During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus . This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain .in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection  The severe acute respiratory syndrome (SARS) emerged in the end of 2002 in China and caused an international epidemic . Its cauIn the context of viral host switching, it is interesting that several SARS-CoV proteins encoded on subgenomic (sg) RNAs seem to be dispensable for virus replication in cultured cells of primate or rodent origin, as well as in rodent models -19. Becain-vitro [E. coli. In-vitro ligation of subgenomic cDNA fragments without the assembly of full-length plasmids has been successfully used to establish CoV reverse genetics [E. coli cell [in-vitro transcription of capped, infectious RNA that is transfected into cells. The latter uses a CMV promoter and relies on the transfection of full-length cDNA into cells, which is then transcribed in the nucleus into infectious RNA. In this study we have implemented a modified approach to CoV reverse genetics by cloning the entire SARS-CoV genome downstream of a T7 RNA polymerase promotor in a BAC. Using the linearized BAC construct as a template for in vitro transcription, this system combines plasmid-based handling of cDNA constructs with direct delivery of genome-like RNA into the cytoplasm.The characterization of virus proteins can be unreliable if only the protein of interest is studied on its own. The study of proteins in the whole virus context reflects virus-host interactions more realistically, and takes into account intraviral protein interactions. Such experiments can be done using reverse genetics techniques which for most plus-strand viruses rely on cloned cDNA copies of the whole RNA genome that can be mutagenized in-vitro -30. Diffgenetics -33. As agenetics ,35. A tholi cell -39. The The novel system was used to characterize a 45 nucleotide in-frame deletion in ORF 7b that is present in the primary isolate of SARS-CoV prototype strain Frankfurt-1 . This spin-vivo. This is relevant for the Frankfurt-1 virus because it has been used as a model virus in several studies on pathogenesis and antiviral drug research . Fin. Finin-vin-vivo replication in Syrian Golden hamsters were determined.In this study, primary clinical samples from the Frankfurt index patient and a secondary case who acquired her infection from him were re-analyzed. Frankfurt-1 viruses with and without the deletion were then reconstructed by reverse genetics. Effects of the deletion on interferon induction and response, on induction of apoptosis, and on The Frankfurt-1 SARS-CoV cell culture isolate contained a 45 nt in-frame deletion within a predicted transmembrane region. A back-translated BLAST search on the nucleotide database (tBLASTn) showed that this deletion was not present in any of > 150 SARS-CoV ORF 7b sequences in GenBank (except in an independent sequence of the Frankfurt strain), and in none of 8 SARS-like bat-CoV sequenced in the ORF 7b region . One whole BAC was digested with Bgl I, which was present at seven positions on the BAC construct. As shown in Figure In order to clone subgenomic portions of the SARS-CoV genome, seven PCR fragments covering the whole genome were generated with primers described by Yount et al. . Fragmenin-vitro transcribed and co-transfected in BHK cells. Because BHK cells did not support SARS-CoV replication, supernatants were transferred to Vero cells susceptible for SARS-CoV infection. Virus progeny was identified by immunofluorescence analysis with anti-SARS-CoV patient serum after 24 h  tag sequence was introduced in the infectious clone prSCV by overlap-extension PCR at the C-terminus of ORF 7b. As shown in Figure et al [It was concluded that the ORF 7b protein of the recombinant viruses was expressed in infected cells, and that its principal properties are not affected by a C-terminal flag-tag epitope. These findings, including the pattern of fluorescence when expressing ORF 7b, were consistent with earlier reports by Pekosz et al ,40.Growth properties of rSCV and r7bΔTMD on different cell lines were compared. Plaque morphology was determined for both viruses, with no discernible differences Figure . BecauseGrowth curves in three different cell cultures were determined next. Virus RNA was measured in supernatant by real-time RT-PCR. A multiplicity of infection (MOI) of 0.001 was used for both recombinant viruses in Vero and CaCo-2 cells. For HuH7 cell, an MOI of 0.01 was used, due to their lower susceptibility to SARS-CoV infection. In Vero cells, very similar increases in RNA concentration were observed with both viruses during 48 hours Figure . In CaCoBecause Vero cells as well as HuH-7 cells are deficient in interferon induction , HEK 293et al. that interferon alpha expression is induced in SARS-CoV-infected 293-lp cells to a certain level [HEK 293-lp cells were used to study release of interferon alpha in the supernatants of infected cells. It has been reported by Spiegel in level . ExactlyTo study the effects of interferon on replication of both viruses, Vero cells were pre-treated with increasing concentrations of interferon beta in order to induce an antiviral state. Cells were infected with either rSCV or r7bΔTMD at an MOI of 0.001. As shown in Figure Programmed, caspase-mediated death of infected cells is an efficient way of controlling virus replication. Several SARS-CoV accessory gene products have been implicated in the induction of apoptosis, including the ORF 7a and ORF 7b proteins as confirmed in this study Figure . Activatin-vivo, both viruses were tested in hamsters. Syrian Golden hamsters have been shown to be an acceptable rodent model for SARS-CoV replication and pathogenicity [4 PFU of either rSCV or r7bΔTMD, and sacrificed on day 1 or 3, respectively. Whole lungs were minced and tested for infectious virus and viral RNA. The deleted virus yielded 95-fold more infectious particles and 23-fold more RNA copies in the lungs on day 1 . By in-vitro and in-vivo. Our initial overexpression experiments pointed to a strong pro-apoptotic effect of intact ORF 7b, which was in concordance with a study by Schaecher et al. who found that sgRNA 7-derived proteins activated caspase 3 if overexpressed [et al. studied a recombinant virus with a double deletion of both ORF 7a and 7b, and this virus induced apoptosis clearly less efficiently than the parent full-length virus [et al. was contributed by ORF 7a rather than ORF 7b.Apoptosis of target cells can limit virus infection xpressed . Complemth virus ,19. The in-vivo, using Syrian Golden Hamsters as a model of human SARS-CoV infection [et al. have described ca. 10-fold more efficient replication of Frankfurt-1 in hamsters as compared to Urbani and HKU-39849 [et al. was observed in our study between two variants of Frankfurt-1 that differed only by the ORF 7b deletion. As the deletion is not present in Urbani or any other prototype strain, this identifies the 7b protein as a potential attenuating factor within the genome of SARS-CoV.Even though the ORF 7b deletion in Frankfurt-1 was not affecting interferon and apoptosis systems, the virus with a deletion seems to have been selected during isolation in cell culture and shows a replicative advantage in two of three cell lines. This is remarkable because SARS-CoV variants with deletions in the ORF 7  gene region have been transmitted and maintained in humans in the late phases of the 2003 epidemic -27. It hnfection ,53. InteKU-39849 ,53. MortKU-39849 ,53. It win-vivo. Because it has been suggested that delayed accumulation of high virus concentrations in infected patients has limited the spread of SARS-CoV in the population, it is tempting to speculate that the occurrence of viruses with deletions in the ORF7/8 region in the late phase of the 2003 epidemic might have added to the efficiency of virus transmission in humans [We have seen in this study that the attenuating effect of ORF 7b was focused on the early phase of infection n humans -69. It wThe original Vero cells on which Frankfurt-1 was primarily isolated , human hepatoma cells HuH7 (ATCC CCL-185), human colonic cancer cells CaCo-2 (ATCC HTB-37) and human embryonic kidney cells HEK 293-low passage  were maThe SARS-CoV Frankfurt-1 isolate ,20,70 wain-vitro transcribed RNA standards was done as described previously [Plaque assays were done with Avicel overlays  as described elsewhere . Immunofeviously .E. coli cells . Prior to digestion with methylation-sensitive endonucleases plasmids were transformed in Sure cells . BAC preparations were done with the Nucleobond®AX-kit  as instructed. Plasmid-based inverse PCR was performed with QuikChange XXL kit . PCR mutagenesis by overlap-extension PCR used Phusion® DNA polymerase and around 50 ng of input plasmid DNA. SARS-CoV coding sequence within constructs was fully sequenced after every mutagenic step.Standard cloning techniques were used. All gel purifications were done with the QIAEX II kit . DNA constructs were electroporated into E. cloni  or Stbl 3 et al. [et al., a Not I restriction site was introduced downstream of the genomic poly-A tail. Figure 5'-TACTAATACGACTCACTATAGATATTAGGTTTTTACCTACCCAGG-3' and A1rev 5'-aatgccagtatgacctgagccaatatc-3' and A2fwd 5'-GATATTGGCTCAGGTCATACTGGCATT-3' and Arev 5'-ACACCATAGTCAACGATGCC-3'. After correction of errors both inserts were amplified from plasmids and used as templates in an overlap-extension PCR. A naturally existing Bgl I restriction site at genome position 1572 was thereby deleted. The extension product was subcloned in pSMART, resulting in clone pA. PCR products B, D, and E were cloned in pCR2.1 (Invitrogen). Fragments C and F were cloned in pSMART Low Copy Kanamycin vectors (Lucigen) after instability was observed in pCR2.1. A 45 nt deletion present in the Frankfurt-1 virus isolate (nt 27654 to 27699 in Genbank Accession No AY310120), was filled in by overlap-extension PCR. A region including restriction sites BamH I (genome position 26045) and Not I (following the 3'end of genome) was amplified from subclone pF in two halves using appropriate