation as well as mechanisms to prevent infected cells from rapid cell division. Our results demonstrate that p30 activates multiple G1/S checkpoints to reduce proliferation. We did not detect any significant change in H2A mRNA levels of expression in p30 expressing cells. We think that this may be due to the fact that p30 targets factors involved in S phase entry and regulated by E2F. In contrast H2A activation is E2F-independent and occurs after cells have entered S phase. Cyclin-dependent kinase inhibitor (CDKI) p21waf expression was increased in p30 expressing cells. We do not know the mechanism by which p30 increases p21waf expression at the moment and additional studies are needed to investigate whether p30 affects the expression of p21waf by increasing its promoter expression or mRNA stability. Alternatively p30 may affect other cellular factors involved in p21waf expression such as p53 or Mdm2. We found a significant reduction in E2F and Cyclin E expression at both the transcriptional and protein levels. We do not think that p30 directly affects E2F promoter, since we did not see significant effects of p30 on E2F-mediated transcriptional activation of an E2F reporter construct. Instead, we found that p30 interacts with both Cyclin E and CDK2 preventing the formation of active Cyclin E-CDK2 complexes. Our data may suggest that like p21waf, p30 may form a ternary complex with these proteins further studies are needed to clarify the mechanism. Whether p30 prevents the formation of Cyclin E-CDK2 complexes by sub-cellular compartmentalization to the nucleolus or recruitment of additional cellular factors is currently under investigation. The effects of p30 on the cyclin E-CDK2 complex was associated with a significant decreased phosphorylation of Rb and delayed entry into S phase.In contrast to a previous study  using a Together our data lead to a model Figure  in whichIn addition to p30, other HTLV-I-encoded small proteins play a critical role in evasion of infected cells to immune defenses and survival of tumor cells. p12 has been shown to down regulate MHC class I from the cell surface and to increase the release of calcium from the endoplasmic reticulum to activate NFAT-mediated transcription ,51-54. FE2F: HTLV-I; PCNA: Rb.The authors declare that they have no competing interests.HB performed experiments for figures


Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F.Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including  Activation of T and B cells by cognate interactions is critical for adaptive immune responses, but improper regulation of this process can drive the development of autoimmune disease. B cell activation is triggered by BCR-mediated binding of antigen in secondary lymphoid structures, where B cells present antigen-derived peptides on HLA proteins and interact with cognate T cells Th17 cells, a CD4 T helper subset characterized by the production of IL-17A, IL-17F, IL-21, and IL-22 cytokines, are implicated in the pathogenesis of many autoimmune diseases Recent clinical studies with B cell depleting antibodies support a role for B cells in the development and survival of Th17 cells in humans. Treatment of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), or Sjögren's syndrome (SS) patients with rituximab, a CD20 antibody that depletes B cells, also decreased the number of circulating Th17 cells and serum IL-17A levels In this study we used a co-culture model of T cell-dependent B cell responses to study the B cell regulation of Th17 cells. IL-17F protein levels, and to a lesser extent IL-17A, were highly increased in co-cultures of peripheral blood mononuclear cells (PBMC) and B cells (BT co-cultures) after 3 days of stimulation with α-IgM and a low concentration of superantigens. In order to investigate the mechanisms that regulate production of IL-17A and IL-17F during B cell-dependent T cell activation, we screened 144 pharmacologic modulators that covered a broad spectrum of biologic mechanisms. We identified a number of pathways or targets that selectively impacted either IL-17A or IL-17F alone, or both together. Thus, the production of IL-17A and IL-17F by BT co-cultures is controlled through distinct pathways that can be independently regulated.Frozen vials of positively selected