For non-specific immunity, NK cell activity increased significantly in a dose-dependent manner in P. vulgaris treated mice (p<0.01), mononuclear-macrophage function in medium and high dose P. vulgaris treated mice were significantly higher than that of the control group (p<0.05). For humoral immunity, no significant differences were observed in terms of the half value of hemolysis (HC50), number of hemolytic plaques and serum IgG level (p>0.05). The percentage of active T and Th lymphocytes of mice peripheral blood in high dose group were significantly higher than that of the control group (p<0.01). There was no significant difference in serum levels of IL-1β, IL-4, IL-10 and IFN-γ among all of the four groups (p>0.05). The data indicated that 0.90 g/kg BW P. vulgaris extract  had some effect on cellular immune function and non-specific immune function in mice.The present study was designed to evaluate the effects of Prunella Vulgaris (P. vulgaris) on the immune function in mice. The mice were randomly divided into one control group and three treatment groups of 10 mice each. The control group received pure water and the treatment groups received P. vulgaris extract at concentrations of 0.15, 0.30 and 0.90 g/kg BW orally for 30 days, respectively. Changes in cell immune function, non-specific immunity and humoral immunity function were evaluated. Active lymphocytes and T lymphocyte subsets were determined by fluorescence-activated cell sorting (FACS). Certain Serum concentrations of cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that, for cell immune function, compared with the control group, foot pad thickness in high dose group increased significantly ( In vivo studies have suggested that herb tea consumption could alleviate the restraint stress-induced reduction of immune function in mice, manifested by alleviated lymphocyte impairment, increased amount and activity of lymphocytes such as natural killer (NK) cells and T lymphocytes In vitro studies revealed that both aqueous and ethanol extracts of P. vulgaris could promote lymphopoiesis and regulate the production of cytokines in vitro, few have been reported in vivo. Therefore, in the current study, we investigated the effect of P. vulgaris on cellular immunity, humoral immunity and non-specific immunity in mice. By measuring the activities of lymphocytes and T lymphocytes subgroups as well as the serum level of humoral immunity-related cytokines such as IFN-γ, IL-1β, IL-10 and IL-4 after P. vulgaris administration, the result may provide some basis for potential mechanisms responsible for immunomodulatory effect of P. vulgaris.P. vulgaris L. (Labiatae) belongs to Lamiaceae family Prunella genus. It is widely distributed in the temperate zone. Its Chinese name, Xia Ku Cao in pingyin, originates from a description that the herb dried and withered after the summer solstice. P. vulgaris has been used as a traditional Chinese medicine for a long time. According to the Chinese Pharmacopoeia This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Ethics Committee of Guangdong Provincial Centre for Disease Control and Prevention. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.The extract of P. vulgaris was provided by Guangzhou Wanlaoji Pharmaceutical Co., Ltd. It was prepared from crude drug through water extraction followed by alcohol precipitation. One hundred g of crude drug can typically generate 11.76 g of extract; in other words, 1 g of extract is equivalent to 8.5 g of crude drug. The extract was maintained at 4°C. According to the Chinese Pharmacopoeia Female BALB/c mice  The mice were randomly divided into one control group and three treatment groups (10 mice in each group). The control group received pure water and the treatment groups received P. vulgaris extract at 0.15, 0.30 and 0.90 g/kg BW, which were 5, 10 and 30 times of recommended dosage and equivalent to 1.25, 2.50 and 7.50 g/kg BW of crude drug respectively. To prepare the test substances for low, medium and high dose groups, 0.15,0.30 and 0.90 g P. vulgaris extract were dissolved in water to a final volume of 20.0 ml, respectively. The test substances were administrated intragastrically (0.2 ml/10 g BW) everyday for 30 days. The body weight was measured weekly. Twenty-four hours after the last administration, the mice were weighed and sacrificed.Fluorescent beads (FITC-labeled), Concanavalin A (ConA), 3--2,5-diphenyltetrazolium bromide (MTT), agar and bovine serum albumin (BSA) were purchased from Sigma . Antibodies against mouse CD3 FITC, CD4 PE, CD8 APC, CD19 PE, CD25 PE-Cy5 and CD25 APC were from eBioscience . Heamolysin was from Becton Dickinson . ELISA kits detecting Mouse IgG, IL-1β, IL-4, IFN-γ and IL-10 were purchased from R&D .2 incubator was from NuAire . Digital sonofier 450 was from Branson (MX). CELL-DYN 3700 hematology analyzer was from Abbott . CASY DT cell counter was from Schärfe System (DE).VL-5BS biological safety cabinet was from Tsaohsin . FACS Calibur flow cytometer was from Becton Dickinson . Varioskan Flash Spectral Scanning Multimode Reader was from Thermo Scientific . AutoFlow COAfter the last measurement of body weight, mice were sacrificed by cervical dislocation. The spleens and thymus organs were removed immediately and the wet weight was measured. The spleen and thymus indexes were calculated as the wet weight of spleen and thymus divided by the body weight, respectively.6 cells/mL. One mL splenocyte suspension was incubated with or without 75 µL ConA solution  in a 24-well plate at 37°C with 5% CO2 for 72 hours. After 68 hours, 0.7 mL media was removed and replaced with 0.7 mL serum-free RPMI 1640 media plus 50 µL 5 mg/mL MTT. Four hours later (t = 72 h), 1 mL acidic isopropanol was added to dissolve the purple formazan product. The final solution was added into a 96-well plate in triplicate. The optical density (OD) value was measured by a microplate reader set to 570 nm.To prepare splenocyte suspensions, spleens were removed in a sterile environment and put into a petri dish with sterile Hank’s buffered salt solution (HBSS). Each spleen was teased apart by sterile forceps and pushed through 200 BSS mesh. The resultant single splenocytes were washed with HBSS and centrifuged  three times. The cells were then re-suspended in 2 mL RPMI 1640 complete media. The live cells were counted by trypan blue exclusion assay (the percentage of which should be above 95%) and the cell number was adjusted to 3×10Twenty-six days into the experiment, mice were immunized by 0.2 mL 2% sheep red blood cells (SRBC) (v/v) via intraperitoneal injection. Four days later (t = 30 d), the thickness of the left hind foot pad was measured by cornier caliper. Each sample was measured three times at the same place and the mean was taken. The spot of measurement then received 20 µL 20% SRBC (v/v) via subcutaneous injection. An additional 24 hours later, the thickness of left hind foot pad was again measured. The difference between two measurements was used as a measure of DTH. The result is considered positive if the value of experimental group is significantly higher than that of control group.50) was calculated as follows:Twenty-six days into the experiment, mice were immunized by SRBC. On the last day (t = 30 d), blood was collected from the angular vein before mice were sacrificed. Serum was separated and diluted with salicylic acid (SA) buffer at a 1∶100 ratio. 100 µL diluted serum was added into a 96-well plate, followed by 50 µL 10% SRBC and 100 µL complement (diluted with SA buffer at 1∶8 ratio). For the blank control, SA buffer was used instead of diluted serum. The plate was incubated in a 37°C water bath for 30 min. After incubation, the plate was centrifuged at 2000 r/min for 10 min. Fifty µL supernatant was transferred into a new 96-well plate, followed by addition of 150 µL buffer A . 12.5 µL 10% SRBC was then added, again followed by buffer A to a final volume of 200 µL. The plate was incubated at room temperature for 10 min. The OD value was measured by a microplate reader set to 540 nm. The half value of hemolysis (HC6 cells/mL) were prepared as described above. Fifty µL 20% SRBC  and 200 µL splenocyte suspension were mixed with 0.5 mL agar , and added into a 6-well plate coated with 1 mL/well agar . Each sample was tested in duplicate. The plate was incubated at 37°C with 5% CO2 for 1 hour. 500 µL diluted complement  was then added into each well, followed by further incubation for another 2 hours. After incubation, hemolytic plaques were counted by automatic image analyzer. The number of hemolytic plaques in each sample is presented as mean of duplicate/106 splenocytes.Twenty-six days into the experiment, mice were immunized by SRBC. On the last day (t = 30 d), mice were sacrificed by cervical dislocation. Splenocyte suspensions , followed by 25 µL enzyme-linked avidin. The plate was covered with adhesive strip and incubated at 37°C for 1 hour. The plate was then washed with pre-diluted washing buffer 5 times and 50 µL each of substrate I and substrate II were added into each well. The plate was incubated at room temperature and protected from light for 15 min. After incubation, 50 µL stop solution was added. The OD value was measured by a microplate reader set to 450 nm within 15 min. The IgG concentration in each sample was calculated according to the standard curve generated by standard solution. The result was considered positive if the value of the experimental group was significantly higher than that of the control group.5 cells/mL in RPMI 1640 complete media. Splenocyte suspensions were prepared as described above. Six µL splenocyte suspension was mixed with 6 mL cell counting buffer for cell number counting by automatic cell counter. The number of splenocytes was then adjusted to 2×107 cells/mL in RPMI 1640 complete media. 100 µL target cells and 100 µL splenocytes (effector cell) (ratio of effector cells to target cell is 50∶1) were added into a 96-well plate. 100 µL target cell plus 100 µL media served as target cell spontaneous release control, whereas 100 µL target cell plus 100 µL 1% NP40 served as target cell maximum release control. The plates were incubated at 37°C in 5% CO2 for 4 h. After incubation, the plate was centrifuged at 1500 r/min for 5 min. 100 µL supernatant was transferred into a new 96-well plate, followed by addition of 100 µL LDH substrate. After 3 min, 30 µL l mol/L HCI was added to stop the reaction. The OD values were measured by a microplate reader set to 490 nm. NK cell activity was calculated and transformed as Formula (2). The result was considered positive if the transformed value of the experimental group was significantly higher than the transformed value of the control group.Routinely subcultured target cells were washed three times with HBSS. Sixty µL target cells were diluted with 6 mL cell counting buffer for cell number counting by automatic cell counter. The target cell number was then adjusted to 4×105 cells/mL) and added into a 6-well plate. The plate was incubated at 37°C for 1 hour. After incubation, the supernatant was removed and the wells were gently washed with PBS. The adherent cells were then collected and filtered onto a 70 µm filter, followed by FACS analysis. The phagocytic activity of peritoneal macrophages was presented as percentage of phagocytosis (which needs data transformation) and phagocytic index calculated as Formula (3) and (4).Twenty-six days into the experiment, mice were immunized by SRBC. On the last day (t = 30 d), the mice were sacrificed by cervical dislocation. To collect peritoneal macrophages, 3 mL HBSS  was directly injected into the peritoneal cavity. After peritoneal massage (40 times), the peritoneal cavity was surgically opened and 2 mL peritoneal cavity fluid was collected. The peritoneal macrophages were then collected by filtration onto a 70 um filter. 