outer primers and overlap-extension primers 5'-TTTCTGCTATTCCTTGTTTTAATAATGCTTATTATATTTTGGTTTTCACTCGAAATCCAGGATCTAGAAG-3' and 5'-ATTATTAAAACAAGGAATAGCAGAAAGGCTAAAAAGCACAAATAGAAGTCAATTAAAGTGAGCTCATTC-3'.Total RNA was extracted from infected Vero cells with the Qiagen RNeasy kit. Using primers described by Yount et al. , cDNA fret al. . In addiGATTACAAGGATGACGACGATAAGTAAACGAACATGAAACTTCTC-3' and 5'-CTTATCGTCGTCATCCTTGTAATCGACTTTGGTACAAGGTTCT-3'.The fragments were overlap-extended, digested with BamH I and Not I, and cloned back into the corresponding restriction sites in clone pF. All clones were verified by sequencing. Using the same technique, a DDDDK (flag-) tag sequence was introduced at the C-terminus of ORF 7b, with overlap-extension primers 5'-GGGCCCTAAGTACTAATACGACTCACTATAGATATTAGGAGTAAT-3' and 5'-ACACCATAGTCAACGATGCC-3', thereby introducing a PspOM I site upstream of the T7-promotor ; and primers 5'-AGCGCAATCGTGCCACACGTGTTGAGTGTA-3'and 5'-TGAACCGCCACGCTGGCTAAACC-3' (3'-part), respectively. Both products were overlap-extended, resulting in a PCR product with a depleted Mlu I site at position 7453. The product also contained a Not I site upstream of the Bsu36 I site at position 6544, introduced by a primer 5'-overhang. The product was Not I and Bgl I digested and ligated to the long EcoR I - Not I fragment of pBeloAd4 . The fragment was ligated to the dephosphorylated short BamH I - EcoR I fragment of pBelodNco   at an input of 1 μg of DNA per 20 μl reaction. A PCR product spanning the nucleocapsid reading frame and the genomic 3'-prime end was generated with primers N-fwd (5'-ATTTAGGTGACACTATAGATGTCTGATAATGGACCCCAATCGGCC), the underlined sequence representing an SP6 promoter) and Frev (5'-TTTTTTTTTTTTTTTTTTTTGTCATTCTCCTAAGAAGC-3'). The product was purified and transcribed with mMessage-mMachine SP6 kit. Transcripts from both in-vitro transcription reactions were quantified photometrically. Genomic transcripts and N transcripts were co-electroporated at a 10:1 ratio into 107 BHK-21 cells, using a GenePulser instrument  with two pulses of 1.5 kV, 25 μF and maximal resistance. Cells were left at room temperature for 10 minutes and seeded in 75 cm2 flasks. In a biosafety-4 laboratory, electroporated BHK-21 cells were incubated at 37°C for 24 hours. Supernatants were serially diluted and transferred to Vero cells. Using 1% SeaPlaque® agarose overlay , three rounds of plaque purification were performed for each recombinant virus.Full-length BAC clones were linearized with Not I, extracted with phenol-chloroform, and transcribed with the mMessage-mMachineCAGCTGCGTGCAAGATCAGT-3') and 27900 rev (5'-CCCTAGTGTTGTACCTTACAAG-3'), thus comprising ORF 7b and yielding a 400 bp fragment for rSCV, while giving a 355 bp fragment for r7bΔTMD. For RT-PCR 2 the identical reverse primer was used but the binding site of the sense primer 27690 fwd (5'-TAGCCTTTCTGCTATTCCTTGT-3') was placed in ORF 7b, recognising the 45 nt only present in rSCV but deleted in r7bΔTMD, hence a PCR product was only obtained for rSCV.To distinguish between the two genotypes, two different RT-PCRs were performed. RT-PCR 1 used primers 27500 fwd (5'-AY310120) was amplified using primers 5'-CACCATGAAAATTATTCTCTTCCTGACA-3' (fwd) and 5'-TCATTCTGTCTTTCTCTTAATGGT-3' (rev), and cloned into pCDNA 3.1. Because of low expression rates of the protein (data not shown) the insert was cloned into the high level expression vector pCAGGS [KpnI and NotI. ORF 7b gene (nts 27623 to 27751) and the deletion mutant ORF 7b del gene (nts 27623 - 27751 deletion of 45 nts 32-76) were amplified using primers 5'-CTAGAATTCCTCGAGACAATGAGAAGTTTCATGTTC-3' and 5'-ATCGTCGACCTCGAGTCACCATTAAGAGAAAGACAG-3', and cloned into pI.18 vector  with a T7 promoter that was inserted by standard cloning procedures. Expression of constructs was verified by coupled in-vitro transcription and translation using the TNT T7 Coupled Reticulocyte Lysate System  and immunofluorescence analysis of transfected cells (data not shown).ORF 7a -stimulated response element (ISRE)-driven firefly luciferase reporter plasmid pHISG-54-Luc , 0.3 μg of the constitutive Renilla luciferase expression plasmid pRL-SV40 (Promega) and 4 μg of the plasmid of interest. 16 μg of herring sperm DNA (Promega) were transfected along with the plasmids to optimize DNA uptake. 24 h post transfection, cells were infected with SeV (20 hemagglutinating units) to induce the type I IFN response or were not infected. At 24 h post infection (p.i.), cells were harvested and lysed in 100 μl of passive lysis buffer . Subsequent luciferase assays were performed by using the Promega DUAL luciferase assay system according to the manufacturer's instructions. Relative renilla luciferase production was used to normalize for transfection efficiency.Transfection of 293 cells was performed using the calciumphosphate transfection kit (Invitrogen) according to the manufacturer's instructions. 10® 8 well, Ibidi, Martinsried, Germany) and infected with SARS-CoV t a multiplicity of infection of 1. After 24 hours cells were washed once with PBS and fixed in ice cold acetone for 15 minutes. Prior to antibody staining, cells were washed three times with PBS. Reconvalescent SARS patient serum from our own diagnostic laboratory was diluted 1:1000 in PBS-T. Rabbit polyclonal antibody against the DDDDK - tag  was diluted 1:10000 in PBS-T. Cells were overlaid with 100 μl of antibody solution, incubated at 37°C for 1 hour, and washed four times for 5 minutes with PBS containing 0.1% Tween 20 (PBS-T). Fluorescein-conjugated goat anti-human or anti-rabbit IgG serum  was diluted 1:10000 in PBS-T and incubated at 37°C for 30 minutes. Cells were washed 4 times with PBS. Chambers were overlaid with 200 μl of PBS and a few drops of mineral oil. Fluorescence was analysed on an inverted fluorescence microscope.Cells were seeded on chamber slides  and blocked for 1 h with 1× RotiBlock . Membranes were washed with PBS and incubated over night with primary antibody at 4°C. Rabbit polyclonal anti-caspase-3 and anti-PARP antibodies  were diluted 1:1000 in PBS-T. Rabbit anti-flag antibody  was diluted 1:5000 in PBS-T. Membranes were washed 4 times for 5 minutes with PBS-T. Horseradish peroxidase-conjugated goat anti-rabbit antibody  was used at 1:2000 dilution in PBS-T and incubated on membranes for 1 hour. Membranes were washed four times with PBS-T before LumiGLO reagent  was added. Membranes were exposed to scientific imaging film  for appropriate times before development.4 cells were transfected with 1 μg of empty pI.18 vector, pCAGGS-ORF 7a, pI.18-ORF 7b or pI.18-ORF 7b del, respectively. At 48 h post transfection, cells were lysed in 50 μl 1× Chaps buffer. Proteins were separated on 15% SDS polyacrylamide gels, transferred onto PVDF membranes and blocked for 1 h with 5% skim milk (w/v) in PBS-T. Membranes were washed with PBS-T and incubated over night with primary antibody at 4°C. Western blot detection was done with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody using an enhanced chemiluminescence detection reagent kit  according to the manufacturer's protocol. Immunoreactive bands were visualized using an Optimax 2010 imaging system  with high performance chemiluminescence films .In overexpression experiments, Vero E6 cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 5 × 10Interferon alpha was detected with the Human IFN Alpha ELISA Kit  according to the manufacturer's instructions. Briefly, 100 μl of supernatant of samples and controls were added to pre-coated microtiter plates and incubated at room temperature for 1 hour, followed by one washing step, addition of antibody solution and another hour of incubation. After three washing steps, 100 μl of HRP conjugate concentrate were added and incubated for 1 hour. The plate was washed four times and TMB substrate was added. After 15 minutes of incubation stop solution was added and absorbance was determined at 450 nm.® reagent  according to the manufacturer's instructions. RNA was quantified photometrically and 150 ng per reaction were analysed by real-time RT-PCR. Interferon beta mRNA was amplified with primers IFNβFwd (5'-GAACTTTGACATCCCTGAGGAGATT-3') and IFNβRev (5'-GGAGCATCTCATAGATGGTCAATG-3'), and 5'-nuclease probe IFNβ-P (FAM-CAGCAGTTCCAGAAGGAGGACGCC-TAMRA). GAPDH mRNA was detected in parallel with primers GAPDHFwd (5'-AGGTGGTCTCCTCTGACTTCAACA-3'), GAPDHRev (5'-AGTGGTCGTTGAGGGCAATG-3'), and probe GAPDH-P (FAM-CACCCACTCCTCCACCTTTGACGCT-TAMRA). Reactions using the OneStep RT-PCR kit  comprised 50°C for 30 minutes, followed by 95°C for 15 minutes and 40 cycles of 95°C for 10 seconds and 58°C for 30 seconds. For both genes standard curves were generated from limiting dilution series of quantified RNA. The dilution end-points were defined as one PCR unit for each gene. Log PCR units for each experimental sample were calculated from the linear equations of the dilution series. Interferon beta quantity was normalised to GAPDH quantity by subtraction of logarithmic quantities (Interferon - GAPDH).Total RNA was prepared from 293-lp cells in 6-well plates with Trizol4 PFU each. 100 μl of virus solution were applied. Hamsters were sacrificed at indicated days post infection. Lungs were prepared in total, weighed, and homogenized. Tissue was suspended to a concentration of 0.5 g/ml in complete DMEM before analysis by RT-PCR or cell culture.Infections were performed with rSCV and r7bΔTMD. Heat-inactivated rSCV served as the mock-control. Syrian Golden hamsters  were infected via the intranasal route with 10The authors declare that they have no competing interests.SP constructed the infectious clone and conducted all experiments with recombinant viruses. VK and EM designed and carried out the overexpression experiments and the reporter assays and/or critically revised the manuscript. VD conducted hamster infections. KG participated in construction of the infectious clone. CD designed the study, participated in the construction of the infectious clone, and wrote the manuscript. All authors took part in manuscript preparation. All authors read and approved the final manuscript.


Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases  catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics. Plants emerged as effective systems for production of pharmaceutically important mammalian proteins, and a number of transgenic plant-based products are currently available in the market . Work inO-glycans are attached primarily to hydroxyproline (Hyp) and serine residues in plant glycoproteins. Typically, non-contiguous Hyp residues are modified with arabinogalactan polysaccharides, whereas contiguous Hyp are exclusively arabinosylated ,8. The eIn contrast to plants, mammalian O-glycosylation is a well understood posttranslational modification. Substitutions of serine and threonine residues with sugars such as fucose, galactose and N-acetylglucosamine are common modification on mammalian proteins , but theN-acetylglucosaminyltransferase are required for synthesis of core 1 O-glycans and core 3 O-glycans, respectively.Mucin type O-linked glycans are synthesized in the Golgi apparatus  in a stein silico analysis and more recent experimental data [A previous study reported attachment of core 1 Galβ1-3GalNAc disaccharide to rice glutelin basic subunits , howevertal data  suggest N. benthamiana and used to demonstrate that mammalian GalNAc-Ts retain their functionality, specificity and localization in plant cells. We further tested the engineered GalNAc-T2 transgenic plants for their ability to produce GalNAc-glycosylated proteins. A model glycoprotein, mucosally targeted mucin 1 (MUC1) tandem repeat-derived peptide, was transiently expressed using the magnICON deconstructed viral vector system to ensure rapid accumulation and high yield [The present study tested, for the first time, the feasibility of generating a plant-based platform for production of Tn antigen-containing proteins/peptides. Many valuable pharmaceuticals, such as immunomodulators and tumor-associated antigens essential for the development of anticancer therapeutic vaccines, are GalNAc -glycosylated ,25. Despgh yield . GlycosyAgrobacterium-mediated transformation of N. benthamiana L. with pH7WG2:GNT2 binary vector , positioned at the C terminus of GalNAc-T2 to avoid interfering with the transmembrane domain (TMD), was used to monitor the localization of mammalian enzyme in tobacco cells. A discrete punctuate pattern was observed by confocal microscopy  as an acceptor peptide. Under the chosen HPLC conditions, the retention time for UDP-GalNAc was 4.09 min, while hCG-β peptide eluted at 14.08/14.43 min as two peaks ("a" and "a' "), as confirmed by MALDI (data not shown). HPLC separation of an aliquot of the enzymatic reaction with the microsomal fraction from control tobacco plants showed the presence of only the non-glycosylated hCG-β peptide  acceptor peptide. This was not surprising since the peak of the acceptor hCG-β peptide (peak "a") partially overlapped peaks "b" and "c". The ions at m/z 2694.4 and m/z 2897.5 were suggestive of two glycosylated forms of hCG-β peptide with attached one or two GalNAc residues. MALDI-TOF-TOF spectra analysis further supported the assumption. The major fragmentation product resulting from loss of one HexNAc residue from the glycosylated peptide containing a single GalNAc was evidenced by the abundant ion at m/z 2491.4  indicated that some annotated plant proteins known to transport through the ER-Golgi pathway do contain potential sites for GalNAc-glycosylation (data not shown). Therefore, we tested for possible attachment of GalNAc to endogenous proteins of N. benthamiana L plants engineered to express human GalNAc-T2. Equal amounts of soluble proteins from the control and transgenic plants were subjected to affinity chromatography on Vicia villosa agglutinin (VVA)-agarose. VVA is a lectin that strongly recognizes terminal GalNAc residues attached to serine or threonine in a polypeptide. The binding buffer was supplemented with 0.1 M NaCl to prevent non-specific interactions, and the resin was thoroughly washed before the elution. Eluted proteins were further subjected to lectin blot analysis with alkaline phosphatase-conjugated VVA. Several positive bands were detected in the transgenic plant sample, but not in the negative control . Following silver staining, the strongest band, corresponding to a 33 kDa polypeptide  containing the modification, thus strongly suggesting attachment of a N-acetylhexosamine residue to the polypeptide chain. It is of interest that cleavage on the carboxy-terminal side of the potentially modified Thr-119 was absent (lack of y3 and b13 ions), possibly as a result of the sugar moiety blocking access of the ionizing mobile proton at that position. Loss of the sugar moiety from glycopeptides is often observed during MS/MS, but in certain cases of low-energy-collision induced dissociation the sugar moiety may remain attached, resulting in fragment ions carrying the modification [The substrate preferences of GalNAc-T2 have been extensively studied and the most favorable amino acids at positions -3, -2, -1, 1, 2, and 3 relative to the site of glycosylation (Ser/Thr), have been determined . The firtrol Fig.. Followification .O. sativa L. japonica class I chitinase , suggests that Thr-119 should be located on the surface of the catalytic domain, therefore accessible for glycosylation. Previous study has shown that Thr-119 is not directly involved in the catalytic act [A homology model of tobacco endochitinase Fig. , built bytic act .E. coli enterotoxin B subunit and human MUC1 tandem repeat (TR)-derived peptide (LTBMUC1) was used for this purpose , but only serine and threonine in GSTAP motif  and threonine in GVTSA motif (Thr-140) have been verified experimentally as target sites of GalNAc-T2 [2+ Sepharose Fast Flow chromatography combined with a His tag-based screen to detect the fractions containing the recombinant protein; (iii) affinity chromatography on VVA agarose to capture the GalNAc-glycosylated population of LTBMUC1, assisted by a His-tag-based and VVA-lectin blot detection to verify the molecular mass of the captured protein; (iv) LC-MS/MS analysis to confirm the identity of LTBMUC1 and determine the sites of glycosylation.The MUC1 TR-derived peptide used as a part of the model LTBMUC1 protein has seven potential O-glycosylation sites predicted alNAc-T2 ,  puet al., submitted). Following protein extraction and ammonium sulfate precipitation, the His-tagged recombinant protein was purified by chromatography on Ni2+ Fast Flow Sepharose. Aliquots of the fractions eluted from the column were analyzed for purity by Ponceau S staining and by Western blotting to detect the His-tag on LTBMUC1. The recombinant protein was not visible on the Ponceau S-stained PVDF membranes, probably because its amount was below the detection limit of the stain (1 μg/5 mm band). It should be noted that the LTBMUC1-positive fractions contained co-eluted endogenous tobacco proteins . These data suggest that plant-expressed LTBMUC1 undergoes glycosylation by endogenous enzyme activities. The amino acid sequence of LTBMUC1 lacks consensus motifs for N-glycosylation (Asn-Xaa-Ser/Thr), but there are several proline residues in a context favoring their hydroxylation and subsequent glycosylation [The theoretical molecular mass of LTBMUC1 is 17.7 kDa, but on the His-tag blots the recombinant protein migrated as a single ~20 kDa band  and GalNAc-T2//UDP-GlcNAc 4-epimerase//UDP-GlcNAc/UDP-GalNAc transporter plants (2') after NiBound proteins that eluted from the VVA-agarose column from both LTBMUC1 samples were analyzed by SDS-PAGE and subsequent silver staining. A strong band with a molecular mass of ~20/21 kDa was visible only in the sample from the triple transgenics Fig. . Peptide/Ser-518 sequence remains unoccupied by N-glycans [et al. [GalNAc-T2 is one of the best characterized members of the GalNAc-Ts family that exhibits a very low level of UDP-GalNAc hydrolase activity . Thus, i-glycans . The hum-glycans . The pla-glycans  at sites-glycans ,42. Spec [et al.  supports [et al. .In silico analysis suggests that plants do not have orthologs of human GalNAc-Ts. By a combination of in vitro radioactive enzyme assay and mass-spectrometry analysis, our work provides direct experimental evidence for a lack of endogenous GalNAc-T2 activity in N. benthamiana L. plants. A similar conclusion was conveyed indirectly by a study showing absence of mucin type O-glycans in the hinge region of maize-produced human IgA1 [H. sapiens and D. melanogaster UDP-galactose transporters [Y. enterocolitica UDP-GlcNAc 4-epimerase and C. elegans UDP-GlcNAc/UDP-GalNAc transporter resulted in the production of LTBMUC1 exclusively as a glycoform. Altered electrophoretic mobility of the model protein, and especially its strong binding to VVA-agarose, are unequivocal proofs for attachment of GalNAc residue(s) to the polypeptide backbone.man IgA1 . Our worman IgA1 . These dman IgA1 . A nucleman IgA1 -46. Simisporters , the latSimilar to previous observations with maize-expressed human IgA1 , tobaccoOur work provides the first experimental evidence that plants can be engineered for production of Tn-antigen-containing therapeutics by "updating" their enzyme machinery with UDP-GlcNAc 4-epimerase, UDP-GalNAc transporter and GalNAc-T. Further genetic manipulations could potentially lead to the synthesis of more complex O-glycans. Thus, plants can become an attractive alternative platform for production of homogenous populations of mucin type O-glycosylated recombinant therapeutics with predictable O-glycan structures. Endogenous plant enzyme activities that target directly the actual sites of O-glycan attachment, serine and threonine residues, are not as abundant as in other heterologous production systems (e.g. O-mannosyltransferase activity in yeasts). However, proline residues are often in proximity to the attachment sites, and some of these prolines might be in a sequence context favoring their plant-specific hydroxylation and subsequent glycosylation. The possibility of "collision" between the plant O-glycosylation process and the engineered GalNAc-glycosylation process requires careful examination of the amino acid sequences of mammalian proteins targeted for plant-based expression, and if necessary and possible, appropriate alterations to eliminate the risky motifs.Homo sapiens GalNAc-T2 (GenBank Accession No BC041120) was amplified with Pfu DNA polymerase  using G1 (5'-CACCATGCGGCGGCGCTCGCGGATGC-3')/G2 (5'-CAGCTACTGCTGCAGGTTGAGCGTGAACTTCCACTGCTGCGAAAGGGCC-3') pair of primers. The product was separated on 1% agarose gel, gene-cleaned  and subcloned in pENTR/D-TOPO vector . Using the Gateway™ technology , the gene was transferred either to pH7WG2, or to pK7FWG2 plant expression vector [A. tumefaciens EHA101 competent cells. Recombinant colonies were selected on LB plates supplemented with 200 mg/l spectinomycin.The coding region of n vector  for natiCaenorhabditis elegans clone (CO3H5.2) coding for UDPGlcNAc/UDPGalNAc transporter [CCATGGATCGAGCTAACGACACGAGCTC-3')/GT2 (CGGTACCTCAGGCATTATGAGCTTCGGCTG-3') set of primers. Yersinia enterocolitica type 0:8 gne gene coding for UDP-GlcNAc-4 epimerase was isolated by a PCR approach using GE1 (5'-CACCTCTAGAATGTCTATATTAATTACTGGTGGTGCTG-3')/GE2 (5'-CCGAGCTCCTAACAGTTATAACCATTAGGATTCATTTTTTGCC-3') pair of primers. Both PCR products were subcloned in