primary normal human CD19+ B cells and negatively selected CD4+ T cells were obtained from AllCells . PBMC were isolated from buffy coats  as previously described A co-culture system that models human B cell-dependent T cell responses (BT BioMAP® system) has been previously described Intracellular cytokine staining and FACS analysis were performed as previously described 2 plate reader  set at 546 nm. Drug effects on cell viability  were measured in a similar fashion by adding AlamarBlue to 18–24 hour cultures. Drug-treated samples showing a decrease in absorbance > Log10 −0.2 compared to stimulated samples without drug were identified as overtly cytotoxic and were not included in analyses.Cytokine concentrations in 72 hour-culture supernatants were measured by ELISA as previously described 10 of the ratio of the average values measured for the positive control stimulation wells was divided by the calculated average values for the DMSO control non-stimulated wells (6 to 12 DMSO control samples from the same plate). Data were generated from multiple pools of donors and significance prediction envelopes (95%) were calculated for historical controls as previously described Pharmacologic agents used for the screen of stimulated BT co-cultures were obtained from the sources listed in P-value for each gene. Gene comparisons were between donor-matched stimulated and non-stimulated control B cell and PBMC co-cultures. Data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus database under accession number GSE42567. For Th17-related gene analysis, B cells and PBMC were cultured as above and CD4 T cells and B cells were isolated by positive FACS sorting using a FACSAria II (BD Biosciences) at the Laboratory for Cell Analysis . Sorted B and CD4 T cells used for gene expression analysis were greater than 98% pure (data not shown). The following antibodies  were used for cell sorting: α-CD19 (clone HIB19), α-CD79b (clone CB3-1), α-CD4 (clone RPA-T4), α-CD3 (clone SK7). Purified cells were immediately lysed in RLT Buffer and stored at −80°C. RNA was isolated and qRT-PCR was performed using a Human Th17 for Autoimmunity & Inflammation RT2 Profiler PCR Array (Qiagen Genomics Services). Fold changes were calculated for each gene by dividing Illumina AU values or RT-PCR expression values (2−Ct) for stimulated samples by the respective values for non-stimulated samples.For microarray analysis, B cells and PBMC were cultured as above for three days with and without α-IgM and SAg stimulation, and then washed twice with PBS and lysed in RLT Buffer . Samples were then amplified and run on an Illumina HumanHT-12 v4 Expression BeadChip (Qiagen Genomics Services). Data was exported from GenomeStudio  and normalized using quantile normalization in GeneSpring software . A paired t Test was performed within the Multiple Array Viewer function of MultiExperiment Viewer software t test. Differences with a P value less than 0.05 were considered statistically significant.Statistical analyses for gene expression studies are described above. For cytokine measurements, paired data were evaluated using a 2-tailed Student's Il17f was the most strongly induced gene in co-cultures after three days of stimulation (We have previously described a co-culture assay with primary human B cells and PBMC stimulated with α-IgM and a relatively low concentration (0.02 ng/ml) of the SEB and TSST-1 superantigens (SAg) that models T cell-dependent B cell activation (BT BioMAP system) mulation . This fiTo determine which cell types in BT co-cultures were producing IL-17 family cytokines, we performed intracellular flow cytometry for IL-17A and IL-17F with cell surface markers specific for CD4 T, CD8 T, B, NK, and NKT cells. Detection of IL-17A and IL-17F by intracellular flow cytometry requires secondary stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin in combination with a protein transport inhibitor, such as monensin Il17f, Il22, Il23r, and Il21 , Il4 (Th2), Il13 (Th2), and Foxp3 (Treg), were either unchanged or significantly decreased compared to non-stimulated cells. Stimulation increased a larger number of genes in B cells, including Ccl22, Csf2 (GM-CSF), Il12rb, Stat4, and Icam1. Although Il17f mRNA was elevated nearly 5-fold in B cells, consistent with the small