100 µL FITC-labeled fluorescent beads was added into 10 mL PBS containing 1% BSA, incubated in a 37°C water bath for 30 min, followed by sonication for 5 min. Pretreated fluorescent beads were then mixed with peritoneal macrophages  Mice were anesthetized and blood was collected via the abdominal vein. Serum was separated by centrifugation and used for the measurement of cytokine concentrations by ELISA according to the product manual. For measurement of IL-4 and IL-10, 100 µL standard solution or serum sample were added to the ELISA plate. For measurement of IFN-γ and IL-1β, 50 µL standard solution or serum sample were added. Then, 50 µL enzyme-linked avidin was added into each well. The plate was covered with adhesive strip and incubated at 37°C for 1 hour. The plate was then washed five times with pre-diluted washing buffer, followed by addition of 50 µL each of substrate I and substrate II into each well and incubation at room temperature with protection from light for 15 min. After incubation, 50 µL stop solution was added into each well. The OD values were measured by a microplate reader set to 450 nm within 15 min. The concentration of each cytokine was calculated according to the standard curve generated by the corresponding standard solution.p<0.05 were considered statistically significant.Values were expressed as mean ± SD  treated mice for 30 days. The spleen and thymus indexes were calculated as the wet weight of spleen and thymus divided by the body weight, respectively. As the result of statistical analysis, the data were expressed as mean±SD of each group (n = 10 per group). There was no significant difference in spleen index  and thymp<0.01) among these four groups. Further analysis revealed that a statistically significant difference was observed between the high dose group (0.75±0.11) and the control group (0.60±0.07) (p<0.01). While there were no significant differences between low or medium dose P. vulgaris exact treated mice with the control group (p>0.05). ConA-induced splenic lymphocyte proliferation was tested by MTT assay. There was no obvious difference in the splenic lymphocyte proliferation ability among the four groups (p>0.05)  and ConA-induced splenic lymphocyte proliferation were tested in mice treated with P. Vulgaris exact  for 30 days. The data were expressed as mean±SD of each group (n = 10 per group). The increase in foot pad thickness of mice immunized with SRBC was examined to represent the DTH. As the result, the thickness of the mice foot pad increased in a dose dependent manner. The increase in foot pad thickness increased significantly ((p>0.05) . The resp<0.05). While there was no obvious difference in the transformed percentage of phagocytosis and the phagocytic index between the low dose group and the control group (p>0.05)  for 30 days. The percentage of phagocytosis (which needs data transformation) and phagocytic index of mononuclear-macrophage of each mice was calculated as Formula (3) and (4) according to the number of macrophages containing fluorescent beads analyzed by FACS. The data were expressed as mean±SD of each group (n = 10 per group).Both the transformed percentage of phagocytosis and the phagocytic index increased in a dose-dependent manner in mice treated with P. vulgaris. The phagocytic index of the medium (1.32±0.19) and high (1.33±0.22) dose group were significantly higher than that of the control group (1.05±0.12) (p<0.05). The transformed percentage of phagocytosis of the medium (0.89±0.06) and high (0.89±0.09) dose group remained significantly higher than that of the control group (0.80±0.05) ((p>0.05) . The resp<0.01). As shown in p<0.01). The transformed NK cell activity of low dose group (0.54±0.03 ) remained significantly higher than that of the control group (p<0.05). The results suggest that low, medium and high dosage of P. vulgaris may have some effect on mice NK cell activity.The mice were treated with increasing concentrations  of P. vulgaris exact for 30 days. Then the NK cell activity was evaluated by the LDH releasing assay. Transformed NK cell activity of each sample was calculated and transformed as the Formula (2) according to the OD values measured by microplate reader. The data were expressed as mean±SD of each group (n = 10 per group).Transformed NK cell activity increased in a dose-dependent manner in P. vulgaris treated mice. Statistically significant differences were observed between all three experimental groups and the control group (50), hemolytic plaques (duplicate/106 splenocytes) indicating antibody production of splenocytes, as well as serum IgG level were evaluated in mice treated with increasing concentrations  of P. Vulgaris exact for 30 days. The data were expressed as mean±SD of each group (n = 10 per group).No significant difference was detected among the three experimental groups and a control group in terms of HC50, the number of hemolytic plaques and serum IgG level (p>0.05)  .+CD25+) and Th cells (CD3+CD4+CD8−CD25+) increased in a dose-dependent manner in P. vulgaris treated mice. In terms of the percentages of active T lymphocytes, the high dose group (CD3+CD25+ = 8.15±0.95% ), but not the low or medium dose group, is significantly higher than the control group (CD3+CD25+ = 6.38±0.66% )(p<0.01). The active Th lymphocytes in high dose group (CD3+ CD4+CD8− CD25+ = 6.53±0.91% ) remained significantly higher than that of the control group (CD3+ CD4+CD8− CD25+ = 3.78±0.57% ) (p<0.01). While there was no significant difference in the percentage of active B lymphocytes (CD19+CD25+) and Tc lymphocytes (CD3+CD4−CD8+CD25+) among the four groups (p>0.05). of P. Vulgaris exact for 30 days Then the active lymphocytes and the T lymphocyte subsets in mice peripheral blood were analyzed by FACS. According to the ratio of the active T, Th and Tc lymphocytes of each sample obtained by the flow cytometry, the detection result for each dose group can then be compared by statistical analysis. The data were expressed as mean±SD of each group (n = 10 per group).Compared with the control group, the percentages of both active T lymphocytes (CD3(p>0.05). The resup>0.05).Serum concentrations of different cytokines including IL-1β, IFN-γ, IL-4 and IL-10 in P. vulgaris exact  treated mice were tested by ELISA according to the manual of ELISA kits. The data were expressed as mean±SD of each group (n = 10 per group).The serum concentration of IL-1β tended to increase in a dose-dependent manner in P. vulgaris treated mice. However, no significant difference in serum levels of IL-1β , IFN-γ , IL-4 F and IL-1Thunb, another species in the same family as P. vulgaris, showed similar ability to enhance phagocytic activity of mouse macrophages in a dose-dependent manner Studies have suggested that the anti-inflammatory and antitumor activities of P. vulgaris are closely related to its immunomodulatory effect The activity of T lymphocyte subsets is an important indicator of immune homeostasis In conclusion, 0.90 g/kg BW P. vulgaris extract  had some effect on cellular immune function and non-specific immune function in mice.Figure S1Flow cytometry detection of the active T lymphocyte subsets. These pictures are the detection results of a sample. I gate in + CD25+), Th(CD3+ CD4+CD8− CD25+) and Tc(CD3+ CD4−CD8+ CD25+) lymphocytes. According to the ratio of the active T, Th and Tc lymphocytes of each sample obtained by flow cytometry, the detection result for each dose group can then be analyzed.(TIF)Click here for additional data file.Figure S2Flow cytometry detection of the active B lymphocyte. These pictures are the detection results of a sample. I gate in + CD25+). According to the ratio of the active B lymphocytes of each sample obtained by flow cytometry, the detection result for each dose group can then be analyzed.(TIF)Click here for additional data file.


In mouse embryonic stem cells (mESCs), transcriptional silencing of numerous class I and II endogenous retroviruses (ERVs), including IAP, ETn and MMERVK10C, is dependent upon the H3K9 methyltransferase (KMTase) SETDB1/ESET and its binding partner KAP1/TRIM28. In contrast, the H3K9 KMTases G9a and GLP and HP1 proteins are dispensable for this process. Intriguingly, MERVL retroelements are actively transcribed exclusively in the two-cell (2C) embryo, but the molecular basis of silencing of these class III ERVs at later developmental stages has not been systematically addressed.Here, we characterized the roles of these chromatin factors in MERVL silencing in mESCs. While MMERVK10C and IAP ERVs are bound by SETDB1 and KAP1 and are induced following their deletion, MERVL ERVs show relatively low levels of SETDB1 and KAP1 binding and are upregulated exclusively following KAP1 depletion, indicating that KAP1 influences MERVL expression independent of SETDB1. In contrast to class I and class II ERVs, MERVL and MERVL LTR-driven genic transcripts are also upregulated following depletion of G9a or GLP, and G9a binds directly to these ERVs. Consistent with a direct role for H3K9me2 in MERVL repression, these elements are highly enriched for G9a-dependent H3K9me2, and catalytically active G9a is required for silencing of MERVL LTR-driven transcripts. MERVL is also derepressed in HP1α and HP1β KO ESCs. However, like KAP1, HP1α and HP1β are only modestly enriched at MERVL relative to IAP LTRs. Intriguingly, as recently shown for KAP1, RYBP, LSD1 and G9a-deficient mESCs, many genes normally expressed in the 2C embryo are also induced in HP1 KO mESCs, revealing that aberrant expression of a subset of 2C-specific genes is a common feature in each of these KO lines.Our results indicate that G9a and GLP, which are not required for silencing of class I and II ERVs, are recruited to MERVL elements and play a direct role in silencing of these class III ERVs, dependent upon G9a catalytic activity. In contrast, induction of MERVL expression in KAP1, HP1α and HP1β KO ESCs may occur predominantly as a consequence of indirect effects, in association with activation of a subset of 2C-specific genes. Endogenous retrovirus-like sequences (ERVs) are fossils of ancient retroviral integrations into the mammalian germline. Multiple independent colonization events have led to the accumulation of over 400 different ERV families with defined transcriptional patterns, often limited to specific developmental stages and cell types. Based on the similarity of their reverse transcriptase genes, ERVs are grouped into three classes: I, II and III, most closely related to exogenous gammaretroviruses, betaretroviruses and spumaretroviruses, respectively . Most ERThe class III MT subfamily of MaLR retrotransposons, for example, which comprise less than 5% of the mouse genome , accountde novo methyltransferases DNMT3A and DNMT3B and the maintenance methyltransferase DNMT1, plays a critical role in proviral silencing in somatic tissues [To minimize the generally deleterious effects associated with retrotransposition, a number of pathways have evolved to inhibit transcription of ERVs. DNA methylation, mediated by the  tissues -15 inclu tissues ,17, as w tissues ,19. Surp tissues , neither tissues ,22, this tissues , indicatIndeed, we recently reported that in mESCs, numerous class I and II ERVs, including MMERVK10C, MusD and ETn elements, are de-depressed in the absence of the H3K9 KMTase SETDB1/ESET, while IAP elements show the highest level of activation in the absence of both DNMT1 and SETDB1 ,24. FurtMest allele in embryonal carcinoma cells [Cbx5), HP1β (encoded by Cbx1) and/or HP1γ (encoded by Cbx3), alone or in combination, does not lead to derepression of class I or class II ERVs in mESCs [Heterochromatin protein 1 (HP1) proteins, which encode chromo- and chromoshadow domains, function in both structural and gene regulatory pathways in eukaryotes -33. Thesma cells . Surprisin mESCs , raisingHere, using genetic knockouts and/or RNAi, we analyzed the roles of KAP1, HP1α, HP1β as well as G9a and GLP, in transcriptional silencing of MERVL elements in mESCs. Our results indicate that MERVL expression is induced as a consequence of both direct and indirect effects, the former due to loss of H3K9me2 and the latter in association with derepression of genes normally expressed at the 2C stage.Setdb1 or Kap1 by RNAi followed by qRT-PCR yielded similar results  datasets ,47. Nume by KAP1 ,26,29. FG9a did not yield efficient depletion of G9a mRNA), with a 14-fold increase in MERVL expression observed 4 days post siRNA transfection . IntrigZfp352 for example, showed an increase in expression of 14- and 28-fold in the HP1α and HP1β KO lines, respectively. To determine whether additional unannotated genic transcripts (that is transcripts not present in the RefSeq or ENSEMBL databases) initiate in MERVL/MT2 LTRs in HP1 mutant cells, we identified all transcripts in our paired-end RNA-seq data in which one of the mate pairs aligns to an LTR element and the other to an annotated genic exon and scored all genes having >5 unique support reads for each in HM1 (wt), HP1α and HP1β KO mESCs. While ERVs of all three classes are represented among constitutively expressed chimeric transcripts in wt mESCs  control immunoprecipitation (IP), consistent with our previous observations  , including Zfp352, Zscan4d2[Zscan4d and Tdpo[Tdpoz3-4  and H3K9me2 is depleted on the paternal genome at this stage [Although the molecular basis of H3K9me2-mediated transcriptional repression of MERVL elements remains to be determined, we propose that this pathway is particularly important in early embryogenesis. Consistent with this model, is stage -67, perhis stage .Zfp352 and Zscan4[Intriguingly, depletion of several chromatin factors in mESCs including HP1α and HP1β (this study), G9a/GLP (this study and ), LSD1 , KAP1 2, RYBP 6 and ZFP4nd Zscan4,68. HereZfp352[Tdpoz4[per se. These alternative explanations are not necessarily mutually exclusive.Complicating interpretation of these observations is the fact that a significant number of genes expressed specifically at the 2C stage initiate transcription within MERVL LTRs, such as Zfp352 and Tdpo52[Tdpoz4, or are 52[Tdpoz4,12. ThusIn summary, our results indicate that G9a and GLP play a direct role in silencing of MERVL ERVs and genes driven by MERVL LTR promoters via G9a-mediated deposition of H3K9me2, while the KAP1-interacting H3K9 KMTase SETDB1 is neither recruited to MERVL elements nor required for their repression. Conversely, the results presented here are consistent with the model that induction of MERVL expression following deletion of KAP1, HP1α and HP1β occurs primarily via indirect effects. Given that expression of MERVL elements, unlike other ERVs, is normally restricted to the 2C embryo, any perturbation of mESCs that induces a nuclear milieu permissive for expression of 2C-specific genes may indirectly induce expression of these ERVs as well as genes driven by MERVL promoters.™ mRNA miniprep kit  and reverse transcribed using SuperScript III  as per the manufacturer’s instructions. Quantitative RT-PCR was carried out using SsoFAST™ EvaGreen Supermix  on StepOne™ Software v2.1 . Data are presented as mean +/- standard deviations of three technical replicates. Primer efficiencies were 95 to 105%. Dissociation curve analysis was performed after the end of the PCR to confirm the presence of a single and specific product. Corresponding ERV primers detect 519 MERVL elements, 202 MMERVK10C elements and 638 IAPEz elements. Primer sequences are listed in Additional file Mouse ESC lines, including HP1α and HP1β KOs and their corresponding wt line HM1  and G9a 5 cells saved at day 1 (30 h) for the RNA analysis of KD efficiency.For RNA collection, 7,000 mESCs per well of a 96-well plate were plated into antibiotic-free ES medium the day before transfection. Transfection was performed according to the manufacturer’s protocol, using 100 nM of each siRNA (Dharmacon siGENOME SMARTpool) and 0.4 μl DharmaFECT 1 reagent per well . The first day after transfection, a fraction of cells was transferred into a 12-well plate into antibiotic-free ES medium, and the transfection was repeated on the third day. The following day (approximately 30 h) after the second KD, most of the cells were collected for RNA for confirmation of KD efficiency (day 1 after the second KD), and the rest were plated onto 3.5-cm dishes for expansion and collection of RNA to monitor ERV derepression on day 4 after the second KD. For ChIP on day 4 after the second KD, the same steps were performed accounting for the increased volumes, with the cells plated onto a 12-well plate for the first transfection, transferred onto two 6 cm dishes for the second transfection and onto 4 to 6 × 10 cm dishes for collection on day 4 post second transfection, with approximately 2 × 107 ES cells for each cell line were resuspended in douncing buffer  and homogenized through a 25 G5/8 needle syringe for 20 repetitions. Following addition of 1.25 μl (50 U/μl) of MNase , the sample was incubated at 37°C for 7 min. The reaction was quenched with 0.5 M EDTA and incubated on ice for 5 min. 1 ml of hypotonic buffer  was added and the sample incubated on ice for 1 h. Cellular debris was pelleted and the supernatant was recovered. Protein A  and G Sepharose   beads were blocked with single-stranded salmon sperm DNA and BSA, washed and resuspended in IP buffer (10 mM Tris–HCl (pH 8.0), 1% Triton X-100, 0.10% deoxycholate, 0.10% SDS, 90 mM NaCl, 2 mM EDTA). Blocked protein A/G beads were added to the digested chromatin fractions and rotated at 4°C for 2 h to preclear chromatin. An aliquot of a 100 μl of precleared chromatin was purified by phenol-chloroform extraction, and DNA fragment sizes were analyzed and confirmed to correspond to 1 to 3 nucleosome fragments. Chromatin was subdivided into aliquots for each IP. Antibodies specific for unmodified H3 , H3K9me1 , H3K9me2  , H3K9me3  , and control IgG  were added to each tube and rotated at 4°C for 1 hour. The antibody-protein-DNA complex was precipitated by adding 20 μl of the blocked protein A/G beads and rotated at 4°C overnight (O/N). The complex was washed and eluted; IP’d material was purified using the QIAquick PCR Purification Kit .For N-ChIP, 1 × 10et al. [7 ES cells were harvested. Chromatin was cross-linked in 10 ml of PBS + 1% formaldehyde for 10 min at room temperature (RT). Reaction was quenched with 0.125 M glycine and incubated at RT for 5 min. Cells were washed two times with PBS and resuspended in 1 ml of collection buffer. The solution was incubated on ice for 10 min, then for 10 min at 30°C. Cells were lysed by vortexing pellets sequentially in 1 ml buffer A, 1 ml buffer B, followed by incubation of cell pellets at RT in 1 ml of lysis buffer for 10 min. Chromatin was sonicated using the Diagenode Bioruptor  at high setting for 18 min at intervals of 30 sec ‘on’ and 30 sec ‘off’ to achieve fragment lengths of 250 to 500 bp. For each IP, 2 × 106 cell-equivalents of precleared chromatin were used and diluted 2.5 times in IP buffer. Antibodies specific for HP1α , HP1β  XP™ rabbit mAb 8676) and control IgG  were added into each IP and rotated at 4°C O/N. To each tube, 40 ul of precleared beads were added, and tubes were rotated at 4°C for 2 h. The complex was washed and eluted off the beads. The cross-links were reversed O/N at 65°C, and the DNA was treated with Proteinase K and RNase A. DNA was purified using the Qiaquick PCR Purification Kit (Qiagen) and analyzed by qPCR with respect to input using SsoFAST™ EvaGreen Supermix (Bio-Rad). Primer sequences are listed in Additional file Cross-link ChIP for HP1 proteins was performed as described by Metivier et al. . Briefly6 TT2 or G9a KO mESCs were harvested cross-linked in 0.75% formaldehyde in PBS for 10 min, quenched with glycine, and lysed in ice-cold radioimmunoprecipitation assay buffer (RIPA) . Chromatin was sonicated with the Bioruptor on 30 sec ‘on’ and 30 sec ‘off’ to achieve 200 to 600 bp fragment size for each sample. The samples were divided into three equal aliquots and incubated O/N at 4°C with 4 μg of purified mouse IgG (Sigma-Aldrich), mouse monoclonal anti-G9a (R&D Systems PP-A8620-00) , or mouse monoclonal anti-KAP1 (Abcam Ab22553). Samples were precipitated with 30 μl protein-G Dynabeads (Invitrogen), washed three times with ice-cold RIPA buffer and eluted by shaking in 0.1 M NaHCO3, 1% SDS, 20 mM DTT for 15 min. Cross-links were reversed by heating samples at 95°C for 5 min in the presence of 300 mM NaCl. DNA was RNase A-treated, purified and qPCR was performed as described above. Primers used or ChIP-qPCR analyses detect: 638 IAP ERVs, 519 MERV-L ERVs, 202 MMERVK10C ERVs, 637 MERV-L pol internal regions, as determined by in silico PCR on the UCSC genome browser.Cross-linked ChIP for G9a and KAP1 were performed similarly with several modifications. Approximately 5 × 10et al. [http://genome.ucsc.edu on 17 August 2011). To quantify expression levels and the strength of KAP1 and SETDB1 marks, we calculated reads per kilobase per million mapped reads (RPKM) [RNA-seq libraries were constructed from mRNA as described in Morin et al.  from 10 et al.  with Smiet al. , RefSeq et al.  and UCSCet al.  (downloas (RPKM) ,78 for gA and RPKMB are RPKMs in the region of interest of A and B samples respectively, and rAB = NA/NB, where Nx is the total number of aligned reads used for normalization.where RPKMIn order to compare the coverage of KAP1 and SETDB1 among all families of ERVs and generate the average density of KAP1 in the genomic regions flanking ERVs, we mined the published KAP1 ChIP-seq data set . SequencThe KAP1 profile was generated at the genomic flanks of intact elements , which satisfied the length criteria, for three ERV families: MMERVK10C, IAPEz and MERVL. The mean density of KAP1 for each family was calculated for 50 bp bins within 6 kb distance at 5′ and 3′ flanks of elements by agglomerating the coverage inside the bins for all elements of one family and dividing this number by the number of elements and total number of aligned reads in the KAP1 IP.Chimeric transcripts containing ERV and genic sequences were identified by exploiting the genomic locations of paired-end reads. Mate-pair reads separated by more than one standard deviation from the mean fragment size were identified, and those mate-pairs containing one read in an ERV located upstream of the first exon of a gene, and the other read in an annotated genic exon of that gene, were enumerated. The number of chimeric mate-pairs was calculated for each chimeric transcript and the transcripts with the number of chimeric mate-pairs >5 were scored as valid transcripts.Whole-cell extracts were prepared by lysing cells in modified RIPA buffer . Nuclear extracts were prepared as previously described  except nhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47370).The data sets supporting the results of this article are available in NCBI's Gene Expression Omnibus repository , GEO SerThe authors declare that they have no competing interests.Zfp352, ChIP-qPCR of G9a and KAP1 and Western blotting, PG performed ChIP-qPCR of HP1s, MK performed bioinformatics analyses, with support from SJ, PS contributed the HP1 KO lines, IM and ML designed the study and wrote the manuscript. All authors read and approved the final manuscript.IM performed most experiments, PT performed qRT-PCR of Figures S1-S3 and supporting figure legends.Click here for fileTable of annotated genic transcripts initiating in a MERV-L LTR. All protein-coding genes that initiate in an annotated MERV-L/MT2 LTR in both RefSeq and ENSEMBL databases were identified and cumulative RPKM values at annotated exons for each gene were quantified for KAP1 [for KAP1  and G9a for KAP1  KO ESCs,Click here for fileTable of primer sequences used in this study.Click here for file


Excellent agreement with the theory was observed. With the integration of a screen-printed heater, sensor elements were operated actively heated in a cold gas stream.A potentiometric sensor device based on screen-printed Nasicon films was investigated. In order to transfer the promising sensor concept of an open sodium titanate reference to thick film technology, “sodium-rich” and “sodium-poor” formulations were compared. While the “sodium-rich” composition was found to react with the ion conducting Nasicon during thermal treatment, the “sodium-poor” reference mixture was identified as an appropriate reference composition. Screen-printed sensor devices were prepared and tested with respect to CO Although optical detection of CO2 using infrared radiation is very exact, a more cost-effective method capable of also working in harsh and dirty environments is needed. To meet these requirements, several potentiometric sensor devices based on electrochemical cells with sodium conducting solid electrolytes such as β‘’-Al2O3 or Na1+xZr2P3-xSixO12  have been investigated  or sodium titanate/titania (Na2Ti6O13/TiO2) mixtures were proposed as a reference system  were dried at 450 °C. The carbonate mixture was heated at 5 K/min to 720 °C, a temperature above the melting point. The melt was retrieved from the furnace and quenched onto a brass plate. The as-obtained material was ground in a mortar.An eutectic mixture of NaIn order to prepare screen-printable pastes, each of the as-prepared powders was sieved (< 200 µm) and mixed with a thixotropic organic binder to form a homogeneous paste.2.22Ti3O7/ Na2Ti6O13 or the Na2Ti6O13/TiO2 reference was printed on one side of the Nasicon film and fired at 950 °C for 5 h. Two gold grid electrodes were printed according to 2CO3/BaCO3 mixture was painted on top of one gold grid and heat treated at 600 °C.The sensor set-up is shown diagrammatically in 2.3.2Ti3O7 with 1 g Nasicon or 0.6 g Na2Ti6O13 with 1 g Nasicon, respectively. While one part of these mixtures was studied directly by XRD, one part was heat-treated at 950 °C for 5 h using the sintering profile of the reference thick films.In order to study compatibility between the reference materials and the solid electrolyte, XRD studies were conducted in the 2θ range from 10 ° to 90 °. Two samples were prepared by mixing either 0.75 g Na2.42 gas with dry air using mass flow controllers. The actual CO2 concentration was monitored by an FTIR  located downstream the sensor chamber. The emf output of the sensor element was monitored using a digital multimeter (Keithley 2700).For tests of the sensor performance, a custom-build test bench similar to the one described in  for hydr3.3.12Ti3O7/Na2Ti6O13 were measured. In emf trace at 500 °C upon CO2 exposure is shown exemplarily. The sensor device presented a stable and perfectly reversible response. An additional variation of the oxygen partial pressure from 0.15 bar to 0.27 bar indicated no cross sensitivity of the sensor to this gas as expected from the literature.In an initial test series, sensor samples with the sodium-rich reference composition Naemf = f (log2))emf=EAs known from the cell reactions (1) and (2), the theoretical electron transfer number equals 2. With the present sensor set up, values of 2.14 ± 0.06  and 2.12 ± 0.06  were determined.emf reading than expected from the thermodynamic calculations and experimental results on the pellet-type sensor discussed in 20]. In p


Due to recent leaps forward in DNA synthesis and sequencing technology, DNA manipulation has been extended to the level of whole-genome synthesis. Bacteriophages occupy a special niche in the micro-organic ecosystem and have potential as a tool for therapeutic agent. The purpose of this study was to carry out chemical synthesis of the bacteriophage G4 and the study of its infectivity.Full-sized genomes of bacteriophage G4 molecules were completed from short overlapping synthetic oligonucleotides by direct assembly polymerase chain reaction and ligase chain reaction followed by fusion polymerase chain reaction with flanking primers. Three novel restriction endonuclease sites were introduced to distinguish the synthetic G4 from the wild type. G4 particles were recovered after electroporation into Escherichia coli and were efficient enough to infect another strain. The phage was validated by electron microscope. Specific polymerase chain reaction assay and restriction analyses of the plaques verified the accuracy of the chemical synthetic genomes.Our results showed that the bacteriophage G4 obtained is synthetic rather than a wild type. Our study demonstrated that a phage can be synthesized and manipulated genetically according to the sequences, and can be efficient enough to infect the Escherichia coli, showing the potential use of synthetic biology in medical application. Bacteriophages, being viruses that infect bacteria, have played an important role in underpinning the development and advancement of the biosciences since the dawn of molecular biology. Bacteriophages, first discovered around 1915, occupy a special place in viral biology. They are perhaps the best understood viruses. Their genome being less than 10,000 bases long is particularly amenable to genetic alterations at the level of whole genome synthesis thanks to the recent advance made in DNA synthesis and sequencing technology. We may now modify genetic information to an extent not possible before. Due to the advances in synthetic biology, it is now possible to obtain large segments of synthetic DNA, assemble them into entire genomes of infectious agents, and boot them to life. Cello J et al Bacteriophage G4, first isolated in 1973 G4 can only multiply within E. coli and kill the cells by lysis. E. coli are the most common etiologic agent clinically associated with Urinary Tract Infection (UTI) which is the most frequently diagnosed kidney and urologic disease All these facts induced us to carry out the synthesis of the ΦX -like phage and the study of its infectivity. We set out to generate a synthetic G4 genome (syn-G4) by whole genome assembly. To tell the difference from the wild type of the phage G4, we genetically alter 3 restriction endonuclease sites during the synthesis of the genome and subsequently generate a mutated G4 genome (m-G4). Two of these infectious circlular genomes produced were electropotated into DH5α and the plaques of syn-G4 and m-G4 were observed after incubation. Both of the phages were confirmed and were able to infect E. coli strain BL21. Our report demonstrates that an infectious phage can be synthesized and manipulated genetically according to the sequence published, and efficient enough to infect the E. coli, showing the potential use of synthetic biology for medical application.PstI and then circularized to produce a circular molecule  were introduced to generate a m-G4 genome. Since the mutation sites are located at the third base of the corresponding codon, minimal structural and functional impact was expected. The oligonculeotides were pooled and gel-purified by Sangon Biotech as described in The oligonucleotides were designed to synthesize a G4 genome with exactly the same sequence reported by Godson et al. in 1978 molecule .Three rePstI and then circularized by ligation with T4 ligase using recommended conditions to generate products of syn-G4 and m-G4 circular molecules, then each 10 µl of ligation product was electroporated into DH5α cells, immediately diluted with 600 µl of liquid Luria-Bertani (LB) medium, and then divided into two screwcapped glass culture tubes. The tubes were rotated at 37°C for 60 min and then 200 µl of culture fluid was stained to LB solid culture medium to stay overnight at 37°C. Phage plaques were visualized after 6–12 h of incubation at 37°C  productions of full-length genomes were cleaved with Synthetic G4 samples were positive stained with 0.2% (w/v) phosphotungstic acid and examined by transmission electron microscopy, as seen in KpnI site. These sequences of m-G4 contained 2 KpnI sites, 1 NcoI site and 1 EcoRI site after genetic alteration shown in red in EcoRI, KpnI and NcoI to excise different segments according to the supplier's recommendations. Subsequently, m-G4 could be digested by EcoRI, KpnI and NcoI respectively, but syn-G4 just could be digested by KpnI  in order to distinguish from the wild type G4 genome. The full-sized genomes of bacteriophage G4 and m-G4 were synthesized and provided by Sangon Biotech Co., Ltd (Shanghai). Basically, for synthesis of the full-sized genomic DNA, each of syn-G4 and m-G4 genomes was divided into 4 segments, including A,B1,B2,and B3. All oligonucleotides were chemically synthesized by Sangon Biotech. The resultant fragments of various lengths with overlap were added together and finally assembled into full-sized genome by direct assembly PCR and ligase chain reaction (LCR) followed by fusion PCR with flanking primers from short overlapping synthetic oligonucleotides. The linear full-sized genomic DNA was circularized by means of enzymatic ligation and the use of preexisted PstI restriction sites for preparation of sticky ends using recommended conditions. Transfection of cells with the circular synthetic genome resulted in appearance