C7L in the NYVAC genome prevents the induction of apoptosis and renders the vector capable of replication in human and murine cell lines while maintaining an attenuated phenotype in mice.The highly attenuated vaccinia virus strain NYVAC expressing HIV-1 components has been evaluated as a vaccine candidate in preclinical and clinical trials with encouraging results. We have previously described that the presence of C7L gene into the genome of NYVAC-B, a recombinant virus that expresses four HIV-1 antigens from clade B  (referred as NYVAC-B-C7L). In the present study, we have compared the in vitro and in vivo behavior of NYVAC-B and NYVAC-B-C7L. In cultured cells, NYVAC-B-C7L expresses higher levels of heterologous antigen than NYVAC-B as determined by Western blot and fluorescent-activated cell sorting to score Gag expressing cells. In a DNA prime/poxvirus boost approach with BALB/c mice, both recombinants elicited robust, broad and multifunctional antigen-specific T-cell responses to the HIV-1 immunogens expressed from the vectors. However, the use of NYVAC-B-C7L as booster significantly enhanced the magnitude of the T cell responses, and induced a more balanced cellular immune response to the HIV-1 antigens in comparison to that elicited in animals boosted with NYVAC-B.In an effort to improve the immunogenicity of NYVAC, we have developed a novel poxvirus vector by inserting the VACV host-range C7L and suggest the use of this modified vector as an improved vaccine candidate against HIV/AIDS.These findings demonstrate the possibility to enhance the immunogenicity of the highly attenuated NYVAC vector by the insertion of the host-range gene  AIDS is one of the greatest pandemics of our time, affecting the health and the social and economic foundations of countries worldwide. An effective human immunodeficiency virus (HIV) vaccine offers the best hope for controlling the spread of the virus. While the immune correlates of protection are not well defined, both antibodies and T-cell responses contribute to control the infection with HIV or the related simian immunodeficiency virus (SIV), as well as disease progression 89.6P,+ T cell response over CD8+ T cells and immunodominance for Env over Gag-Pol-Nef antigens, it suggest that to obtain a more balanced response to HIV-1 antigens with the DNA/NYVAC immunization protocol, further improvements of the NYVAC vector are desirable. One way to achieve this goal might be through genetic modifications of the NYVAC vector.Indeed, poxvirus vectors have emerged as prominent vehicles for delivering antigens of HIV-1. Different strains of Vaccinia Virus (VACV) expressing HIV-1 antigens such as Env, Gag, Pol and Nef or other components of HIV-1 have been evaluated in non-human primate C7L host range gene http://www.asia-lifesciences.com, January 10, 2008).NYVAC was derived from Copenhagen strain by the precise deletion of 18 open reading frames encoding functions involved in the pathogenicity of the virus as well as in host-range regulatory functions governing the replication competency of the virus on cells derived from certain species, including human and mouse C7L under the control of the synthetic early/late virus promoter into NYVAC-B, a recombinant virus which expresses four HIV-1 antigens from clade B . Previously we showed that insertion of C7L into NYVAC genome prevented virus-induced apoptosis and the phosphorylation of the translational initiation factor eIF-2 alpha, allowing virus multiplication in human cells while the virus remains attenuated in infected mice + and CD8+ T cells, and these responses are multifunctional, persistent, durable and directed to the different vaccine-encoded HIV-1 antigensTo improve the immunogenicity of the NYVAC based HIV/AIDS vaccine candidate, we have developed a novel vector by inserting the host range gene VACV C7L host range gene in the attenuated NYVAC-B recombinant which expresses four HIV-1 antigens from clade B . As shown in C7L gene inserted into the HA locus (A56R) of NYVAC-B genome under the transcriptional control of the synthetic early/late (E/L) virus promoter and preserves, as its parental NYVAC-B, the HIV-1 genes inserted in the TK locus (J2R). The correct insertion of heterologous genes in the recombinant virus as well as the purity of the stocks was confirmed by PCR (data not shown).In an effort to further improve the immunogenicity of NYVAC based vaccine candidates against HIV/AIDS, we have generated a