pENTR/D-TOPO vector . The internal XbaI site of the gne gene was eliminated by a site-directed mutagenesis  using GE3 (5'-CTGAACTAGCACATCGAGAGTTAGGTTGGTATGC-3') primer. A shuttle pBY210:GNE:GT vector containing both C. elegans UDPGlcNAc/UDPGalNAc transporter gene and Y. enterocolitica gne gene was generated using a two-step strategy. First, the gne gene was ligated as an XbaI/SacI fragment into pBYsE1bE2T210  digested with the same restriction enzymes. The generated vector was then cut open with NcoI and KpnI for ligation of the transporter gene taken from pENTR/D:GT as an NcoI/KpnI fragment. The cassette with the two genes was further excised with AscI and FseI, and ligated into AscI/FseI digested pBYR-H2gpKDEL-K3 , thus producing pBYR:GNE.GT vector. The vector was electroporated into A. tumefaciens LB4404 competent cells. Recombinant colonies were selected in the presence of 200 mg/l streptomycin.nsporter , was puret al., submitted). The vector is the 3' module of the magnICON deconstructed viral expression system , and is delivered simultaneously with the integrase module pICH14011 and the 5' module pICH15879. The system, based on the assembly of functional viral replicons in planta, is described in details in [A. tumefaciens GV3101 competent cells and selected in the presence of 50 mg/L carbenicillin and 60 mg/L rifampicin.The construction of pICH11599:LTBMUC1 vector is described elsewhere , or OD600 = 0.033 (for each of the three magnICON module vectors). Infiltrated plants were kept in growth chambers  until ready for leaf harvest.Transient transformation A PCR screen of the putative GalNAc-T2 transgenic plants was performed with GoTaq DNA Polymerase , 1 μg genomic DNA  as a template, and the same primers and PCR program used for GalNAc-T2 gene amplification.C. elegans UDP-GlcNAc/UPD-GalNAc transporter transcript, and GE4 5'-GTCTATATTAATTACTGGTGGTGCTGGATATATAGG-3')/GE5 (5'-CAGTTATAACCATTAGGATTCATTTTTTGCCATTTCCAAGCGTCC-3') pair for amplification of 1.07 kb of Y. eneterocolitica gne transcript. The PCR and RT-PCR samples were separated on agarose gels along with BenchTop 1 kb DNA ladder  or GeneRuler 1 kb DNA ladder , and visualized by staining with ethidium bromide.Total RNA was isolated with Promega's SV Total RNA Isolation System. RT-PCR was performed using a TITANIUM One-Step RT-PCR Kit , and 500 ng of template. The following pair of primers were used: G3 (5'-GGTGATCACGTTTCACAATGAAGCCAGG-3')/G4 (5'-AGTGTCGAGGCAGTTAGTTCCCTGCTGC-3') pair for amplification of a 0.96 kb fragment of the human GalNAc-T2 transcript, GT3 (5'-GACACGAGCTCCAATCTGAAGCTCATCTC-3')/GT4 (5'- CAGGCATTATGAGCTTCGGCTGGTGTCG-3') pair for amplification of a 1.04 kb of For Western blot analysis, the hydrophobic proteins from the transgenic and control plants' leaves were isolated with a Hydrophobic Protein Isolation Kit . SDS samples were prepared with 5×SDS sample buffer  SDS, 0.05% (w/v) bromophenol blue) and loaded on 4-20% gradient gels  next to Kaleidoscope Prestained Standards . Separated proteins were electro-blotted onto 0.45 μm Immobilon-P PVDF membranes  and blocked overnight at 4°C in TBST buffer  Tween-20) containing 5% (w/v) dried milk. Following two washes with TBST for 15 minutes each, the membranes were probed overnight at 4°C with mouse monoclonal IgG antibodies (10 μg CB-4 in 5 ml TBST containing 1% (w/v) dried milk) raised against human GalNAc-T2  as originally described in . Next da2, and 0.25% (v/v) Triton X-100. At the completion of the reaction, the mixture was filtered sequentially through Ultrafree-MC centrifugal filter  and Nanosep 30 K filter spin columns .Plant leaves were homogenized with a mortar and pestle in cold 50 mM Tris HCl buffer pH 7.4 containing 330 mM sucrose, 3 mM DTT, 3 mM EDTA, 5% polyvinyl pyrrolidone and 1 mM PMSF. The homogenate was filtered through cheesecloth and centrifuged at 10 000 g for 25 minutes at 4°C. The supernatant was then centrifuged at 100 000 g for 2 hours (Beckman Coulter Optima L-100 XP Ultracentrifuge). The pellet representing microsomal membranes was resuspended in 50 mM Tris HCl pH 7.4, and protein concentration was measured according to the assay described in . The Galm/z from 1046.54 (Angiotensin II) to 3147.47 (Somatostatin). The acceleration voltage was as follows: ion source 1 - 25.00 kV, ion source 2 - 21.90 kV, lens - 9.50 kV, reflector 1 - 26.30 kV, reflector 2 - 13.80 kV. Glycosylated hCG-β peptides' sequencing (TOF/TOF MS spectra) provided clear spectra with readily available -b/-y-ions that were analyzed with the Brukers' BioTools version 3.0 software.An aliquot of the reaction mixture was subjected to reverse-phase HPLC  using Luna column . The compounds were eluted at a constant flow rate of 4 ml/min with a linear (10-45%) gradient of solvent B (0.1% trifluoroacetic acid (TFA), 90% acetonitrile in water) in solvent A (0.1% TFA in water) for 20 minutes, followed by a linear gradient (45-95%) of solvent B in solvent A for 10 min. The elution of the peptides was monitored by diode-array detector at 214 nm. Fractions of 2 ml were collected throughout and monitored for incorporated radioactivity on a LS 6500 Multipurpose Scintillation Counter . Alternatively, the peaks were collected and subjected to a matrix-assisted laser desorption ionization time of flight (MALDI -TOF) MS. One microliter from each peak fraction was mixed with 9 μl matrix solution (a saturated solution of α-cyano-4-hydroxycinnamic acid and 0.1% TFA in 50% acetonitrile in water), and 1 μl of this mixture was spotted on a stainless steel plate. Mass spectra were acquired in positive ion mode with the reflector engaged on Bruker Daltonix Ultraflex III TOF/TOF mass spectrometer . The Nd:YAG laser (355 nm) intensity was adjusted for optimum sensitivity and resolution. Single stage MALDI-TOF mass spectra were externally calibrated with a mixture of 7 peptides supplied by Bruker, ranging in monoisotopic 2, 3 mM DTT, and 1 mM PMSF. The homogenate was run through cheesecloth and centrifuged at 10 000 × g for 15 minutes at 4°C. The supernatant was saturated with (NH4)2SO4 to a final concentration of 75% (w/v) and incubated overnight at 4°C. The precipitated proteins were collected by centrifugation at 15 000 g for 45 min and 4°C, and the pellet was dissolved in a small amount of 50 mM Tris HCl buffer pH 7.4 containing 1 mM MgCl2. The samples were desalted using PD10 columns , supplemented with NaCl to a final concentration of 0.1 M, and subjected to chromatography on Ni2+ Fast Flow Sepharose  according to manufacturer's recommendations. The eluted fractions were screened for the presence of the recombinant LTBMUC1 protein by SuperSignal West HisProbe Kit . Blots were stained in parallel with Ponceau S .LTBMUC1 fusion protein was extracted from tobacco leaves by homogenization with cold 50 mM Tris HCl buffer pH 7.4 containing 0.1 M NaCl, 1 mM MgCl4)2SO4-precipitated as described above. After desalting using desalting spin columns , 150 μl samples of total soluble proteins or purified LTBMUC1 normalized by Bradford assay [Vicia villosa (VVA) immobilized lectin gel  pre-equilibrated with 50 mM Tris HCl buffer pH 7.4 containing 0.1 M NaCl and 1 mM MgCl2. The suspension was incubated overnight at 4°C on a Thermoline Labquake shaker. The non-bound proteins were then collected and the column was washed with 20 column volumes of buffer. Bound proteins were eluted with 50 mM Tris HCl buffer pH 7.4 containing 0.1 M NaCl, 1 mM MgCl2 and 0.3 M GalNAc, and used for further analyses.Soluble proteins from tobacco leaves of the wild type or transgenic plants were extracted and , probed with alkaline phosphatase-conjugated VVA  following a protocol of Roche Molecular Biochemicals, or subjected to His-tag-based detection using SuperSignal West HisProbe kit .http://www.thegpm.org[Following SDS-PAGE and silver staining as described above, the bands of interest were excised and digested with chymotrypsin (10 μg/ml) at 37°C overnight. LC-MS/MS analyses of in-gel chymotrypsin digested protein bands  were carhegpm.org and withhegpm.org,60. Pepthegpm.org and the 4, or in the same buffer supplemented with 10 μg/ml Brefeldin A . Microscope slides were prepared with small segments of transformed leaves cut with a razor blade and mounted in water. Imaging was conducted on the Leica SP2 Multi-photon Scanning Laser Microscope  housed in the W.M. Keck Bioimaging Facility at Arizona State University. Multiple lasers allowed for simultaneous imaging of GFP fusion protein , and auto-fluorescence given off by the chloroplasts . The detection channels (Ch) were set to the following ranges: Ch 1: 490-510 nm and Ch 2: 600-720 nm. Settings for gain and offset of the detectors were kept constant for all experiments. Using a 40× oil objective, images were scanned at 0.6 μm intervals in the z-axis. The post-acquisition image processing was performed using the Leica SP2 software  and Adobe Photoshop CS3 software .Leaves from tobacco plants expressing GalNAc-T2:GFP fusion protein were cut in half and incubated for 1 h at room temperature either in 50 mM Pipes buffer, pH 6.9 containing 5 mM EGTA and 5 mM MgSOhttp://www.ncbi.nlm.nih.gov was accessed in February, 2008 - May, 2009. Localization of proteins was predicted with PSORT software http://psort.ims.u-tokyo.ac.jp/form.html. Predictions of signal sequences and potential mucin type O-glycosylation sites were done with SignalP 3.0 http://www.cbs.dtu.dk/services/SignalP/ and NetOGlyc 3.1 http://www.cbs.dtu.dk/services/NetOGlyc/, respectively. A homology model of the tobacco endochitinase (GenBank Accession No AAA34070) was built based on the 3 D structure of O. sativa L. japonica class I chitinase (pdb ID 2DKV) using Geno3 D automatic molecular modeling tools http://pbil.ibcp.fr/htm/index.php and Jmol software http://molvis.sdsc.edu/fgij/index.htm for visualization.A GenBank/NCBI database The authors declare that they have no competing interests.in silico analyses, LTBMUC1 purification and the subsequent molecular analyses, and drafted the manuscript; JR constructed pK7FWG2:GNT2 expression vector, carried out the non-radioactive enzyme assays, the molecular analyses of the transgenic GalNAc-T2 plants, and helped to draft the manuscript; ZC carried out the characterization of the reaction products of the enzymatic reactions by MS and peptide sequencing; SO participated in lectin chromatography and lectin blot analysis, prepared the endochitinase sample, and maintained the transgenic plants; GT performed nano LC/MS-MS analyses; HM constructed pBYsE1bE2T210 and pBYR-H2gpKDEL-K3 vectors, and provided magnICON 5' and integrase modules; LL constructed pH7WG2:GNT2 plant expression vector, generated stably transformed GalNAC-T2 plants, participated in molecular analyses, and conceived the study. All authors read and approved the final manuscript.SD carried out the construction of pBYR:GNE.GT expression vector, transient plant transformations, radioactive enzyme assays, confocal microscopy,