of infective plaques.The oligonucleotides were designed to generate a syn-G4 genome according to the sequence reported by Godson PstI and then circularized by ligation with T4 ligase using recommended conditions. The ligation mixture was stayed overnight at 22°C in preparation for infectivity testing.PCR products of full-sized genomes were pooled and confirmed by sequencing. The linear DNA products were cleaved with One microliter of syn-G4 ligation product was electroporated into DH5α cells , immediately diluted with 600 µl of liquid LB medium, and then divided into two screwcapped glass culture tubes. The two tubes were rotated at 37°C for 60 min and then 200 µl of culture fluid was stained to LB medium A to stay overnight at 37°C. Another microliter of m-G4 and pure water underwent the same procedure and each of 200 µl of culture fluid was stained to LB medium B and C respectively, to stay overnight at 37°C. Phage plaques in A and B were visualized after 6–12 h of incubation at 37°C and recorded by photography at 8 h. No plaque was observed in C.glutaraldehyde was added for fixation before examination. A concentrated phage sample was negatively stained with 0.2% (w/v) phosphotungstic acid on a carbon-coated grid and examined by JEM100CXII (Japan) transmission electron microscope  at an accelerating voltage of 80 kV. Electron micrographs were taken at a magnification of 800,000×. The phage size was determined from the average of two independent measurements (Anteroposterior diameter and left-right diameter).Several plaques were picked up to 1.5 ml Eppendorf and 0.5 ml of 2.0% (w/v) 2O up to 1 L), gently shaken and maintained in the refrigerator at 4°C for 3 min. After adding a few drops of chloroform, the plate is manually shaken. The phage suspension was centrifuged (5000×g for 10 min) to remove DH5α and debris. The 200 µl of the supernatant was retrieved and diluted to the proper concentration (10−7/L∼10−5/L) for infectivity test. E. coli strain BL21  colony was picked up to 5 ml LB culture medium to prepare log phase of BL21. BL21 were grown in LB broth without antibiotic selection at 37°C until an OD600 of 0.4 was reached. Bacterial cells were diluted in LB broth to a final density of 106 CFU/ml. An aliquot of cells  was added with 100 µl phage suspension with a diluted concentration of 10−5/L and mixed with 0.7% soft agar, slightly shaken, then the mixture was plated on LB plates, following overnight incubation at 37°C. Phage plaques were visualized after 6∼18 h of incubation at 37°C.A confluent plaque was picked up and covered with 50 µl SM buffer (50 ml 1 M tris-cl(PH 7.5), 2 g MgSO4, 5.8 g NaCl, 5 ml 2% gelatin and ddH2, 2 µl of 2.5 mmol/L dNTP, 1.0 µl of each 20 pmol/L primer, 0.4 µl of 5 U/L Taq polymerase (Takara), 25 of primerstar, and ddH2O up to total 50 µl. PCR of NLE-pCMV10 for NLE gene was also performed as the control group (date not shown). w The conditions for PCR were as follows: 99°C for 2 min, followed by 30 cycles of 98°C, 10 s, 68°C, 40 s, 72°C, 10 min and a final extension of 72°C for 7 min. The PCR was performed as previously described. The PCR products were analyzed using a Rapid Agarose Gel Electrophoresis System  in 1.0% agarose gels in 5.0 µl loading buffer (10 min at 90 V). The genome was gel-purified and sequenced on a 3730 XL sequencer  with depth of 2-fold. Sequencing of the cloned amplification product confirmed that it was identical to part of the syn-G4 and m-G4.Specific primers for syn-G4 and m-G4 were synthesized by Jierui Biotech co., Ltd. (Guangzhou), and their sequences were listed in EcoRI, KpnI and NcoI) according to the supplier's recommendations . Digestion of syn-G4 and m-G4 with 2.0 µl of EcoRI restriction endonuclease  was done at 37°C in 10 m M Tris-HCl, 50 mM NaCl, 15 mM MgCl2, pH 7.5. DNA was digested with 2.0 µl of KpnI endonuclease  at 37°C in 10 mM Tris-HCl, 10 mM MgCl2, 0.02% Triton X-100, 0.1 mg/ml BSA. Digestion of DNA with 2.0 µl of NcoI restriction endonuclease  was done at 37°C 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% glycerol. The digested DNA was examined by gel electrophoresis after 10 hours digestion. The resultant fragments were separated and purified as described above.For restriction analyses, 10 µl of PCR products with expected length of 645 bp were digested with restriction endonucleases  with 0.5 µmg ml−1 ethidium bromide. Electrophoresis was performed at constant voltage 90 V) for 30 min and visualized by UV light (300 nm) after staining with ethidium bromide (1 Ag/ml). This procedure was followed for all the experiments except where stated differently. The relative sizes of the DNA fragments were estimated by comparing their electrophoretic mobility with that of the standards run with the samples on each gel (D2000marker).DNA was analyzed using a 6.0% (w/v) agarose gel in TAE buffer Click here for additional data file.File S1Trace files for specific PCR fragments, syn-G4 genome and m-G4 genome.(RAR)Click here for additional data file.


The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the β(1→3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the β(1→3) ψ dihedral (as previously predicted by simulation), while the β(1→4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties.The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-strucutre at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without  Depending on chemical features, they can be further sub-typed as hyaluronan (the only unsulfated GAG), chondroitin sulfate and heparin sulfate,Glycosaminoglycans (GAGs) are extracellular matrix polysaccharides that play crucial mechanical and regulatory roles in vertebrate physiology. The majority of GAGs share an anionic disaccharide repeat, comprising one uronic acid  comprises repeating disaccharides of In order to improve our understanding of GAG 3D-structure and relate this to physical properties and biological function, we have previously conducted experimental and theoretical studies on defined oligosaccharides from hyaluronan and heparan sulfate. In this latest study, we turn our attention to the 3D-structure of CS, which provides the heterogeneous sulfation found in CS proteoglycans (CSPGs).The primary barrier to structural studies of CS, and sulfated GAGs in general, has been the high level of chemical degeneracy and hence the unavailability of homogeneous material.Escherichia coli (O5:K4:H4) strain, which is identical (apart from a labile fructose decoration) to the unsulfated carbohydrate backbone of CS, as a preparative starting point.15N). Here we report production and purification of natural abundance isotope and 15N-enriched unsulfated chondroitin oligosaccharides from K4 antigen polysaccharide, up to decasaccharides, with slight modifications to the existing protocols in order to achieve milligram quantities of homogeneous defructosylated material in minimal media. Furthermore, the similarity of pathogenic polysaccharides to GAGs is more than a coincidence,We used capsular polysaccharide K4 from the 15N-isotopic enrichment, which resulted in novel sequence-specific NMR data. Again, using a similar strategy, we report a full triple-resonance  chemical-shift assignment of the chondroitin tetrasaccharide, and provide sequence-specific insights for larger oligosaccharides. Additionally, we used ultra-high-field (900 MHz) NMR to determine a set of nuclear Overhauser effect measurements, which were used to calculate a 3D-structural model for the unsulfated chondroitin hexasaccharide in an aqueous solution.We previously demonstrated the value of homogeneous hyaluronan oligosaccharides,Based on our findings, we discuss whether there is a major 3D-structural difference between the carbohydrate backbones of hyaluronan and chondroitin and consider the conformational effects of sulfation, by comparison with previous literature data. The results help clarify the role of GAG  sulfation in molecular interactions and hence vertebrate physiology and provide the basis for structure-based design of novel medicines and medical devices.22.11H frequency of 600 MHz were sufficient to assign all resonances  within the chondroitin tetramer (CN4) using a 15N-enriched sample. It was necessary to record additional data at 900 MHz to measure some scalar couplings and obtain a well-resolved NOESY spectrum on a natural abundance isotope sample for 3D-structure elucidation.Spectra obtained with a 4 all-atom assignment was achieved by taking advantage of chemical shift perturbations caused by end-effects . The 1H and 13C atoms were fully assigned  to d-glucuronic acid-4 (GlcA-4) of CN6 are available as The CNgned see  using -HSQC spectra to be recorded rapidly, as we have shown previously in other GAGs.1H, 15N]-HSQC spectra for the tetra-, hexa- and octa-saccharides  and the 1H and 15N chemical-shift assignments for CN4, CN6, CN8 and CN10 are detailed in 15N nuclei were resolved at 600 MHz, apart from resonances in the N7 and N9 residues of CN8 and CN10 (they could not be significantly improved at 900 MHz). End-effects manifested as two distinct regions in the 600 MHz -HSQC NMR spectra, which are exemplified in the broken-axis CN6 spectrum shown in 1H dimension compared to the internal residues, which are magnified in panels B, C and D  in order to illustrate the resonance overlap that is a result of the very similar chemical environment of these atoms.Enrichment with 1H and 13C resonances of CN6 and longer oligosaccharides are indistinguishable at 600 MHz, and the fact that the 15N chemical shifts of the internal residues of CN6 and CN8 are almost identical . The amide region is magnified in 1JH,N and 3JHN,H2 coupling constants, measured from 600 MHz [1H]–1D spectra of the type illustrated in 6, CN8 and CN10, due to resonance overlap in 600 and 900 MHz [1H]–1D spectra. Inspection of the 2D -HSQC spectra (with 15N-decoupling during acquisition) for each oligosaccharide revealed similar 3JHN,H2 coupling constants to the tetrasaccharide but these measurements are significantly less accurate and are thus not reported here.δ/ΔT) of the exchangeable GalNAc amide proton chemical shifts in CN4, CN6, CN8 and CN10, which is a useful indicator of inter-residual hydrogen bonding, as we showed previously.