percentage of B cells that expressed IL-17F by FACS  and IL-17F (1549±206 pg/ml) after stimulation with α-IgM and SAg for three days . We thenWe next screened a panel of 144 pharmacologic modulators representing 91 different targets or pathways to determine their effects on IL-17A and IL-17F production in this co-culture system. Since small molecule pharmacologic inhibitors can often inhibit multiple targets, whenever possible multiple chemically unrelated compounds specific for the same target were used. Biologics, including cytokines and antibodies, were also used to test some mechanisms. These screening results suggest that a number of pathways or targets are involved in the regulation of IL-17A and IL-17F production . Inhibit3 , a vitamin D receptor agonist (0.15–4.1 nM), significantly decreased IL-17A and IL-17F production in the BT co-culture system, consistent with the reported effects of these agents in CD4 T cells The diversity of patterns observed in the regulation of IL-17A and IL-17F are illustrated in 1 receptor agonist, selectively stimulated IL-17A and IL-6 production (1 (PGE1) and PGE2 (EP receptor ligands), stimulated IL-17A production but inhibited IL-17F . We show that significant amounts of IL-17A and IL-17F are produced and secreted by CD4 T cells in these stimulated BT co-cultures and that both B and T cells are required for optimal production of IL-17 cytokines. Previous studies examining the role of Th17 cells in B cell activation focused on either the activity of Th17 cells or IL-17A and IL-17F cytokines on B cell function Several studies in transgenic mice have examined the role of IL-17 family cytokines in B cell activation and subsequent formation of GC in vivo. IL-17A-deficient mice show impaired antigen-specific Ig production upon immunization compared to normal control mice, although the deficient mice still form normal GCs Il17a mRNA in purified CD4 T cells. Importantly, this finding is in line with previous work showing that human B cells are capable of producing IL-17A and IL-17F Rorc mRNA in CD4 T cells, this finding is consistent with reports that IL-17A and IL-17F can be independently regulated from RorcRorc or Il17a were regulated on a time course not examined in this study. CD4-CD8- T cells, which are known to produce IL-17A under some conditions, may have also contributed to the production of IL-17A or IL-17F measured in culture supernatants Our data and the data of others indicate that CD4 T cells are the predominant source of IL-17A and IL-17F in the BT co-cultures, but other cell types may have also contributed to amounts measured in culture supernatants Ccl22, the most highly induced gene in B cells isolated from BT co-cultures, is typically produced by macrophages and dendritic cells and is found at elevated concentrations in the CNS of multiple sclerosis (MS) patients CSF2 (GM-CSF), the second most highly induced Th17-related gene, was recently reported to mark a novel B cell subset critical for innate immune responses A surprising number of genes related to Th17 biology were up regulated in B cells after BT co-culture and stimulation with α-IgM + SAg. To our knowledge, these B cell genes have not been previously implicated in B cell regulation of Th17 differentiation. For example, As an initial step to identify pathways important in the B cell regulation of IL-17A and IL-17F production by T cells, we screened a broad panel of diverse pharmacologic agents. Regulation of IL-17A and IL-17F production by CD4 T cells has been both expected and observed to occur predominantly through shared pathways due to the proximity of the IL-17A and IL-17F genes on chromosome 6 and parallel H3 histone hyperacetylation at multiple conserved noncoding sequence sites within the IL-17A-IL-17F locus CP-690,550, the same JAK inhibitor used in our screen, has been shown to block IL-17A and IL-17F production when Th17 cells are differentiated in the presence of IL-6 and IL-23, but to enhance IL-17A and have no effect on IL-17F when TGFβ is added to the differentiation media We here identify novel pharmacologic agents that regulate IL-17A or IL-17F production. Several microtubule inhibitors, including paclitaxel, epothilone B, and picropodophyllin, all preferentially inhibited IL-17F over IL-17A, but did