Mitochondria are semi-autonomous, semi-self-replicating organelles harboring their own DNA , and their dysregulation is involved in the development of various diseases. While mtDNA does not generally undergo epigenetic modifications, almost all mitochondrial proteins are encoded by nuclear DNA. However, the epigenetic regulation of nuclear-encoded mitochondrial genes (nuclear mt genes) has not been comprehensively analyzed.We analyzed the DNA methylation status of 899 nuclear mt genes in the liver, brain, and heart tissues of mouse, and identified 636 nuclear mt genes carrying tissue-dependent and differentially methylated regions (T-DMRs). These nuclar mt genes are involved in various mitochondrial functions and they also include genes related to human diseases. T-DMRs regulate the expression of nuclear mt genes. Nuclear mt genes with tissue-specific hypomethylated T-DMRs were characterized by enrichment of the target genes of specific transcription factors such as FOXA2 in the liver, and CEBPA and STAT1 in the brain.A substantial proportion of nuclear mt genes contained T-DMRs, and the DNA methylation status of numerous T-DMRs should underlie tissue-dependent mitochondrial functions. Mitochondrial dysfunction is a common cause of human diseases ,2, and tOct-4 and Nanog, are hypomethylated only in a few cells [Methylation of nuclear DNA is a major component of epigenetic system in mammalian cells, and is involved in silencing of gene transcription and maintaining genomic stability ,7. Hypomew cells ,13, the ew cells ,14. Tissew cells ,11,15.Ant4, which encodes mitochondrial outer membrane protein, contains T-DMRs which is specifically hypomethylated in the testis [A nuclear mt gene, e testis ,17. Alsoe testis . HoweverTo investigate the DNA methylation status of nuclear mt genes in the liver, brain (cerebrum), and heart tissues, we conducted pairwise tissue comparisons using model-based analysis of tiling-array (MAT) along with D-REAM analysis ,18. DiffP < 0.05, Fisher's exact test; Figure Otc and Lrpprc . Sixty-four out of 123 nuclear mt genes with liver-hypo T-DMRs were the targets of FOXA2 .Genomic DNA was extracted as described previously . BrieflyWe used T-DMR profiling with restriction-tag mediated amplification (D-REAM) analysis  to obtaiD-REAM analysis was performed twice for each of the biological duplicates of heart, and once for the liver and brain in this study. For the liver and brain, we added single D-REAM data set from our previous study using tissues from different individual . CorrelaGenomic DNA was digested with HindIII (Takara). Digested DNA (5 μg) was denatured with 0.3 M NaOH. Sodium metabisulfite (pH 5.0) and hydroquinone were added to a final concentration of 2.0 M and 0.5 mM, respectively. The reaction mixture was incubated under following conditions: 15 cycles of 95°C for 30 s and 50°C for 15 min. Next, 1.77 volume of QG buffer was added to the reaction mixture, and DNA was purified using a Quiagen gel extraction kit (Qiagen), and eluted with 100 μl of elution buffer (EB). DNA was treated with 0.3 M NaOH at 37°C for 15 min, precipitated using 6 M ammonium acetate (pH 7.0) and ethanol, and dissolved in 200 μl TE (pH 8.0). For each bisulfite PCR, 2 μl of DNA solution was used as the template, and BIOTAQ HS DNA polymerase (Bioline) was used for amplification. PCR was performed under the following conditions: denaturation at 95°C for 10 min followed by 43 cycles, each cycle comprising 95°C for 30 sec, 60°C for 45 sec, 72°C for 30 sec, followed by 10 min at 72°C. All primers used in this experiment are listed in Additional file Total RNA was prepared using TRIzol reagent (Invitrogen). Before synthesis of first-strand cDNA, the RNA preparation was treated with RNase-free DNase I (Invitrogen) to eliminate any residual genomic DNA. The total RNA was then converted into first-strand cDNA using random hexamers and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). The obtained cDNA were amplified and quantified in triplicates by using the Quantitect SYBR Green PCR Kit (Qiagen) with ABI 7500 Real Time PCR system (Applied Biosystems). PCR was performed under the following conditions: Incubation at 95°C for 10 min followed by 40 cycles of PCR, each cycle comprising 95°C for 15 sec and 60°C for 1 min. All primers used in this experiment are listed in Additional file MAT  was usedn = 2 for each tissue) were downloaded from Gene Expression Omnibus (accession no. GSE10246). The array image data (.CEL files) was processed by the factor analysis for robust microarray summarization algorithm (FARMS) with quantile normalization [For expression analysis, data from the GeneChip Mouse Genome 430 2.0 Array of liver, heart, and cerebral cortex tissues of C57BL/6N male mice (8-10 week old; lization .Enrichment analysis of specific transcription factor targets was performed using oPOSSUM program . This prMT and KS designed this study. KH performed D-REAM. MT performed COBRA and data analysis with help of SY, and wrote the paper with SY and KS. All authors read and approved the final manuscript.Figure S1 Differential methylation levels of HpyCH4IV sites and the corresponding MATscores.Click here for fileTable S1 Primers used for COBRA, and the result of the experiment.Click here for fileTable S2 Genomic locations of T-DMRs.Click here for fileTable S3 Genomic locations of hypo T-DMRs.Click here for fileFIgure S2 Expression ratio of nuclear mt genes with hypo T-DMRs.Click here for fileTable S4 Genes with liver-hypo T-DMRs and FOXA2-binding sites.Click here for fileTable S5 Genes with hypo T-DMRs and overrepresented transcription factor binding sites.Click here for fileTable S6 Primers used for real-time PCR.Click here for fileFigure S3 Distribution of MATscores calculated from D-REAM data of each tissue.Click here for file


Polymorphisms within the insulin gene can influence insulin expression in the pancreas and especially in the thymus, where self-antigens are processed, shaping the T cell repertoire into selftolerance, a process that protects from β-cell autoimmunity.We investigated the role of the -2221Msp(C/T) and -23HphI(A/T) polymorphisms within the insulin gene in patients with a monoglandular autoimmune endocrine disease , those with a polyglandular autoimmune syndrome type II  as well as in healthy controls .CC" -2221Msp(C/T) and "AA" -23HphI(A/T) polymorphisms than the HC .T1D patients carried significantly more often the homozygous genotype "The distribution of insulin gene polymorphisms did not show significant differences between patients with AD, HT, or APS-II and HC.C" of the -2221Msp(C/T) and "A" -23HphI(A/T) insulin gene polymorphisms confer susceptibility to T1D but not to isolated AD, HT or as a part of the APS-II.We demonstrate that the allele " Type 1 diabetes (T1D) has a multifactorial pathogenesis, partly caused by environment but also by genetic predisposition. The association of the MHC class II genes with T1D is well known, but there are also non- MHC genes such as the insulin gene, that confer susceptibility to T1D  as well as in healthy controls (HC).All patients were recruited from the endocrine outpatient clinics at the University Hospitals Frankfurt am Main, Freiburg, Würzburg, Munich and Düsseldorf (Germany).Altogether 1248 individuals were genotyped for the -2221 (C/T) polymorphism within the promoter region of the insulin gene .T1D was diagnosed according to World Health Organization criteria. The median age of disease onset was 10 years (range 1–50 years). From the 213 T1D families, 17 offspring had T1D as a part of the APS-II. There was no evidence for MODY- or other rare forms of insulin-deficient diabetes, furthermore all were simplex cases.Patients with isolated T1D may develop other autoimmune diseases in the future. Nevertheless, at the point of this study, they had the diagnosis of isolated T1D.Addison's disease was diagnosed by primary adrenal failure without evidence of tuberculosis or adrenoleukodystrophy. Adrenal autoantibodies were detected with indirect immunofluorescence on cryostat sections; these were confirmed to be directed against 21-hydroxylase in a subgroup of patients by radioimmunoassay. No neurological deficits could be detected. No patients with AD without T1D had antibodies against β-cell antigens (GAD-Ab or IA-2 Ab).The diagnosis of HT was established by positive thyreoglobulin (Tg) and/or thyroid peroxidase antibodies, reduced echogenicity on thyroid ultrasound, and normal or elevated thyrotropin (TSH) levels.Patients with an APS-II (n = 62) were diagnosed as a combination of T1D or AD and other of the above mentioned disorders.Among the patients with APS-II, there were 22 patients with the diagnosis T1D and autoimmune thyroidtis (AIT) (15 with T1D and HT and 7 with T1D and GD), 33 with AD and AIT (23 with AD and HT and 10 with AD and GD) as well as 7 with T1D, AD and HT.The diagnosis of GD rested on autoimmune hyperthyroidism with elevated TSH receptor autoantibodies and/or ophthalmopathy.Age- and sex-matched HC were volunteer blood-donors from the Red Cross Blood Transfusion Centre in Frankfurt am Main (Germany), staff personal or medical students from the University Hospital Frankfurt am Main (Germany) without family history of T1D, HT, GD or AD. Although the adrenal function of the controls was not formally assessed, they had normal thyroid function and were thyroid autoantibody negative and did not report clinical symptoms.All individuals were of German origin and were inhabitants from the surrounding area of Frankfurt am Main (Germany).The male: female ratio of patients with T1D, AD, HT, APS-II and HC was 1:0.91, 1:2.14, 1:4.56, 1:3.89 and 1:0.48, respectively. The median age of the patients with T1D, AD, HT, APS-II and HC was 30 (range 5–81), 57 (range 22–92), 52 (range 25–95), 46 (range 15–76) and 40.5 (range 23–83), respectively.The study protocol was approved by the Ethics Committee of the University Hospital, Frankfurt am Main and written informed consent was obtained from all patients and controls.DNA was extracted from whole blood according to standard protocols and polymorphisms were typed according to the following protocols:Patients with T1D, HT and APS-II and HC were studied for the insulin gene promotor polymorphism -2221 (C/T) using polymerase chain reaction (primers: 5'-ACCCCACTACACGCT GCTG-3' and 5'-CCCTTCAGAGACACCCCCA-3') followed by digestion with the restriction enzyme Msp I  and separated on 4% agarose gel.