new vector by inserting the VACV C7L gene was determined by measuring the viral growth efficiency of the recombinant viruses in three different cell lines of human (HeLa), mouse (3T3) and monkey (BSC40) origin. Monolayers of cells were infected at 0.01 PFU/cell with NYVAC-B or NYVAC-B-C7L, and at 0 and 48 hours postinfection the cells were collected with the media and virus titers in cell homogenates were determined by plaque assay. As shown in The functionality of Bx08gp-120 as a protein secreted from cells and III-BGPN as an intracellular fusion polyprotein of about 150 kDa Bx08gp-120 and III-BGPN expression levels at late times post-infection were higher in cells infected with NYVAC-B-C7L than in cells infected with NYVAC-B. The protein expression levels were similar when infected cells were treated with the DNA synthesis inhibitor Ara C, suggesting that differences in antigen expression between both viral recombinants are mainly a late event. Similar results for enhanced expression of gp120 and of GPN by NYVAC-B-C7L were observed in infected monkey BSC40 and mouse 3T3 cells . At 18 hours post-infection, cells were harvested, stained for HIV-1 p24 Gag or for late structural VACV proteins with the corresponding polyclonal antibodies, and we analyzed the frequency of Gag and VACV expressing cells as described in  p24 Gag 3A or VA p24 Gag  in compa p24 Gag . The redC7L gene into NYVAC genome affects the virus distribution and heterologous antigen expression in vivo, BALB/c mice were inoculated i.p. with a dose of 2×107 PFU of NYVAC-Luc, NYVAC-C7L-Luc or WR-Luc which is one of the most effective routes for systemic virus dissemination C7L gene into the NYVAC genome allows the recombinant virus to express luciferase to levels higher than the parental virus, mainly in peritoneal cells, ovaries and spleen  followed by an i.p. injection of 1×107 PFU of NYVAC-B or NYVAC-B-C7L. Adaptive immune responses were measured 10 days after final immunization using a fresh IFN-γ ELISPOT assay with splenocyte stimulation using pools of overlapping peptides that span the HIV-1 antigens included in the immunogens.BALB/c mice, 4 in each group, were immunized according to the schedule shown in the diagram of As shown in 6 splenocytes, p<0.005).Similarly, the magnitude of the total HIV-1 response , determiInterestingly, when calculating the average proportion of the response to the HIV-1 vaccine-expressed genes 5D, it wOverall, the combination of a DNA-B prime followed by the NYVAC-B-C7L vaccine construct boost induced a more balanced, broader, and higher IFN-γ HIV-1-specific T-cell responses as compared with that elicited by a DNA-B/NYVAC-B immunization protocol.+ and CD8+ T cells. As illustrated in + T and CD8+ T cells. For the GAG response the CD8+ T cell compartment was enhanced 4 fold in NYVAC-B-C7L in comparison to NYVAC-B (data not shown). With regard to ENV- and GPN-specific responses, NYVAC-B-boosted animals responded mainly to Env and Gag antigens, and the response was mediated by both CD4+ and CD8+ T-cell populations (+ T cell populations (+ compartment was responsible for most (>90%) of the total IFN-γ and TNF-α responses and the IL-2 secretion was mainly mediated by CD4+ T cells in DNA-B/NYVAC-B-C7L group after ENV stimulation. In contrast, in the DNA-B/NYVAC-B immunization group, the CD4+ compartment was responsible for more than 60% of the production of the three cytokines.To determine the phenotypic characteristics of the T-cell populations activated after immunization with the DNA/Pox combination, we employed multiparameter intracellular FACS analysis to identify the HIV-1-specific T-cell responses. Splenocytes were cultured overnight and then stimulated with ENV, GAG or GPN peptide pools for 6 hours in the presence of brefeldin A. Representative functional profiles from splenocytes of animals vaccinated with DNA-B followed by NYVAC-B or NYVAC-B-C7L stimulated with ENV pool are shown in ulations . The CD8+ and CD8+ T-cell responses. On the basis of the analysis of IL-2, IFN-γ and TNF-α secretion, seven distinct HIV-1 specific CD4+ and CD8+ T-cell populations were identified. To further characterize the immunogenicity triggered in each immunized group, we assessed polyfunctional T-cell responses. The ENV-specific CD4+ and CD8+ T cell responses elicited by DNA-B/NYVAC-B or DNA-B/NYVAC-B-C7L groups were polyfunctional, as more than 70% of CD8+ T cells and 80% of CD4+ T cells had two or three functions