δ/ΔT values were in the range −6.8 to −8.4 ppb/°C.2.31H, 1H]-NOESY spectrum of a natural abundance isotope CN6 sample was recorded at 900 MHz and used to measure nuclear Overhauser enhancements (NOEs) between protons close in spatial proximity. The ultra-high-field spectrum was used to gain extra resolution compared to 600 MHz and also to shift our NOE data further from the zero-NOE point. At 900 MHz all the NOEs were observed to be negative . We were particularly cautious only to include high-quality unambiguous NOE data in our structure calculations , calculated using a restrained molecular dynamics simulated annealing protocol : βN1 8.98 (10.5), βN3 9.75 (7.8) and βN5 n/d (7.8), 6 3D-model to be trans orientated (with respect to H-2) and is consistent with the observed experimental NMR data.Of note in lues see . Further33.115N-enriched) have been prepared previously using a similar method to that described here.1H, 13C]-HSQC NMR spectra (previous studies used HPLC and mass spectrometry). To our knowledge, NMR studies to date on K4 polysaccharide and its defructosylated derivative have been restricted to polymeric material using basic 1D NMR spectra,Oligosaccharides of defructosylated K4 , this work contributes a major advancement in our knowledge of unsulfated chondroitin properties.In light of these comparisons with the literature data that are consistent with expectations and which support that unsulfated K4 material was produced and purified in this work, we propose that our assignments could be used with confidence to predict the chemical shifts for any sulfation pattern in any of the CS variants. Indeed, by providing a full triple-resonance assignment and vicinal 3.24 and previous literature data for HA4. Resolved CN4 and HA41H and 13C chemical shifts are compared in 4 hexosamine 4-position 1H chemical shifts were downfield (deshielded) compared to the HA4 values, by approximately 0.6 ppm. This observation was understood to be the manifestation of the different stereochemistries at this site. By inspection of 4 hexosamine 13C chemical shifts were comparatively more sensitive to this structural perturbation than the 1H values. Significant differences in 13C chemical shifts (between CN4 and HA4) were evident in all hexosamine 13C atoms except C-1, notably the ring carbon furthest from H-4. In contrast, the C-1 atoms of both GlcA residues were shifted downfield by approximately 1 ppm in CN4 relative to HA4. This observation suggests that the stereochemical  hydroxyl rearrangement at C-4 exerts an influence on the neighbouring sugar residue, perhaps by electronic effects that are propagated through either the glycosidic linkage or hydrogen bonds.Owing to the paradoxical structural similarity and significant functional differences of chondroitin (CN) and hyaluronan (HA), we undertook various direct comparisons of our data for CN3JH,H) coupling constants can report on carbohydrate ring conformation. By inspection of 3J3,4 and 3J4,5 coupling constants being approximately 5-8 Hz smaller in CN4 than in HA4. This was consistent with the small scalar coupling and gauche torsions (≈60°) in GalNAc residues of CN relative to the large scalar coupling and trans torsions (≈180°) in the N-acetyl-d-glucosamine (GlcNAc) residues of HA. The observed CN values were consistent with the 4C1 chair conformations of both GalNAc and GlcA, thereby justifying the use of 4C1 torsional restraints in the CN6 structure calculations  of exchangeable-proton  chemical shifts correlate with the presence or absence of both intra- and inter-molecular through-space interactions.δ/ΔT values for CN oligosaccharides are compared with previous work on HAδ/ΔT values for the CN oligosaccharides were only slightly less than those for amide protons in free exchange with water (≈−11 ppb/°C).6 are not persistent but rather transient and short-lived in an aqueous solution.Temperature coefficients  and psi (ψ) torsions: φ1–4 = O5(N)–C1(N)–O1(N)–C4(U), ψ1–4 = C1(N)–O1(N)–C4(U)–C5(U) [GalNAc–GlcA β1→4 linkages] and φ1–3 = O5(U)–C1(U)–O1(U)–C3(N), ψ1–3 = C1(U)–O1(U)–C3(N)–C4(N) [GlcA–GalNAc β1→3 linkages].The experimental data were measured and used to generate physically allowed 3D-models of CN45 and octasaccharides.13C -isotopic labelling8Presently, most 3D-structures of CN and CS comprise short oligosaccharides co-crystallised with GAG-degrading lyasesφ and ψ) from all previous studies considered with the exception of the X-ray crystallographic data . It has been speculated previously that crystal packing and the presence of Ca2+ in the small molecule structure likely explain its deviation from other literature values considered here.φ1–3 values of our 3D-model are again similar to all other CN, CS and HA data considered, differing at most by 11°.By inspection of ψ1–3 torsions. Again, excluding the X-ray data, our values for this torsion were most similar to those of the combined NMR and molecular modelling study of a CS pentasaccharide,ψ1–3 torsion. Our ψ1–3 torsion data for the CN6 model differed most significantly from the solution structure of HA8.ψ1–3 torsion value in CN would be approximately 30° less than that in HA, due to the lack of a hydrogen bond (and incorporation of a dynamic water bridge) between the hexosamine OH-4 atom and the glucuronic acid O-5 atom across the β(1→3) linkage but that the β(1→4) linkage would remain largely unchanged. The predicted sign and magnitude of the ψ1–3 torsion difference in CN and HA is largely consistent with our data (cf. the ψ1–3 torsions of CN6 and HA8 in The largest differences in glycosidic linkage geometries were observed in the 6 generated in this work (panel B) and in the aqueous 3D-structure of hyaluronan (panel C).6 3D-models and published structures of CS  may also not be attributable to GalNAc 4-position sulfation but rather to differential solvation. Therefore, we now hypothesise that chondroitin is amenable to sulfation because the GalNAc residue of both OH-4 and OH-6 atoms are available (solvent exposed). In contrast, the hyaluronan GlcNAc 4-hydroxyl may not be suited to sulfation because it is involved in an intra-molecular hydrogen bond with the adjacent sugar residue .We also note that the glycosidic linkage geometry in heparin is hypothesised to be unaffected by sulfate substituents.In summary, the 3D-structure of the unsulfated chondroitin backbone represents a significant milestone in understanding the biological function of CS and CSPGs in the extracellular matrix. In particular, it provides an experimentally based 3D-structural platform for the investigation of all CS sulfation patterns and their interactions with proteins in physiological processes  and disease pathologies . The 3D-model is also the basis of structural and computational strategies for developing new medicines and medical devices. In order to realise these objectives, we are working towards coarse-grained models of CS and CSPGs, which will link our microscopic 3D-structural data to macroscopic biological observations.44.1E. coli (05:K4:H4) strain in much the same way as described previously for the K5 antigen.kfoC gene encoding the K4 chondroitin polymerase gene using the primers 5′-CGGGATCCCGATGAGTATTCTTAATCTAAG3-3′ and 5′-GGAATTCCGGCCAGTCTACATGTTTATCA-3′. The nucleotide sequence of the PCR amplicon confirmed the presence of the kfoC gene. Bacteria were cultured to late logarithmic phase on M9-minimal media. Cells used as the source of 15N-enriched material were cultured on media containing 2.5 g/l 15N-ammonium chloride  as the only nitrogen source. In the current work 3 g/l ferrous sulfate heptahydrate (Sigma Aldrich) was added and the glucose component was raised from 20% to 60% to increase productivity in minimal media. The K4 polysaccharide was defructosylated by acid treatment in a slightly modified version of an existing protocol.15N-enriched oligosaccharides of length 4, 6, 8 and 10 were separated (using anion-exchange chromatography) and desalted, as described previously.The K4 polysaccharide was produced from cultures of the 4.22O, 0.02% (w/v) azide (NaN3) 0.3 Mm and 5% DSS . Approximate sample dry weights (mg) were: natural abundance isotope CN6, 6.6 and 15N-enriched CN4, 6.2; CN6, 8.2; CN8, 1.8; CN10, 7.6. Samples were adjusted to pH 6.0 to allow the observation of amide protons. The final concentration of all samples was calculated to be in the range 5–10 mM.Oligosaccharide samples for NMR spectroscopy were prepared from lyophilised material reconstituted in 500 μl MilliQ water comprising: 10% (v/v) D4.31H frequency of 600 MHz on a Bruker spectrometer equipped with a z-gradient TXI cryoprobe. The 15N-enriched tetra- and hexasaccharide [1H]-1D spectra were recorded with 16,384 complex points , an acquisition time of 2280.7 ms and a dwell time of 69.6 μs. At 900 MHz (Varian INOVA 900), these data were recorded with 32,768 points, an acquisition time of 2949.17 ms and a dwell time of 45.5 μs. No 15N-decoupling was applied during acquisition.All spectra, with the exception of the temperature coefficient data, were recorded at 25 °C. Through-bond experiments were conducted with a 1H, 13C]-HSQC and -HMBC spectra were standard experiments recorded on natural abundance tetra- and hexasaccharides. The -HSQC spectra were collected with acquisition times of 584.96 ms and 4096 points for 1H and acquisition times of 266.67 ms and 160 points for 15N. The dwell time and carrier frequency were set to 71.4 μs and 122.75 ppm, respectively, for these experiments. 15N-decoupling was applied during acquisition.The heteronuclear -NOESY spectrum of the unlabelled CN6 sample was recorded with a 1H frequency of 900 MHz and a mixing time of 600 ms seconds, known to be appropriate from our previous work on HA samples of the same length.A 2D [4.4Spectra were processed with NMRPipe4.5I) to proton–proton distance (r) using the relationship k) was then derived from a linear plot of I) for the five reference NOEs. This was employed to compute distance restraints for the eight conformationally dependent NOEs , which precluded the formation of non-4C1 ring conformations during high temperature dynamics, were set alternately to 60 ± 20° and −60 ± 20°. All restraints were applied fully during heating, holding and subsequent cooling phases of molecular dynamics.Parameters of the shake algorithm was applied to all bonds involving hydrogen atoms. Energetic and geometric data were recorded each ps.Due to the fact that explicit solvent is incompatible with the high temperatures inherent in simulated annealing, a modified generalised born continuum modelglycam 06The starting conformation was allowed to evolve from its initial configuration during 1 ns dynamics, throughout which it was heated linearly from 1 K to 500 K. Following thermalisation, 1 ns holding dynamics was done at 500 K, and the hexasaccharide was thereby allowed to explore regions of conformational space inaccessible at room temperature. The simulation was subsequently cooled linearly to 0 K for a further 1 ns. Final structures were geometry optimised (without restraints) using 5000 steps of steepest-descent energy minimisation followed by 5000 steps of conjugate gradient energy minimisation. Final energies were evaluated by a single point calculation using the