not impair production of IL-2 or have significant cytotoxic effects at the doses tested. Interestingly, we found that erythromycin, a macrolide antibiotic, blocked IL-17A and IL-17F without affecting B cell proliferation or production of IgG, IL-2, IL-6, or TNFα in stimulated BT co-cultures. Erythromycin inhibits NF-κB signaling in T cells, which may account for the effect of this antibiotic on IL-17A and IL-17F in vivo is not well understood, but the compounds and agents identified here with activity on IL-17A versus IL-17F should help address this question.In this study we present data that indicates primary human B cells contribute to the polarization of CD4 T cells toward a Th17 phenotype in a model of T cell-dependent B cell activation. This finding adds to a growing body of evidence implicating IL-17A and/or IL-17F production by CD4 T cells in the development of antibody-mediated immune responses and the formation of GCs. Understanding the cellular interactions and signaling processes that govern how B cell responses are regulated and GCs are formed should benefit the development of therapeutics for autoimmune diseases. The approach described here utilizing a T cell-dependent model of B cell activation that also reflects B cell-dependent differentiation of Th17 cells provides a useful screening system for the identification of targets, pathways, and small molecule inhibitors that selectively act on IL-17A or IL-17F production. The importance of selectively targeting IL-17A or IL-17F Table S1Microarray data set for genes significantly increased or decreased (P≤0.05) in stimulated BT co-cultures. Microarray analysis in BT co-cultures stimulated with α-IgM and SAg for three days compared to the same cells co-cultured for three days without stimulation. Means and standard deviations are from 3 independent replicates with 3 different donor pools.(DOC)Click here for additional data file.Table S2Quantitative RT-PCR Th17 Array data set for purified CD4 T cells and B cells isolated from stimulated and non-stimulated BT co-cultures. Quantitative RT-PCR gene expression data from CD4 T and B cell populations purified by FACS after three days in co-culture with α-IgM and SAg stimulation. Data normalized to b-actin expression levels and presented as fold change (above) and raw Ct values (below) are shown. Gene expression data are from 3 independent donor pools of stimulated BT co-cultures compared to cells isolated from non-stimulated BT co-cultures.(DOC)Click here for additional data file.Table S3Screen of agents in stimulated BT co-cultures. Agents screened are listed with the concentrations tested, supplier, and putative mechanism of action. Data for each agent screened on B cell proliferation, PBMC cytotoxicity, IgG, IL-17A, IL-17F, IL-2, IL-6 and TNFα. Data are presented as the Log10 ratio of values from agent-treated stimulated BT co-cultures to values from control stimulated BT co-cultures.(DOC)Click here for additional data file.Table S4Lists of agents and their concentrations that regulated IL-17A and/or IL-17F in stimulated in BT co-cultures. Agents are listed that decreased both IL-17A and IL-17F, decreased IL-17A but not IL-17F, decreased IL-17F but not IL-17A, and increased either IL-17A or IL-17F. Agents were only included if they inhibited readouts greater than Log10 ratio −0.2 or increased readouts greater than Log10 ratio −0.15 at 2 or more non-cytotoxic concentrations.(DOC)Click here for additional data file.


Entamoeba histolytica have uniquely evolved and compartmentalized a sulfate activation pathway. Although this confined metabolic pathway is the major function in E. histolytica mitosomes, their physiological role remains unknown. In this study, we examined the phenotypes of the parasites in which genes involved in the mitosome functions were suppressed by gene silencing, and showed that sulfate activation in mitosomes is important for sulfolipid synthesis and cell proliferation. We also demonstrated that both Cpn60 and unusual mitochondrial ADP/ATP transporter  are important for the mitosome functions. Immunoelectron microscopy demonstrated that the enzymes involved in sulfate activation, Cpn60, and mitochondrial carrier family were differentially distributed within the electron dense, double membrane-bounded