based on IFN-γ, TNF-α and IL-2 secretion. However, some differences were observed between both groups. GPN-specific CD8+ and CD4+ T-cell responses were highly polyfunctional in the DNA-B/NYVAC-B immunization group, while in DNA-B/NYVAC-B-C7L only CD8+ T cells were multifunctional. Examination of the relative contributions of individual cytokines versus polyfunctional responses showed that NYVAC-B-C7L improves the quality of the response in CD8+ T cell compartment but not in the NYVAC-B group, with a proportion (1–5%) of the HIV-1-specific CD8+ T cells expressing simultaneously the three cytokines,. In contrast, no improvement was found in the CD4+ T-cell compartment in terms of the quality of the response . While the Gag- and GPN-specific responses were also higher in NYVAC-B-C7L group, the differences were not statistically significant. For CD8+ T cells, higher levels of both IFN-γ and IL-2 were produced in mice primed with DNA-B and boosted with NYVAC-B-C7L than when boosted with NYVAC-B (2 fold). Interestingly, the production of IFN-γ by these cells was considerably higher than the production of IL-2. In fact, a high frequency of total IL-2 production by CD8+ T cells (more than 90%) resulted also positive for IFN-γ secretion representing 15% of the total Env-specific CD8+ T cell response. However, the Env-specific CD4+ T cells generated a higher frequency of IL-2 than IFN-γ response in both immunization groups and the percentage of multifunctional Env-specific CD4+ T cells was about 26% of the total response.As shown in These findings demonstrate that both NYVAC-B and NYVAC-B-C7L induced long-lived HIV-1 specific T-cell responses but the magnitude and quantity of these responses were significantly enhanced by the use of NYVAC-B-C7L in the booster.in vivo is limited which curtails the amount of antigen that can be produced in cells Different viral vectors of RNA and DNA origin are being considered as HIV/AIDS vaccine candidates, but to date only the poxvirus vector ALVAC in combination with a purified gp-120 prime boost has shown some efficacy in a phase III clinical trial C7L gene into the genome of NYVAC-B, a vector that expresses gp-120 and Gag-Pol-Nef antigens of HIV-1 from clade B. The C7L gene was selected for insertion into NYVAC-B genome because it is a VACV host range gene C7L insertion into the parental NYVAC genome restores the viral replication cycle in cultured human and murine cells by preventing eIF2-α phosphorylation and the induction of apoptosis C7L in the NYVAC-B genome improved significantly the level of expression of HIV-1 antigens as a result of enhanced translation, thus increasing viral replication and cell spreading. An increase in antigen expression observed in culture cells infected with NYVAC-C7L was also observed in vivo. As expected, the levels of luciferase activity were the highest in tissues from mice infected with WR-Luc, were moderate in NYVAC-C7L and the lowest in NYVAC-immunized mice, correlating luciferase levels with the replication capacity of each vector. We have previously described the safety profile of NYVAC-C7L, which remains replication competent in murine and humans cells but does not cause body weight loss or death after intranasal challenge in mice J2R, A56R, N1L or K1L which have been described to enhance the virulence and replication of VACV in vivoM2L, which are involved in the regulation of inflammatory responses and severity of VACV infection C7L rescues, in part, the in vivo replication capacity of NYVAC-C7L providing higher antigen production during infection than NYVAC and, importantly, the vector remains safe.In an effort to improve the immune potency of the NYVAC vector, we have constructed a new vector with replication competence in human cultured cells. This was done by the insertion of VACV + and CD8+ T cells, were directed to the four vaccine-encoded HIV-1 antigens, and were multifunctional (producing more than one cytokine simultaneously) and durable. NYVAC-B-C7L elicited significantly stronger HIV-1-specific cellular immune responses than replication-restricted NYVAC-B.When we compare the immune responses elicited in mice by NYVAC-B and NYVAC-B-C7L after DNA prime/poxvirus boost immunization approach, the replication-competent NYVAC-B-C7L was clearly superior to NYVAC-B in the induction of specific cellular immune responses against HIV-1 antigens. The data obtained by ELISPOT and ICS revealed that the combination of DNA-B/NYVAC-B-C7L