®). Primer and probe sequences are as follows: forward primer: 5'-TGAGTAGGAGTTTGCCAGTGTTG-3'; reverse primer: 5'-GCTGCTGGGATCCTGGAT-3'; probe for T allele 5'-VIC-TCAGCTCCCTGGCCGA-3' and probe for C allele 5'-FAM-AGCTCCCCGGCCGA-3'.DNA of patients suffering from AD as well as of 100 samples of the above mentioned (which were genotyped by restriction enzyme and chose at random to confirm the accuracy of the method) were analyzed by real-time PCR using ABI 7300 (Applied biosystems®). In order to check the accuracy of the method 100 DNA-samples – chosen at random- were genotyped according to a previously published [All samples were genotyped by real time PCR by using C_1223317-Assay  [Patients and controls were compared using allele-wise and genotype-wise ChiGermany) .The transmission disequilibrium test (TDT) was used for the family analysis to detect preferential transmission of the alleles to affected offspring  as well In order to detect a statistical difference (p < 0.05) at an OR 1.8 (as seen for T1D) we calculated for the -2221Msp(C/T) SNP that 276 chromosomes (138 patients vs. 138 HC) and for -23HphI(A/T) 224 chromosomes (112 patients vs. 112 HC) are necessary to reach a power of 80%- using the software version 2.1.30 .P values were corrected by the number of polymorphisms (n = 2). A corrected p (pc) < 0.05 was considered significant.We found a significant difference in the genotype and allele distribution of both -2221Msp and -23HphI polymorphisms between patients with T1D and HC.CC" of the -2221MspI and "AA" of the -23HphI polymorphisms, respectively than the HC , matching our findings from the case-control analysis.Heterozygous parents in T1D families transmitted significantly more often the allele "The -23HphI polymorphism showed the strongest association to T1D in both T1D-family and case-control analysis.Neither genotype frequencies nor allele wise comparison differed between patients with HT, AD, APS-II and HC table  and 2. NC" of the -2221MspI and "A" of the -23HphI polymorphisms within the insulin region to confer susceptibility to T1D in German patients. Therefore our data are in line with previous reports, which found that the -2221 MspI SNP and the -23HphI SNP are associated with T1D, where the -2221 MspI SNP was only part of a susceptibility insulin gene haplotype and the -23HphI SNP was found with the strongest association to T1D [In the present study, we demonstrate the allele "n to T1D ,9.Other studies found no evidence of excessive transmission of the -23HphI polymorphism  in patients with GD . Also noC" allele of -2221 MspI has been associated with a decreased insulin expression in the thymus, leading to less efficient deletion of autoreactive insulin-specific T-cells [T" [Class III alleles of the insulin VNTR region are associated with substantially higher insulin mRNA levels than VNTR class I in the thymus presumably, causing a more efficient deletion of insulin autoreactive T cells , explain T-cells . This maThis association appears to be confined to patients with T1D only and larger numbers are needed to rule out an effect in patients with other immune components of T1D associated with the APS-II. So far this locus may help to differentiate the susceptibility to isolated T1D in comparison to T1D predisposition loci that are shared with endocrine autoimmune disorders.C" of the -2221MspI and "A" of the -23HphI polymorphisms within the insulin region confer susceptibility to T1D but not to isolated AD, HT or as a part of the APS-II.We demonstrate the allele "T1D: Type 1 diabetes; VNTR: variable number of tandem repeat; AD: Addison's disease; HT: Hashimoto's thyroiditis (HT); APS-II: Polyglandular autoimmune syndrome type II; GD: Graves' disease; HC: Healthy controls; TDT: Transmission disequilibrium test; HLA: Human Leukocyte Antigen.The authors declare that they have no competing interests.ER–L and BL carried out the genotyping of the patients and controls. MP–M carried out the re-genotyping of the patients and controls to confirm the accuracy of the method. HK participated in the statistical analysis GM, HSW, NR, SH and JS made the diagnosis and collaborated in collection of samples. HK carried out statistical analysis. ER–L and KB conceptualized and designed the study.The pre-publication history for this paper can be accessed here:


Specimens of formalin-fixed, paraffin-embedded non-small-cell lung carcinomas  were analysed immunohistochemically for expression of cyclin A. The analysis was intended to determine whether cyclin A has additional prognostic value for predicting patients' survival and drug response. Of the 187 NSCLCs, 141 cases (75%) showed expression of cyclin A. Patients with cyclin A-positive carcinomas had significantly shorter median survival times than patients with cyclin A-negative carcinomas . Similar results were obtained with more homogeneous groups of patients: patients with only T3 tumours, patients with epidermoid carcinomas and patients with lymph node involvement. The clinical parameters  had no influence on expression of cyclin A. A direct correlation between cyclin A and the proportion of S-phase cells (P = 0.08) and an inverse relationship between cyclin A and the proportion of G0/G1-phase cells (P = 0.04) were found. Furthermore, a significant correlation between the expression of cyclin A and the response of NSCLC to doxorubicin in vitro was detected (P = 0.026).


Such observations suggest a contributing role of contaminant-induced suppression of cell-mediated immunity leading to decreased host resistance. Here, we assessed the effects of the prototypic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (B[a]P), and two polychlorinated biphenyls (PCBs), CB-156 and CB-80, on the T-cell proliferative response to mitogen in harbor seal peripheral lymphocytes. Despite the variability associated with our samples from free-ranging harbor seals, we observed a clear suppressive effect of B[a]P (10 uM) exposure on T cell mitogenesis. Exposures to 10 uM CB-156 and CB-80, and 1.0 and 0.1 uM B[a]P, did not produce significant depression in lymphoproliferation. Exposure to the model PAH at 10 uM resulted in a 61% (range 34-97%) average reduction in lymphoproliferation. We were able to rule out a direct cytotoxic effect of B[a]P, indicating that observed effects were due to altered T cell function. Based on our


P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.We screened 50 glioblastomas for  P53 gene, heterozygous mutations have also been detected. Typical mutations of this gene are of the missense type, leading to P53 protein gain of function  or translation  , 0.5 mM dNTP, 40 units RNase inhibitor and 200 units M-MLV reverse transcriptase (Promega).The investigations were performed using snap-frozen tissues stored at −80°. DNA was isolated from tumour tissues and blood samples from each patient. DNA and RNA were coextracted by means of Macherey-Nagel DNA/RNA purification kit. RNA samples were treated with DNAase. RNA and DNA concentrations were measured spectrophotometrically. In all 100 ng of total RNA was reverse-transcribed into single-stranded cDNA in a final volume of 40 μl containing 50 mLoss of heterozygosity (LOH) and microsatellite instability (MSI) analyses were performed using paired tumour specimens and corresponding peripheral blood samples, to recognise tumour samples with minimal contamination by normal cells. The following LOH and MSI markers were used: D1S2734, D1S197, D1S162, D1S156, D9S319, D9S319, D9S162, D10S587, D10S1267, D17S1828, AFM119, BAT25, BAT26, BAT40. Forward primers were 5′ end fluorescence-labeled. PCR was performed in thermocycling conditions individually established for each pair of primers. PCR products were denatured and gel electrophoresis in LiCor automatic sequencer system was applied to the separation and analysis of PCR-generated alleles.P53 gene were amplified by PCR as described before and sequenced using the dideoxy termination method and SequiTherm Excel DNA Sequencing Kit (Epicentre Technologies) (Exons 5–8 of the p53: 5′-GTGCAGCTGTGGGTTGATT-3′ (sense) and 5′ GCAGTGCTCGCTTAGTGCTC-3′ (antisense); annealing temperature was 53°C. The sequencing primers were: p53 exon 5–8: 5′-GCCATCTACAAGCAGTCACA-3′ (sense), and p53 exon 8–5: 5′-CCCTTTCTTGCGGAGATTCT-3′ (antisense); annealing temperature was 55°C. LiCor automatic sequencer system was applied to the separation and analysis of PCR-sequencing products.The primers used for the PCR amplification of cDNA sequences were: To verify the results, a semi-quantitative densitometric analysis was performed in which wild and mutated band intensity was estimated, and then compared to a neighbouring band in the same sequencing lane for reference.P53 promoter, and DNA from peripheral blood leukocytes was used as the control for unmethylated alleles of P53. The MSP was performed as previously described . CpGenome Universal Methylated DNA  was used as a methylation-positive control for the methylated P53 mutations. Mutations of P53 were detected in 16 cases, eight of these being heterozygous , cDNA sequencing revealed a decreased amount, or lack of, the wild (nonmutated) template when compared to the genomic DNA sequencing . The third case also presented a heterozygous mutation of P53 – but without any decrease in the amount of wild-type cDNA template – as shown by cDNA vs genomic DNA sequencing  which would allow for either (1) the silencing of wild-type P53 transcription, (2) the degradation of wild-type P53 mRNA, or (3) the selective overproduction of mutated P53 mRNA. Moreover, our results show that heterozygous mutations of P53 gene, elimination of wild-type P53 mRNA, or selective production of mutated mRNA can occur during glioblastoma tumorigenesis. An extremely important question thus arises – that is, ‘what mechanism(s) is (are) responsible for favouring the mutated P53 mRNA over the wild-type ones?’Heterozygous mutations of P53 mutation present P53 promoter methylation  responsible for the elimination of wild-type P53 mRNA requires more research. Obviously, the elimination of wild-type P53 may have resulted from a mechanism other than epigenetic changes of P53 DNA regulatory elements. Nonetheless, uncovering of this mechanism can be very important for the development of new anti-tumour therapeutics.A methylation of DNA regulatory elements – mainly promoters, is one of the mechanisms of tumour suppressor genes silencing (P53 employ some sort of mechanism(s) to positively select mutated P53 mRNA. We offer a relatively easy procedure for determining whether similar situations also occur in other cancers. The precise mechanism(s) for favouring the mutated type of P53 mRNA – however, still remains to be discovered.In conclusion, we show in this article that glioblastomas presenting heterozygous mutations of


Specific risk factors leading to this poorer outcome have not been identified. In the present study, we hypothesized that iron deficiency, a common ailment in young women, contributes to the poor outcome by promoting the hypoxia inducible factor-1α  formation. This hypothesis was tested in an Human breast cancer MDA-MB-231 cells were transfected with transferrin receptor-1 (TfR1) shRNA to constitutively impair iron uptake. Cellular iron status was determined by a set of iron proteins and angiogenesis was evaluated by levels of VEGF in cells as well as by a mouse xenograft model. Significant decreases in ferritin with concomitant increases in VEGF were observed in TfR1 knockdown MDA-MB-231 cells when compared to the parental cells. TfR1 shRNA transfectants also evoked a stronger angiogenic response after the cells were injected subcutaneously into nude mice. The molecular mechanism appears that cellular iron deficiency elevates VEGF formation by stabilizing HIF-1α. This mechanism is also true in human breast cancer MCF-7 and liver cancer HepG2 cells.Cellular iron deficiency increased HIF-1α, VEGF, and angiogenesis, suggesting that systemic iron deficiency might play an important part in the tumor angiogenesis and recurrence in this young age group of breast cancer patients. Young women (< 45 years old) diagnosed with breast cancer are well known to have a higher risk of dying from the disease than older patients because of greater early recurrence rates, increased aggressiveness, and mostly estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) negative or triple negative breast tumors ,2. The sUsing data initially collected from the New York University Women's Health Study, we observed that serum iron and ferritin levels are significantly lower in pre- than in postmenopausal women . A typicIron is an essential metal for