organelles. The importance and topology of the components in E. histolytica mitosomes reinforce the notion that they are not “rudimentary” or “residual” mitochondria, but represent a uniquely evolved crucial organelle in E. histolytica.Mitochondrion-related organelles, mitosomes and hydrogenosomes, are found in a phylogenetically broad range of organisms. Their components and functions are highly diverse. We have previously shown that mitosomes of the anaerobic/microaerophilic intestinal protozoan parasite  Entamoeba histolytica, which causes intestinal and extraintestinal amebiasis in humans, possesses a highly divergent form of mitochondrion-related organelles, named “mitosomes.” Previously, we demonstrated that sulfate activation is the major function of mitosomes in E. histolytica. As the sulfate activation pathway was discovered only in the cytoplasm and plastids in other eukaryotic organisms, its compartmentalization to mitosomes is unprecedented. In this study, we showed that this pathway is important for sulfolipid synthesis and cell proliferation in E. histolytica. Together, we infer that E. histolytica mitosomes are not just rudimentary or residual mitochondria, but important for proliferation of E. histolytica. Thus, E. histolytica represents a useful model to understand evolutionary constraints of mitochondrion-related organelles in eukaryotes.The mitochondrion and its related organelles are ubiquitous in all extant eukaryotic cells. The mitochondria are believed to have originated from the endosymbiosis of α-proteobacteria in an ancestral eukaryote, and show diverse structures, contents, and functions. Evolution and diversification of mitochondrion-related organelles remains one of the central themes in biology.  Mitosomes and hydrogenosomes are mitochondrion-related organelles and found in a phylogenetically broad range of eukaryotes. Since organisms that possess hydrogenosomes or mitosomes do not cluster together in eukaryote phylogenies, it is suggested that secondary losses and changes in mitochondrial functions have independently occurred multiple times in eukaryote evolution Entamoeba histolytica, a widespread intestinal protozoan parasite E. histolytica mitosomes Mastigamoeba balamuthi, a free-living amoeba that is distantly related to E. histolytica, also possesses mitochondrion-related organelle. An expressed sequence tags (EST) project showed that the organism has enzymes for sulfate activation, and one of the enzymes has the putative mitochondrial targeting signal at the amino terminus . Trichomonas vaginalis, Giardia intestinalis, and Cryptosporidium parvum, which also possess mitochondrion-related organelles, i.e., hydrogenosome and mitosome, apparently lack genes encoding these enzymes. Phylogenetic analyses further revealed that E. histolytica appears to have acquired enzymes involved in sulfate activation from distinct prokaryotic and eukaryotic lineages by lateral gene transfer E. histolytica and M. balamuthi belong to. Although iron sulfur cluster biosynthesis is shared by aerobic eukaryotes and highly divergent G. intestinalis mitosomes and T. vaginalis hydrogenosomes E. histolytica and M. balamuthiE. histolytica genome contains 10 potential genes encoding for sulfotransferases, but lacks the enzymes for sulfate reduction E. histolyticaMycobacterium tuberculoris, respectively M. tuberculoris. However, the structure and function of sulfolipids in E. histolytica remains largely unknown.Sulfate is generally activated in two steps. Inorganic sulfate is converted to adenosine-5′-phosphosulfate (APS), in a reaction catalyzed by ATP sulfurylase (AS), and further converted to 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in a reaction catalyzed by APS kinase (APSK). Pyrophosphate concomitantly produced in the first reaction needs to be decomposed to phosphates by inorganic pyrophosphatase (IPP). PAPS acts as a sulfuryl donor to transfer the sulfuryl moiety to various acceptors by sulfotransferases, resulting into the formation of sulfurylated macromolecules such as mucopolysaccharides, sulfolipids, and sulfoproteins E. histolytica mitosomes E. histolytica by using the parasites in which genes for the enzymes involved in sulfate activation, MCF, and Cpn60 were knocked down by gene silencing. We