Lrmp, Hcls1 and Prkrir, were up regulated and one, i.e., Ptp4a3, was down regulated in the affected tumor. Next, we investigated the role of PTP4A3 in human AML and we show that PTP4A3 expression is a negative prognostic indicator, independent of other prognostic parameters. In conclusion, our novel strategy has identified PTP4A3 to potentially have a role in AML, on one hand as a candidate HIG contributing to leukemogenesis in mice and on the other hand as a prognostic indicator in human AML.Acute myeloid leukemia (AML) results from multiple genetic and epigenetic aberrations, many of which remain unidentified. Frequent loss of large chromosomal regions marks haplo-insufficiency as one of the major mechanisms contributing to leukemogenesis. However, which haplo-insufficient genes (HIGs) are involved in leukemogenesis is largely unknown and powerful experimental strategies aimed at their identification are currently lacking. Here, we present a new approach to discover HIGs, using retroviral integration mutagenesis in mice in which methylated viral integration sites and neighbouring genes were identified. In total we mapped 6 genes which are flanked by methylated viral integration sites (mVIS). Three of these, i.e.,  EVI1, FLT3, RAS, RUNX1, CKIT, WT1, CEBPA and NPM1Acute myeloid leukemia (AML) is a complex disease driven by multiple cytogenetic abnormalities, such as inv(16), t, t, 3q abnormalities, deletions of (the q-arms) of chromosome 5 and 7 and by aberrant expression and/or mutations of genes e.g., Retroviral insertion mutagenesis in mouse models has been used to discover novel genes involved in the development of different types of cancer Lrmp, Hcls1 and Prkrir were transcriptionally up regulated and Ptp4a3 was transcriptionally down regulated. Further studies in human AML samples revealed a negative prognostic value of PTP4A3 expression levels, independent of other prognostic indicators. In conclusion, by mapping DNA methylated viral integration sites in murine leukemias induced by retroviral integration mutagenesis followed by comparative analysis in human AML, we identified PTP4A3 not only as a candidate HIG contributing to leukemogenesis in mice but also as an independent prognostic indicator in human AML.To discover potential HIGs relevant for human AML, we used murine leukemia samples induced by Graffi 1.4 Murine Leukemia Virus (Gr1.4 MLV), classified as mixed lineage or myeloid leukemias by immunophenotyping In this study murine leukemia samples induced by Gr1.4 MLV were analysed ActB) and the hemi-methylated imprinting control region 1 (ICR1) of H19. As expected, H19 enrichment scores were high and ActB enrichment scores were low in all categories  may be down regulated due to the proximity of a methylated regulatory sequence, and, their transcriptional down regulation may contribute to murine leukemogenesis. Therefore, after showing that a proportion of viral integration sites are subject to DNA methylation, we set out to identify genes flanking these viral integration sites. To this end, iPCR, to amplify regions flanking viral integration sites, and MeDIP, to enrich for DNA methylated fragments, were combined to amplify regions flanking mVIS . AmplifiTaf12 and Ranbp3. Of the other 4 genes, Ptp4a3 expression was 2–3 fold reduced in the respective tumor . Survival analysis with these probesets gave similar results; all results shown are based on expression levels of probeset 206574_s_at. PTP4A3 expression levels were negatively correlated with prognostic outcome both for overall survival  and event-free survival . Kaplan-Meier curves are shown in PTP4A3 expression with event-free survival was independent of other prognostic parameters, i.e., age, white blood cell count, cytogenetic risk, CEBPA mutation status and +FLT3ITD−NPM1 status (The human orthologue of murine − status .Lrmp (lymphoid-restricted membrane protein), Hcls1 (hematopoietic cell specific Lyn substrate 1) and Prkrir ) were up regulated and Ptp4a3 (protein tyrosine phosphatase type IVA), a phosphatase also known as Prl3 (phosphatase of regenerating liver 3) was down regulated in the respective murine tumor. These results indicate that a flanking methylated viral integration site does not necessarily lead to transcriptional repression. As 1 out of 4 genes flanked by a mVIS was transcriptionally down regulated and expression of the 2 other genes could not be investigated, the efficiency to detect potential HIGs by identifying mVIS would approximately be 17–25%. However, the number of analysed tumors is too small to allow an accurate estimation of the efficiency.We designed a strategy to identify candidate HIGs in AML using retroviral integration mutagenesis, by mapping DNA methylated proviral integrations. By using HAT Ptp4a3 expression is controlled by p53 induced after DNA damage in mouse embryonic fibroblasts (MEFs) and its activity is involved in inducing a G1 cell cycle arrest in these cells PTP4A3 expression PTP4A3 may have both positive and negative effects on cell cycle regulation. Hence, PTP4A3 haplo-insufficiency, but not its complete loss, may lead to an impairment of cell cycle arrest after DNA damage. Dosage effects of PTP4A3 expression in relation to cellular responses may be more complex, particularly in cancer cells. For example, in carcinoma cell lines PTP4A3 expression may lead to down regulation of p53 PTP4A3 expression has been linked to increased tumor aggressiveness in different types of solid tumors, e.g., melanoma, gastric cancer, colon cancer, hepatocellular carcinoma and breast cancer PTP4A3 expression leads to increased epithelial-mesenchymal transition PTP4A3 in hematopoietic malignancies has not been studied as extensively as in carcinoma. Only a few studies report differences in expression levels of PTP4A3 in ALL and myeloma subgroups, based on gene expression profiling PTP4A3 has been proposed to have a role in drug-resistance in AMLs with internal tandem duplication of FLT3 (FLT3ITD) PTP4A3 expression negatively correlates with prognostic outcome, indicates that PTP4A3 might be a potential therapeutic target in AML.The role of PTP4A3 as a potential haplo-insufficient gene with an independent prognostic value in human de novo AML. Challenges for the future are to determine the dose-effect of PTP4A3 expression in myeloid development and to extend the screens to additional myeloid neoplasms, e.g., myelodysplasia, therapy-related AML, AML secondary to bone marrow failure and myeloproliferative disorders.In conclusion, using a retroviral mutagenesis screen in which we enriched for DNA methylated viral integration sites we identified For this study no novel murine leukemias were generated, all experiments described were performed on material generated in a previous study All human cell samples were obtained after written informed consent and stored anonymously in a biobank. The study was performed under the permission of the Institutional Review Board of the Erasmus MC, registration number MEC-2008-387.DNA and RNA samples from a previously generated panel of Gr1.4-induced leukemia's Primer and probe sequences are shown in H19 with H19ICR1fw × H19ICR1rv, ActB with ActBfw × ActBrv and the LTR with LTRfw × LTRrv using (q)PCR. Primer sequences are shown in Ten µg genomic DNA was digested overnight with 100 U of DpnII . Four µg digested DNA was denatured for 10′ at 95°C and incubated with either 2.5 µg anti-5-methylcytidine  or mouse pre-immune IgG  in 500 µL IP-buffer (PBS with 0.05% Triton X-100) for 2 hrs at 4°C, followed by incubation with 30 µL of washed beads  for 2 hrs at 4°C. Beads were washed 3 times with 700 µL IP-buffer. As a 10% input reference, 400 ng digested DNA not subjected to MeDIP was used. Beads and the 10% input reference DNA were resuspended in 100 µL IP-buffer and incubated for 3 hrs at 50°C after adding 20 µg proteinase K . Supernatants, containing immunoprecipitated DNA, and the input DNA were purified using the MinElute Reaction Cleanup Kit  and were eluted in 40 µL elution buffer. Two µL immunoprecipitated DNA was used to amplify the imprinting control region 1 (ICR1) of ActB enrichment (less than 10% of input) were selected for inverse PCR. Eight µL MeDIP-DNA was denatured for 3′ at 95°C, renatured by a temperature decrease of 0.1°C/sec to 20°C, and ligated for 45′ at room temperature using a rapid DNA ligation kit . Two µL out of 20 µl ligated product was amplified with primers mL1 and mL2, followed by a nested PCR with primers mL1N and mL2N using 2 µL of the first PCR product. Cycling conditions were 30″ at 95°C, 30″ at 60°C (first PCR) or 56°C (nested PCR) and 3′ at 72°C for 30 cycles. In the nested PCR 10 mM dCTP, dATP, dGTP, 8 mM dTTP and 2 mM dUTPs were used.Primer sequences are shown in PCR products of 10 nested PCR reactions were purified with a PCR purification kit  and pooled. A total of 7.5 µg of these amplified fragments was fragmented and labeled using the GeneChip WT Double-stranded DNA terminal labeling kit . Fragmentation to 66 bp was checked on a Bioanalyser . Labeled DNA was hybridized to mouse promoter 1.0R arrays  for 16 hrs at 45°C. Arrays were washed with the FS_450_0001 protocol using the Fluidics Station 450 , followed by scanning. Probe values were normalized with model-based analysis of tiling-arrays (MAT) corresponding gene) × LTRfw2, for Lrmp a nested PCR was performed with VIS(Lrmp_nested) × LTRfw. As input, 200 ng of the corresponding tumor DNA was used; cycling conditions were 30″ at 95°C, 30″ at 58°C and 45″ at 72°C for 30 cycles. Products were purified using the Multiscreen HTS 66-well filtration system . Sanger sequencing was performed with primer LTRfw according to the manufacturer's protocol .Primers are shown in H19 ICR1 (H19ICR1fw × H19ICR1rv) and ActB (ActBfw × ActBrv) were amplified. Cycling conditions were 30″ at 95°C, 30″ at 58°C and 45″ at 72°C for 30 cycles. Amplification products were analysed using gel electrophoresis.Primers are shown in Tbp) was analysed. Cycling conditions were 3″ at 95°C and 30″ at 60°C for 45 cycles. Amplification products were quantified using Fast SYBRgreen Master mix . Expression levels relative to Tbp were calculated.RNA of murine samples was isolated using Trizol  according to the manufacturer's protocol. One µg of RNA was used for cDNA preparation, using SuperScript II Reverse Transcriptase  according to the manufacturer's protocol. One µl cDNA was used as input for the qPCR. Genes of interest were amplified with their respective forward and reverse primers , as an iPurified AML blasts were obtained following informed consent as described Ptp4a3, univariate and multivariate survival analyses were performed using expression levels of probesets 206574_s_at or 209695_at in a Cox regression model. In the multivariate analysis age, white blood cell count, cytogenetic risk group, NPM1+−FLT3ITD status and CEBPA mutation status were used as additional prognostic parameters. We recognised the following cytogenetic risk groups: favorable = t, inv(16) and t, unfavorable = t, inv(3), −7/7q-, −5/5q-, complex karyotype, t(11q23) except t, t and t, intermediate = all other cases with known cytogenetics. Kaplan-meier graphs were generated by dividing the AML cohort in 2 groups of equal sample size based on PTP4A3 expression of probe 206574_s_at. Analyses were performed in SPSS .For PTP4A3) was considered significant. This analysis was repeated 100.000 times, followed by calculating the frequency, i.e., probability, of observing a significant p-value for both OS and EFS. Analyses were performed in Matlab .For the permutation test, all probesets with an annotated gene symbol  were selected. Next probesets with expression levels <30 in all 454 patients were discarded, leaving a total of 40720 probesets. The permutation test was performed by randomly selecting 6 probesets (representing 6 mVIS), followed by randomly selecting 1 out of these 6 probesets (representing 1 down regulated gene). For this probeset a univariate Cox regression analysis was performed for overall survival (OS) and event-free survival (EFS). A p-value of <0.0001 Click here for additional data file.Table S2Primers and probes.(XLS)Click here for additional data file.


Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore,  Termination of dopamine (DA) neurotransmission is accomplished primarily through a plasma membrane sodium-dependent re-uptake process mediated by the dopamine transporter (DAT). The physiological contribution of DAT to the control of DA homeostasis has been suggested by decades of pharmacological studies and further substantiated by genetic approaches Because DAT plays a critical role in the control of DA homeostasis, much attention has been placed on mechanisms regulating DAT activity and trafficking. Signaling pathways involved in rapid regulation of DAT include the activation of G protein-coupled receptors, intracellular second messenger systems, and the effect of protein-protein interactions We recently identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein 2 decreases VMAT2-mediated vesicular DA uptake, although it is not known whether this effect is the result of a direct interaction between the vesicular transporter and the G protein. Interestingly, the reduction of VMAT2 activity by G proteins was dependent on the vesicular content of monoamines 2 subunits. Because G subunits are associated with synaptic vesicles VMAT2-mediated DA transport into synaptic vesicles has been shown to be regulated by G proteins Motivated by these provocative findings describing a functional interaction between VMAT2 and synaptic vesicle-associated G proteins, we examined the possibility that DAT-mediated uptake might also be regulated by G proteins. In the present study, we identify a novel interaction between DAT and Gβγ subunits that result in modulation of DAT activity.2 incubator. MN9D cells were provided by Dr. Alfred Heller  and maintained in DMEM high glucose, supplemented with 10% FBS, at 37°C in a humidified, 5% CO2 incubator. The human DAT cDNA was cloned into pcDNA3.1(+) using KpnI and XbaI sites and used to transfect HEK293 or MN9D cells with Lipofectamine 2000 (Invitrogen) combined with a CombiTag Magneto transfection reagent (OZ Biosciences). DAT-expressing single clones (HEK293-DAT or MN9D-DAT cells) were selected with G418 (Gibco), verified by DAT immunoblot and immunofluorescence, and maintained in appropriate media containing 0.5 mg/ml G418. Cells were transiently transfected using Lipofectamine 2000 (Invitrogen).HEK293 cells were obtained from the American Type Culture Collection. HEK293 cells were cultured in MEM supplemented with 10% fetal bovine serum (FBS), 1 mM glutamine, and 50 µg/ml each penicillin and streptomycin at 37°C in a humidified, 5% CO1 , DAT2 . Immunoblotting was performed with a third DAT antibody, DAT3  and a Gβ pan-antibody .C57BL/6 mice were purchased from Jackson Laboratories, whereas DAT knockout mice tissue was a generous gift from Dr. Marc Caron (Duke University). All procedures were carried out in accordance with the National Institute of Health's Guide to the Care and Use of Laboratory Animals, and were approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee (IACUC) (Protocol number: IC-IS00000171-1) and in the case of DAT knockout mice brain samples, procedures were approved by the Duke University IACUC (Protocol number: A183-10-07). Striatum and cerebellum from wild-type and DAT knockout mice, HEK293-DAT, MN9D-DAT, or control cells were lysed in buffer containing (in mM): 20 HEPES, 125 NaCl, 1 EDTA, 1 EGTA and 10% glycerol containing protease inhibitors. After homogenization, 1% Triton X-100 was added, incubated for 1 h at 4°C, and centrifuged at 12000×g for 15 min at 4°C to remove cellular debris. Protein concentration was determined using the Dc Protein Assay kit (Bio-Rad Laboratories). Immunoprecipitations were carried out using 1 mg of total protein as described previously N (amino acids 1 to 60); (2) GST-DATL, (amino acids 119–139); and (3) GST-DATC (amino acids 582–620) of DAT. For pull-down experiments, 5–20 µg of GST fusion proteins were incubated with striatum lysates for 30 min at 22°C. GST fusion proteins and subsequent interacting proteins were isolated with 40 µl of Gluthatione-resin and samples were analyzed by SDS-PAGE and Western blotting. Direct interactions were examined by Immuno-Far Western. Briefly, 5 µg of GST fusion proteins were loaded into SDS-PAGE gels and electro-transferred to nitrocellulose membranes. Membranes were then washed, blocked overnight with PBS containing 1% BSA and 0.05% Tween-20, and incubated with purified Gβγ protein from bovine brain (EMD Biosciences) for 30 min. After washing membranes with 50 mM Tris, 150 mM NaCl, pH 8.0 four times, Western Blot was performed with specified antibodies.cDNA fragments coding for intracellular domains of DAT were amplified by PCR and subcloned into the pGEX4T-1 vector. Plasmids construction, sequencing, and characterization have been described previously Xenopus laevis oocytes using a nanoliter injector , and oocytes were kept at 18°C in ND-96 buffer  supplemented with 2.5 mM sodium pyruvate and 100 µg/ml gentamycin sulfate. Experiments were performed 2–3 days after cRNA injection. Intracellular injections of 50 nl of 100 µM GDP-β-S or GTP-γ-S (Sigma-Aldrich) were performed 15 min before the uptake experiment. Control experiments were performed with intracellular injections of H2O. After treatment with GTP analogs, oocytes were incubated for 10 min in 1 ml of ND-96 buffer containing 0.2 µM of [3H]-DA and 9.8 µM of DA or 0.2 µM of [3H]-Glutamate (PerkinElmer). Oocytes were transferred to non-radioactive ND-96 buffer, and washed three more times with ice-cold stop solution. Individual oocytes were lysed with 1 ml of 1% SDS for at least 1 h before adding the scintillation counting solution. For experiments with the mSIRK peptide, oocytes expressing DAT or EAAT1 were incubated for 30 min with ND-96 solution containing 10 µM of the peptide or 0.1% DMSO as control.Capped RNAs (cRNA) encoding human DAT or human excitatory amino acid transporter 1 (EAAT1) were synthesized from SmalI-linearized pOTV-hDAT or pOTV-hEAAT1 using a MESSAGE machine kit (Ambion). Synthesized cRNA was resuspended in 10 µl of water and stored in 2 µl aliquots at −80°C until use. 50 nl of cRNA was injected into 2, 1.2 MgSO4, 1 ascorbic acid, and 5 glucose, pH 7.4). For HEK293-DAT cells, 20 nM of [3H]DA   and increasing concentrations of cold DA ranging from 0.1 µM to 30 µM were used. After rinsing with 1 ml of NaCl-free uptake buffer, cells were solubilized in 0.5 ml of 1% SDS and the radioactivity incorporated into the cells was measured by liquid scintillation counting. Nonspecific uptake was determined in the presence of 300 µM cold DA. Data are presented as the mean ± SE. For experiments with the impermeable GTP analog GTP-γ-S, HEK293-DAT cells were permeabilized with streptolysin-o  according to the method described in 2HPO4, 0.44 KH2PO4, 1.0 MgSO4, 4.2 NaHCO3, 5 Glucose, and 30 HEPES, pH 7.2). Cells were incubated for 20 min at 37°C with 100 ng/ml SLO containing 50 µM GTP-γ-S and 1 mM Dithiotrietol, followed by incubation with ice-cold HBSS containing 1.4 mM CaCl2 and 30 mM HEPES for 2 h. In other experiments, cells were incubated with the mSIRK peptide (myr-SIRKALNILGYPDYD)  or the scramble version (scb-mSIRK) (myr-SLYRLISLAPRGDYD) (NeoBioScience) prior to uptake. In these experiments, control cells were incubated with 0.1% DMSO. Uptake was normalized to protein concentrations determined using the Dc protein assay kit.The conditions to examine DAT-mediated uptake in cultured cells have been described previously 2, 10 triethanolamine, pH 7.8). The reaction was quenched by incubating the cells for an additional 10 min with 50 mM glycine in PBS. Cells were then washed with PBS and incubated in radioimmune precipitation assay buffer (RIPA)  at 4°C for 1 h. Each sample was divided into two aliquots. One aliquot was used for isolation of biotinylated proteins with ultralink-immobilized avidin beads (Pierce). The second aliquot was used to determine total DAT levels. Samples were analyzed by SDS-PAGE and Western blotting with the anti-DAT antibody  and an HRP-conjugated secondary antibody (Jackson Immunoresearch Lab). Densitometry analysis of bands was performed with ImageJ software .Transfected cells were washed three times with PBS and then incubated with gentle agitation for 30 min at 4°C with 1 ml of 1.5 mg/ml sulfo-NHS-SS-biotin prepared in Biotinylation buffer  for 8 min. Data are expressed as percentage of control values (20205±2065 dpm). Nonspecific [3H]-DA uptake was determined in the presence of 10 µM nomifensine. Kinetic analysis of the synaptosomal [3H]-DA uptake was determined in the absence (control) or presence of 5 µM mSIRK. Synaptosomes were preincubated with or without mSIRK at 34°C for 10 min followed by the addition of one of eight mixed concentrations of the [3H]-DA. In parallel, nonspecific uptake at each concentration of [3H]-DA in the presence of 10 µM nomifensine, was subtracted from total uptake to calculate DAT-mediated uptake.Synaptosomes were prepared from rat striata as described in vitro or in vivo. The center-to-center distance between the microelectrode and the micropipette ejector was ∼200 µm. The micropipette was filled with DA (200 µM), mSIRK, or scrambled peptide (both 100 µM). The electrode/micropipette assembly was lowered into the striatum . Drug application was accomplished using a Picospritzer II (Parker Hannifin Corporation) in an ejection volume of ∼20 nl for DA to deliver ∼4 pmol and attain signals at the recording electrode in the range of 0.5 to 1.0 µM, and 50 nl for peptides to deliver 5 pmol (5–25 psi for 0.25–3 s) with an estimated concentration of peptide reaching the recording electrode in the range of 0.5–10 µM Clearance of exogenously applied DA from the striatum of anesthetized male C57Bl/6 mice was measured by high-speed chronoamperometry using the FAST-12 system (Quanteon) as previously described max and Km values were estimated by fitting the data to the Michaelis-Menten equation and represent the means from three independent experiments ± S.E.M. Statistics for uptake experiments were performed using a non-paired t-test with an accepted significance level at p<0.05. In vivo clearance was analyzed with ANOVA followed by Bonferroni or Newman-Keuls post-hoc comparisons.Functional experiments and densitometry data were analyzed with SigmaPlot (Systat Software Incorporation). Data are presented as the mean of at least three independent experiments with the standard error of the mean (S.E.M.) value. For uptake experiments, the VTo begin to investigate the possibility that G proteins regulate DAT activity, we first examined whether there is a physical association between the transporter and the subunits of G proteins. We generated three lines of evidence supporting a physical interaction between DAT and G protein βγ subunits. First, immunoprecipitations with two antibodies directed against different DAT epitopes resulted in co-precipitation of Gβγ subunits from mouse striatum, while no bands were detected in samples precipitated using the corresponding control IgGs . In mousXenopus laevis oocytes expressing human DAT where the GTP analog can be injected directly into the cytoplasm. Injection of GTP-γ-S (10 µM) resulted in 30.5±6.1% reduction of [H3]-DA uptake in Xenopus laevis oocytes expressing human DAT , but not in the affinity (Km) of the transporter for DA  or the carboxy-terminal domain of G protein–coupled receptor kinase 2 (GRK2ct), which are both known scavengers of Gβγ subunits t or GRK2ct significantly increased DAT activity   . These rXenopus laevis oocytes expressing DAT, incubation with 5 µM or 25 µM of mSIRK resulted in a 34.0±8.0% and 57.3±6.3% decrease in DAT activity, respectively  failed to alter DA uptake (50 = 13.2±1.2 µM). Kinetic analysis revealed that mSIRK incubation resulted in a reduction of Vmax without changes in the Km of DAT  without changes in Km , G proteins dissociate into active Gα and Gβγ subunits. Originally, the Gα subunits were believed to transduce cellular responses, while the Gβγ subunits were regarded to function solely as negative regulators of Gα-mediated signaling. Investigations over the last 20 years have revealed that the activation and function of G proteins is far more complex than previously anticipated. There is now evidence for both, receptor-dependent and -independent Gα- and Gβγ-mediated responses Additionally, evidence has accumulated indicating that Gβγ subunits also transduce cellular signals. In fact, Gβγ subunits have been reported to directly regulate a diverse array of effector molecules including ion channels, enzymes, and intracellular regulators Despite the fact that there is much to learn regarding the modulation of DAT by Gβγ subunits, the identification of DAT as a novel Gβγ effector will open new avenues to our understanding of DA homeostasis regulation. DAT plays a crucial role in the control of DA homeostasis and has been implicated in a variety of psychiatry disorders and drug addiction