all living organisms participating in cellular processes, such as DNA synthesis, enzyme functions, and oxygen transport. Cellular iron metabolism is homologous among most cell types; cellular iron homeostasis is primarily mediated by transferrin (Tf), transferrin receptor-1 (TfR1), and ferritin -13. Tf iIron depletion in cells by iron chelator desferoxamine mesylate (DFO) has been shown to increase hypoxia-inducible factor-1alpha (HIF-1α) ,16. HIF-e.g., L1, 2, 3, a mixture of the three oligonucleotides). Effective TfR1 knockdowns as confirmed by Western blot were shown in Figure Cellular iron deficiency was achieved by either transiently or stably transfecting MDA-MB-231 cells with TfR1 shRNA expression vectors of L1, L2, L3, or their mixtures . Hypoxia significantly induced VEGF production in both parental and TfR1 knockdown L3 cells. DFO also increased VEGF in the TfR1 knockdown cells, although the difference was not statistically significant. Figure Considering the high prevalence of iron deficiency in young women and the high tumor recurrence in young breast cancer patients ,19, assoin vivo anemic condition, in vitro hypoxia at 1% O2 was used. Its effects on cellular iron homeostasis were studied in various non-transfected cancer cells, which were more pathophysiologically relevant than the TfR1 knockdown cells. Figure vs 21.2 ± 0.8 nmol/mg protein).Anemia is marked by decreased numbers of red blood cells and often results in hypoxia in the breast . To mimiFigure Figure Figure Figure Estrogen and family history are two of the most significant and well-characterized risk factors for breast cancer. However, family history and estrogen cannot fully explain the higher breast cancer recurrence rates and greater aggressiveness with mostly triple negative breast tumors in young, pre-menopausal women as compared to the older post-menopausal women -24. BecaTo date, most research involving iron in cancer has been focused on iron overload causing oxidant-mediated cancer promotion . Our conin vitro culture conditions or even 3 D tissue culture can reach iron equilibrium in the in vivo environment once these cells are injected into the animals [It is well known that when a new vasculature forms and penetrates into the tumor core, primary tumor cells can enter into the circulation and form secondary tumor in distant organs. Once the tumors recur, the disease becomes incurable resulting in a high death rate. To test our hypothesis , triple  animals . In thisin vivo [HIF-1α and VEGF were used as the endpoints of the present study because either or both can elicit an array of effects, from initiating angiogenesis to tumor metastasis, ,34. Overin vivo . Patientin vivo . VEGF anin vivo  and incrin vivo . Additioin vivo ,39. WhetAnemia is functionally defined as having inadequate numbers of red blood cells to maintain tissue oxygenation. It has been shown that hypoxia is more intensified in young and anemic breast cancer patients . This leTo confirm that the observed changes in ferritin and TfR1 are a result of hypoxia-mediated iron decrease, IRP binding to IRE was determined Figure . Our resin vitro angiogenesis , β-mercaptoethanol (ME), agar, and DNA ligase kit LIG-1 were purchased from Sigma . Other reagents were: SDS-Tris-HCl or non-denaturing polyacrylamide gels, nitrocellulose membranes, ; M-PER mammalian protein extraction reagent, calcein (Molecular Probes); mini-Prep kit for vector DNA isolation (Qiagen); anti-human TfR1 antibody ; Lipofectamine™ 2000 ; anti-HIF-1α antibody ; shRNA vector sequences, pSilencer expression vector kit, RNA isolation kits, DNA-free and RNAqueous .DFO, ferric ammonium citrate (FAC), ferrous sulfate heptahydrate . Bovine capillary endothelial (BCE) cells, a kind gift from Dr. Paolo Mignatti, were used for in vitro angiogenesis . Growth of each cell line was initiated with the recommended culture conditions from the supplier. All cell lines were then adapted to alpha-minimal essential medium (α-MEM) supplemented with 5% FBS, 2 mM L-glutamine, and 1X antibiotic/antimycotic solution. This measure was taken to prevent confounding outcomes resulting from changing medium during co-culture experiments. α-MEM was the main culture medium because α-MEM is essentially iron-free, thus providing maximal control of the iron levels (by supplementation) in the culture environment.Human triple negative breast cancer cell line MDA-MB-231 was used for shRNA knockdown. For comparison, human breast cancer ER2. Normoxia exposures were carried out in the same incubator, while hypoxic exposures were performed in a specially designed exposure system with hypoxic gas .Unless otherwise noted, the initial growth medium was removed 18-24 h prior to experiments and replaced with the iron-free α-MEM containing serum-replacement supplement at 5 μg/l selenium, 10 mg/l insulin, 5.5 mg/l transferrin, and 2 mg/l ethanolamine. These additives allowed cells to be maintained and even propagated without serum for periods ranging from 72 h to over a week. Cells were maintained in a humidified tissue culture incubator at 37°C under 5% COThree sequences of shRNA duplexes for human TfR1 as well as control shRNA were purchased from Ambion Inc. . The TfR1 shRNA oligonucleotide sequences were as follows:L1: 5'-GATCCGGCCTTAATATGTTAACCTTTCAAGAGAAGGTTAACATATTAAGGCCTTA-3'L2: 5'-GATCCGGCCAATGTCACAAAACCATTCAAGAGATGGTTTTGTGACATTGGCCTT A-3'L3: 5'-GATCCGGTGTAGTGGAAGTATCTGTTCAAGAGACAGATACTTCCACTACACCTT A-3'pSilencer 4.1-CMV neo vector, MDA-MB-231 cells were seeded into 24-well plates and cultured until about 50% confluent, then transfected with 80 pmol TfR1 shRNA in Lipofectamine™ 2000 for 24 h according to the manufacturer's instructions. Cells treated with Lipofectamine™ 2000 alone and control non-silencing shRNA were used as mock-transfected controls. After 24 h, transiently-transfected cells were used for some biochemical measurements and the remaining cells were transferred to 25-cm2 flasks for selection. Stably-transfected TfR1 knockdown cells (L1-L3) as well as nonsense control cells (L-) were selected with neomycin at 2 mg/ml [After ligating into a  2 mg/ml . To veri6 cells were suspended in 0.2 ml of 1:1 (vol:vol) mixture of PBS and the same batch of Matrigel™. Cells were inoculated subcutaneously (0.2 ml) into both flanks of the nude mice. Seventy-two hours post-inoculation, the subcutaneous Matrigel plugs and skin surrounding the injection site were excised, trans-illuminated and photographed.Three groups, each consisting of four 6-week old female nude mice  were treated as follows: parental MDA-MB-231, MDA-MB-231 cells stably-transfected with nonsense shRNA vector (L-), and MDA-MB-231 cells stably-transfected with TfR1 shRNA vector (L3). After harvesting, 2.5 × 102, 40 mM KCl, 5% glycerol, 0.3% NP-40, and a complete protease inhibitor cocktail. The 32P-labeled IRE probe was synthesized by in vitro transcription using the linearized pSPT-fer plasmid as the template , which contains human ferritin H-chain IRE. After dividing samples into two equal parts, 2-mercaptoethanol (2-ME) was added to one part of the sample at a 2% final concentration. The addition of 2-ME creates a reducing environment, which facilitates maximal binding of all present IRP proteins, and was used as the internal control for IRP binding normalization. The two samples  were incubated at room temperature with an excess amount of 32P-labeled IRE probe (4 × 104 cpm) in 20 μl reaction buffer . After 20 min incubation, 1 unit RNase and 100 μg heparin were added, and the mixture was incubated for an additional 10 min period. The reaction mixture was separated on a 5% non-denaturing polyacrylamide gel. The gel was dried and the amount of radioactive material assessed by exposure to X-ray film. The IRP binding was calculated by comparing the band density of the sample without 2-ME to the sample with 2-ME.The binding activities of IRPs to IRE were examined by EMSA ,47. FollLevels of LMW iron in cells were determined using an iron-sensitive fluorescent calcein as previously described . BrieflyTfR1 levels were measured by Western blot as follows: cell lysates (30 μg protein) were subjected to 12% SDS-polyacrylamide gel electrophoresis. After transferring onto nitrocellulose membranes, the membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20, probed together with antibodies against TfR1 (1:500) and β-tubulin (1:3000), and then visualized with peroxidase-conjugated anti-mouse antibody using Western Lightning Plus Chemiluminescence Reagent (Perkin-Elmer). Levels of ferritin in cell lysates were determined by enzyme-linked immunosorbent assay (ELISA) according to the method previously developed in our laboratory . Total pLevels of VEGF in the tissue culture media were measured using pre-coated kits  following the Manufacturer's instruction. Data were expressed as pg VEGF/ml culture media.Cells were initially cultured separately in serum-containing growth medium; cancer cells in 24-well culture inserts containing a 0.4 μm porous membrane, and BCE cells in a 24-well, gelatin-coated plate. After cells were grown to 75% confluence, the medium was replaced with serum-free medium, and the cells were grown for an additional 24 h. To initiate the co-culture, inserts containing cancer cells were placed into wells containing BCE cells. The porous membrane kept cells physically separated while allowing secreted factors to pass through the membrane and be mixed with the medium covering the BCE cells. Prior to the co-culture, photographs of BCE cells were taken for a reference. Twenty four h after exposures to the factors generated by cancer cells grown on the inserts, BCE cells were photographed again.t-test. Graphed data represent the means ± SD of at least three experiments except that the IRP binding assays were done twice. A confidence level of p < 0.05 was taken to represent a significant difference in all cases.The experimental differences were determined by two-tailed Student's 2-ME: 2-mercaptoethanol; BCE: bovine capillary endothelial cells; DFO: deferoxamine; EMSA: electro-mobility shift assay; ER: estrogen receptor; FAC: ferric ammonium citrate; HER2: human epidermal growth factor receptor 2; HIF-1α: hypoxia inducible factor-1 alpha; HPO: hypothalamic-pituitary-ovarian; IDA: iron deficiency anemia; IRE: iron responsive element; IRP: iron regulatory protein; LMW: low molecular weight; PR: progesterone receptor; TF: transferrin; TFR1: transferrin receptor; SHRNA: small interfering RNA; VEGF: vascular endothelial growth factor.The authors declare that they have no competing interests.in vivo and in vitro angiogenesis and helped to draft the manuscript; JD, JJ, and QY evaluated cellular iron status in all three types of cells, JW and HC performed the IRP binding assay, MC and KF participated in the design of the study and helped to draft the manuscript, XH conceived of the study and drafted the manuscript. All authors read and approved the final manuscript.JE carried out shRNA knockdown and the


Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu157 to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential LeuThe present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation. Glaucoma, a major blinding disease worldwide 50→Lys (E50K), Met98→Lys (M98K), and Arg545→Gln (R545Q) in optineurin have been reported. The E50K mutation, found in Caucasian and Hispanic populations Recent genetic studies have demonstrated that POAG is highly heterogeneous, caused by several susceptibility genes and perhaps also environmental factors The human optineurin gene encodes a protein that contains multiple coiled-coil domains, at least one leucine zipper (amino acids 143–164) and a C-terminal zinc finger Optineurin shares a 53% amino acid homology with NEMO  and was identified as a NEMO-related protein Optineurin has also been demonstrated to interact with transcription factor IIIA Protein trafficking by the endocytic pathway is an essential cellular mechanism critical for many functions including cell signaling, nutrient acquisition and maintenance of the plasma membrane Previously, studies using small interfering RNA (siRNA) have suggested that optineurin is involved in the exocytosis of vesicular-stomatitis-virus G protein WT-GFP in the perinuclear region, colocalizing with a population of TfR, a marker for endosomal vesicles WT-GFP or pEGFP-N1  were incubated with Texas red-conjugated Tf (TR-Tf) at 37°C. After 15 min, intense accumulation of red-fluorescent TR-Tf in perinuclear areas were seen in nontransfected (non-green) and GFP mock-transfected green cells  level was used as a protein loading control. In addition, the level of the endogenous optineurin and the OPTNWT-GFP fusion protein was also determined to confirm that the cells were indeed transfected. Results in WT-GFP was similar to that of the mock control. The reduction of Tf uptake in pOPTNWT-GFP-transfected cells was thus unrelated to the expression level of total TfR.The TfR protein level in pEGFP-N1 (mock control)- or pOPTNIn GFP-expressing as well as nontransfected cells, punctate TfR staining was seen distributing largely diffusely in the cytoplasm . After oE50K, induced foci formation more prominently than that seen in the wild type E50K-GFP-expressing cells was much reduced compared to that in the neighboring nontransfected and GFP control (data not shown) cells. Quantitative analyses (E50K-GFP (approximately 70–80% reduction) than OPTNWT-GFP  in the optineurin sequence. OPTNL157A construct in pEGFP vector (pOPTNL157A-GFP) was made. When transfected, two patterns of OPTNL157A-GFP distribution were noted in human RPE  at the N-terminus and Rab8 has been suggested to interact with optineurin through the N-terminal region (amino acid residues 141–209) that includes the leucine zipper domain uman RPE , TM  or optineurin siRNA was transfected into RPE cells for 2 days. These cells were then incubated with TR-Tf. Fluorescent microscopy showed that the amount of Tf internalized after 15 min in optineurin-depleted cells was considerably lower than that in mock controls . The fluOptineurin is a gene linked to glaucoma. The present study demonstrates that overexpression of wild type optineurin results in an impairment of the Tf uptake in both RPE and RGC5 cells . Such a The uptake defect manifested by optineurin overexpression can be rectified by co-expression of TfR, but not by myosin VI or Rab8 , all of 50→Lys mutation results in no particular changes either in the structure or the conformation of optineurin (data not shown). Nevertheless, patients who have E50K mutation in clinical setting are reported to suffer glaucomatous defects more severe than those without this mutation E50K is a mutation prevalent in patients with normal tension glaucoma L157A-GFP , the change of Leu157 to Ala in the optineurin sequence may lead to obliteration of the leucine zipper (data not shown). The much reduced ability of L157A mutant to interact with Rab8 , impairment of Tf uptake, and cell death Using siRNA techniques, a previous study As both the overexpression and the suppression of optineurin cause a reduction in the Tf internalization, a role of optineurin in this process is implicated. In the literature, there have been reports of various genes E50K produces a more prominent foci formation It is further of note that while the E50K finding has pathologic significance, the wild type optineurin overexpression results are also of physiologic relevance. Optineurin, for example, has been shown to be upregulated by proinflammatory cytokines tumor necrosis factor-α (TNF-α) The RPE cell line ARPE-19 obtained from American Type Culture Collection  and RGC5 cells Transient transfection was carried out using FuGENE6  or lipofectamine LTX and Plus reagent  as previously described WT-GFP inducible stable RGC5 cell line was established as previously described E50K-GFP and OPTNL157A-GFP RGC5 cell lines were in addition created following the same procedures and strategies. The only exception was that the OPTNWT-GFP fragment was replaced with OPTNE50K-GFP or OPTNL157A-GFP fragment during the first cloning step WT-GFP, OPTNE50K-GFP, and OPTNL157A-GFP, cells were treated for 16 h with doxycycline (1 µg/ml)  in DMEM complete medium.Tet-on OPTNWT-GFP, pOPTNE50K-GFP, pTarget-FLAG-OPTNWT, pTarget-FLAG-OPTNE50K, pMyocilin-GFP, pRab8Q67L-EGFP, and pTfR-EGFP were constructed as previously described L157A-GFP was generated by QuikChange II Site-Directed Mutagenesis Kit . The sense primer sequence used for mutagenesis was GCGTTGGGCATCGTGTC-3′5′-CAGAGAAGGCAGAC. The antisense primer sequence was the reverse. To generate open reading frame of Rab5, Rab7, Rab8 and Rab11, cDNAs were synthesized from total RNA of RPE cells by Superscript II cDNA synthesis kit (Invitrogen). Rab4 cDNA was obtained from University of Missouri-Rolla cDNA resource center . The cDNAs were used as templates for PCR amplification with primers: 5′-GGCGAATTCTATGTCGCAGACGGCCATGTC-3′ and 5′-GGCGGATCCCTAACAACCACACTCCTGAGCG-3′ for Rab4; 5′- GGCGAATTCTATGGCTAGTCGAGGCGCAAC-3′ and 5′-GGCGGATCCTTAGTTACTACAACACTGATTCCTGG-3′ for Rab5; 5′- GGCGAATTCTATGACCTCTAGGAAGAAAGTG-3′ and 5′-GGCGGATCCTCAGCAACTGCAGCTTTCTG-3′ for Rab7; 5′- GGCGAATTCTATGGCGAAGACCTACGATTA-3′ and 5′- GGCGGATCCTCACAGAAGAACACATCGGA-3′ for Rab8; 5′- GGCGAATTCTATGGGCACCCGCGACGACGA-3′ and 5′- GGCGGATCCTTAGATGTTCTGACAGCACTGCACC-3′ for Rab11. The resulting PCR products were digested with EcoRI and BamHI and cloned in frame into pEGFP-C1  at the same restriction sites to yield constructs pGFP-Rab4, -Rab5, -Rab7, -Rab8 and -Rab11. Plasmids pOPTNWT-His, pOPTNE50K-His and pOPTNL157A-His were made by adding a His tag to the C-terminal of optineurin by PCR using pOPTNWT-GFP, pOPTNE50K-GFP or pOPTNL157A-GFP as the template and the subsequent subcloning of OPTNWT-His, OPTNE50K-His or OPTNL157A-His into EcoR V linearized pcDNA3.1z vector (Clontech). Sequencing was followed to verify all the constructs.Plasmids pOPTNThe following antibodies were used: rabbit polyclonal anti-optineurin , anti-GFP , anti-GAPDH , anti-His (Santa Cruz), and anti-β1-integrin , and mouse monoclonal anti-human TfR , anti-FLAG , anti-EEA1 (BD Biosciences), anti-myosin VI (sigma), anti-Rab8 (BD Biosciences), anti-His (Sigma) and anti-GFP (BD Biosciences).2 and 1 mM MgCl2, incubated with cold acid buffer  and ice-cold PBS, fixed, and mounted in Vectashield with DAPI . Photography was carried out on a Leica SP2 confocal system  with a 63× oil objective using sequential scanning to minimize the bleed through. Confocal images were captured using the same resolution, zoom, pinhole size, and amplitude offset. All cells were captured at the same gain. TR-Tf internalization at each time point was quantified as previously described Cells grown on glass chamber slides were washed in phosphate-buffered saline (PBS) and incubated in DMEM containing 0.2% bovine serum albumin (BSA) (DMEM-BSA) for 1 h to deplete serum. The cells were transferred to fresh DMEM-BSA containing 25 µg/ml TR-Tf (Invitrogen) and incubated for the indicated time periods. The cells were then placed on ice, washed with cold PBS containing 0.2% BSA, 1 mM CaClTM-M cell lysis reagent (Sigma) containing protease inhibitor cocktail . The lysates were centrifuged at 12,000× g for 10 min at 4°C. Protein concentration in lysates was determined using the bicinchoninic acid (BCA) assay  per the manufacturer's protocol. Aliquots of total cell lysate were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto Protran BA83 nitrocellulose membrane . After blocking for 1 h with 5% nonfat dry milk in a buffer containing 20 mM Tris buffer (pH 7.4), 150 mM NaCl and 0.1% Tween 20, the membrane was incubated with primary antibody for 1 h at room temperature or overnight at 4°C. The blot was further incubated for 1 h with horseradish peroxidase-conjugated secondary antibody . Protein bands were detected using SuperSignal Substrate (Pierce). For repeated probing, the blot was stripped for 1 h at room temperature with ImmunoPure IgG Elution buffer (Pierce).Cells were washed with ice-cold PBS and harvested in CelLyticCells  on Lab-Tek 8-well CC2 glass chamber slides  were transfected and fixed with 4% paraformaldehyde in PBS for 15 min. After permeabilization in 0.2% Triton X-100 for 5 min, the cells were blocked with 3% BSA for 30 min, and incubated with primary antibody in blocking solution for 1 h at room temperature. They were further incubated for 45 min with Cy3- or FITC-conjugated secondary antibody , and mounted. Confocal images were captured.Cells plated on 6-well plates were transfected with indicated plasmids for 16 h. For inducible cell lines, the cells were treated with 1 µg/ml of doxycycline for 16 h. After washes with ice-cold PBS, the cells were harvested in IP buffer  and homogenized by ten strokes in a pre-chilled dounce homogenizer on ice. The homogenates were clarified by centrifugation at 12,000× g for 10 min at 4°C. Protein concentration in homogenates was determined by BCA assay. Aliquots of the homogenates were subjected to immunoblotting or IP reaction using Catch and Release v2.0 kit  according to manufacturer's protocol with minor modifications. Briefly, 500 µg of the homogenates were incubated overnight with 4 µg of indicated rabbit polyclonal antibodies and 10 µl of Antibody Capture Affinity Ligand (Millipore) on a rotary shaker with gentle shaking at 4°C. The mixture was transferred to a spin column containing agarose resin slurry and further incubated for 30 min on a rotary shaker at room temperature. Columns were washed three times with 1× wash buffer (Millipore). Protein was eluted with 1× denaturing elution buffer (Millipore). The eluates were subjected to SDS-PAGE and immunoblotting.5′-GAAGCCATGAAGCTAAATA-3′ and matched to 166–184 bp of ORF. As a control, a 19-nucleotide scrambled generic control siRNA (Dharmacon) was used. In some experiments, cells were transfected with scrambled or optineurin siRNA along with Cy3-labeled control siRNA (Dharmacon) to assess transfection efficiency.RPE cells were plated in complete media at 80 to 90% confluency overnight. The cells were transfected with 50 nM of siRNA duplex  for 2 days using TransIT-TKO transfection reagent  according to the manufacturer's protocol. Target sequence of optineurin siRNA was WT-GFP or pOPTNL157A-GFP for 16 h. Images were taken using a 63× oil objective. The number of foci formed was counted in at least 60 transfected cells both manually and through the use of Metamorph software. The significance of the data was determined by Students' t tests. Three sets of independent experiments were analyzed.RPE and TM cells were transfected with pEGFP-N1, pOPTNSurface biotinylation was performed as described