showed that these mitosomal proteins are indeed important for sulfolipid production and cell growth. We also demonstrated the localizations and topologies of the enzymes involved in sulfate activation, MCF, and Cpn60 in mitosomes by immunoelectron microscopy.While the sulfate activation has been demonstrated as the major metabolic pathway in ap-a gene was amplified from psAP-2 using CCGCGGCGGCTTGCTGCACCCTTTG-3′5′-AGCTCTAGA  and GAGCTCGTTTAAAGGCCT<$>\raster="rg1"<$>CATGATTGTTTGTAAGATAT G-3′5′-CTCT . PCR product was digested with SacI and SacII, and ligated into SacI- and SacII-double digested psAP-2 vector to produce psAP-2-Gunma. Approximately 380-450-bp fragments corresponding to the 5′ end of the open reading frame of Cpn60, Mcf, As, Apsk, and Ipp genes, respectively, were amplified by PCR using the following primers sets :G3 strain and psAP-2 plasmid were kindly given by David Mirelman, Weisman Institute, Israel AGGCCTATGCTTTCATCTTCAAGTCATT-3′5′-AA (forward) and GAGCTCTTTGTAATTTTCTTTAATAC-3′5′-GGG (reverse) for Cpn60; AGGCCTATGATACAAGGTATGACTTATAAACG-3′5′-CATC (forward) and GAGCTCCTAGCAGTACCAAAGAATGTATC-3′5′-ACGC (reverse) for Mcf; AGGCCTATGAGCATTCAAGAAAACTTAAACAAC-3′5′-CATC (forward) and GAGCTCGGTCAATTTCAATAGTTCCTGAG-3′5′-ACTT (reverse) for As; AGGCCTATGGCTACTGCTAAGATTGCTG-3′5′-GATC (forward) and GAGCTCGAGGTGGTGGTTCAACAAATTC-3′5′-GACT (reverse) for Apsk; AGGCCTATGTCAATTACTTCTATTGTCCCC-3′5′- CATC (forward) and GAGCTCATCAATTGGATCATTATCTCCAGG-3′5′-CACC (reverse) for Ipp. The PCR fragments were digested with StuI and SacI, and ligated into the StuI- and SacI-double digested psAP-2-Gunma to produce the plasmids used for gene silencing. Lipofection of trophozoites and selection of transformants were performed as previously described Cpn60, Apsk, and Ipp was PCR-amplified with primers containing a BamHI restriction site, digested with BamHI, and ligated into BamHI-digested pCOLD1 to yield pCOLD1-Cpn60, pCOLD1-APSK, and pCOLD1-IPP, respectively. These constructs were introduced into E. coli BL21 (DE3) cells. Expression and purification of the recombinant proteins were performed as previously described E. coli pellet was suspended in 20 ml of lysis buffer  containing 1% Triton X-100 (v/v), 100 µg/ml lysozyme, and 25 U/ml benzonase. After 15-min incubation at 4°C, the cells were sonicated on ice and centrifuged at 12,000×g for 20 min at 4°C. The supernatant was applied on 50% Ni2+-NTA His-bind slurry . The recombinant protein-bound resin was washed three times with buffer A  containing 20–50 mM of imidazole. The bound proteins were then eluted with buffer A containing 100 mM imidazole. Rabbit anti-Cpn60, APSK, and IPP antisera were custom made by Operon Biotechnologies .The open reading frame of Whole cell lysates of each gene-silenced strain were analyzed by SDS-polyacrylamide electrophoresis (PAGE) and immunoblot analysis as previously described Rnapol) was used as a house-keeping reference gene. Total RNA was extracted using TRIzol® reagent . The synthesis of cDNA was performed using the SuperScript III First-Strand Synthesis System (Invitrogen). qRT-PCR was performed using the following primers: 5′-CCTATGAAAATCGATTGGACATTCTATTGCC-3′ (forward) and 5′-GCATCACCAGTAGCAAACTTTGTAACTTG-3′ (reverse) for As; 5′-GCCCCAATTGCACCATATCGTGAAATTAG-3′ (forward) and 5′-GCACATTGATCAACAGACTTACCAGCAG-3′ (reverse) for Apsk; 5′-GATCCTCTTGCTCAAAACCATTACATCTG-3′ (forward) and 5′-GTCTAACGCCAATTTTGATAACTTCTTTTGAG-3′ (reverse) for Ipp; 5′-GCATGTTTTGATTTTGTTGCTCCATTAGTTCC-3′ (forward) and 5′-CACTGACTAATGGAACAACTTTGACAAATCC-3′ (reverse) for Mcf; 5′-GATCCAACATATCCTAAAACAACA-3′ (forward) and 5′-TCAATTATTTTCTGACCCGTCTTC-3′ (reverse) for Rnapol. qRT-PCR was performed using StepOne Plus Real-Time PCR System (AB Applied Biosystems) with the following cycling conditions: 95°C for 20 s, followed by 40 cycles of 95°C for 3 s, and 60°C for 30 s. All reactions were run in quadruplicate, including reverse transcriptase-minus and cDNA-minus controls. Quantification for each target gene was determined by the ΔCt method with Rnapol as reference gene.The Fast SYBR® Green Master Mix  was used for qRT-PCR. RNA polymerase II gene (5 trophozoites were labeled with [35S]-labeled sulfate (25 mCi/mmol) in 1 mL of the BI-S-33 medium either continuously for 2, 4, or 8 h, or labeled for 4 h and chased for 4 or 24 h after removing [35S]-labeled sulfate. Cells were collected and lipids were extracted with 0.5 mL of methanol and separated on a silica high-performance thin-layer chromatography plate in 35∶65∶8 (vol/vol/vol) methanol/chloroform/28% (w/w) ammonium hydroxide Metabolic labeling was performed as previously described E. histolytica transformants expressing epitope-tagged mitosomal proteins were previously established 5 trophozoites were resuspended in 2 ml BI-S-33 medium and seeded onto a molybdenum disk  in a well of a 24-well plate. After 15-min incubation at 35.5°C, the molybdenum disk that amoebas adhered to was removed and immediately immersed in liquid propane at −175°C. The disk was further fixed and sectioned as previously described E. histolytica strains in which AS, APSK, IPP, MCF, and Cpn60 genes were knocked down by gene silencing To investigate the role of the mitosomes and, more specifically, mitosome-localized sulfate activation pathway, chaperones, and ADP/ATP transporter, we established the E. histolytica trophozoites, the majority of activated sulfate are incorporated into sulfolipids 35S]-sulfate, the sulfate activation activity in ASgs, APSKgs, and IPPgs was decreased to 54.1±11.0, 49.1±15.9, and 24.0±0.6%, respectively, as compared to the control , a known inhibitor for AS in mammals and fungi E. histolytica (IC50 = 10.5 mM). At this concentration, the sulfate activation activity  was decreased to 22.5% of the control. Furthermore, the apparent IC50 of the recombinant E. histolytica AS by chlorate was determined to be 3.55±0.25 mM. These results support the premise that sulfate activation plays an important role for proliferation.Chlorate . Immunoelectron microscopy revealed that all the proteins examined are confined to the electron dense double-membrane-surrounded organelles, the size of which are 150–400 nm in diameter . Double E. histolytica uniquely possess sulfate activation pathway, while they have lost most of the functions shared by other aerobic eukaryote mitochondria including TCA cycle, electron transport, oxidative phosphorylation, and β-oxidation of fatty acids E. histolytica mitosomes E. histolytica remains unknown In our previous report, we showed that mitosomes of E. histolytica. Further supporting the specificity of chlorate to AS, two amino acid residues (Asn198 and His201) of Saccharomyces cerevisiae AS, which were implicated in the chlorate binding E. histolytica AS .In this report, we have provided several lines of direct and indirect biochemical and cell biological evidence that the sulfate activation pathway plays an important role in the production of sulfolipids and the growth of trophozoites. Consistent with this premise, the AS inhibitor, chlorate, inhibited the sulfolipid production in and the growth of In addition to the importance of the enzymes in the sulfate activation pathway, we demonstrated that MCF, and Cpn60 also play an important role in cell proliferation. The phenotype of Cpn60gs is most likely attributable to multiple defects as Cpn60 is required for the folding and quality control of mitosome-targeted proteins E. histolytica phosphate transporter (EhPiC) has been identified and proposed to transport phosphate released from ATP through hydrolysis by chaperones, i.e., Cpn60 and mitochondrial HSP70 Immunoelectron microscopy revealed that MCF is mainly localized on the inner mitosome membrane. Although membrane topology may need to be further verified, the observed localization of MCF agrees well with its biochemical characteristics, previously demonstrated: ATP import and ADP export E. histolytica mitosomes revealed by immunoelectron microscopy was far different from the typical eukaryotic mitochondria, it was somehow similar to the mitochondrion-related organelles, described as densely-stained double membrane-bound organelles lacking the typical cristae, in M. balamuthi, in shape, apparent size, number per cell, and structure M. balamuthi possesses genes involved in sulfate activation, it is possible that sulfate activation is a unique feature shared only by E. histolytica and M. balamuthi.While the structure of E. histolytica.The number and structure of mitosomes demonstrated in this study was slightly different from previous reports