induced T-cell responses mediated by both CD4in vitro expression levels of non-replicating C7L in the NYVAC-B genome enhanced significantly the antigen production in vitro and in vivo, correlating to an improvement in the immunogenicity elicited by the replicating NYVAC-B-C7L in comparison to the replication-restricted NYVAC-B. The frequency of total cytokine-producing CD8+ T cells was significantly higher in the spleens of animals that were boosted with NYVAC-B-C7L than in those mice boosted with DNA-B/NYVAC-B. Interestingly, the frequency of total cytokines producing CD4+ T-cell response was similar in both groups, regardless of the ability of each vector to replicate, indicating that the presence of C7L mainly improved the activation of CD8+ T-cell responses.The immunological differences between NYVAC-B and NYVAC-B-C7L in the magnitude of vaccine-elicited immune responses could be associated with the kinetics of antigen expression. Bachmann et al. showed that antigen load influences the stimulatory signals provided to T cells by antigen presenting cells (APCs) and subsequently influences the magnitude of the elicited immune response + T-cell activation site in shaping the response by modulating cell function + T-cell response can control or eradicate viral infections. CD8 T cells appear to be particularly important in the immune response to chronic infections and in the long-term control of latent and reactivating viruses + T-cell responses correlate with acute viral control and long-term nonprogression + T-cell response induced by NYVAC-B-C7L makes this vector an attractive candidate as HIV/AIDS vaccine.Several studies have emphasized the influence of antigen dose, microenvironment, and specificity of the APCs recruited to the CD8+ T-cell populations polarized toward a low average functional avidity peptides One interesting observation of this study is the change in immunodominance during NYVAC-B or NYVAC-B-C7L immunization. The DNA/NYVAC immunization regimen expressing HIV-1 Env and Gag/Pol/Nef antigens has previously shown an Env-dominant response in animal models and in phase I clinical trials +T cells with enhanced effector functions have been demonstrated after NYVAC vaccination in non-human primates and humans + and CD8+ T cells that were mainly IFN-γ+TNF-α+. Of note, Env- and GPN-specific CD4+ and CD8+ T-cell responses were also able to secrete some amounts of IL-2. In fact, a high proportion (10-16%) of the HIV-1-specific CD4+ T cells expressing simultaneously the three cytokines were found in both immunization groups while the percentages of this population in the CD8+ T-cell compartment were lower (1–5%) and only observed in animals boosted with NYVAC-B-C7L, suggesting that good quality of both T-cell responses was induced by the vectors. The presence of polyfunctional CD4+ T cells after immunization with both vectors could be favorable for the induction and maintenance of CD8+ T-cell memory responses + response is a hallmark of chronic HIV-1 infection, and adversely influences the development and functioning of the CD8+ T-cell response With regard to correlates of protection in HIV-1 infection, although the breadth and magnitude of response to viral antigens are considered important markers in HIV-1 vaccine development, a series of recent studies in mice, non-human primates and humans using multiparameter flow cytometry provide compelling evidence that the quality of the T cell response is a crucial factor in defining a protective T-cell response + and CD8+ T-cell compartments after Env stimulation as compared to NYVAC. Hovav and colleagues described that the priming and boosting vectors influenced the magnitude of the secondary CD8+ T cell response, but that the ultimate differentiation of these cells to memory cells was exclusively shaped by the second immunogen + T cells, as this process begins only after most of the antigen is cleared + T-cell development C7L insertion with regard to cytopathic effect, apoptosis induction or viral clearance An important consideration in HIV-1 vaccine development is the establishment of memory responses. Previous studies had shown the induction of long lasting T cell immunity in most of the human volunteers vaccinated with DNA-C prime/NYVAC-C boost + and CD8+ T cells, which are polyfunctional, and more importantly, these immunization regimens induce long-lasting T cell immunity. Furthermore, we showed that insertion of C7L in NYVAC-B genome clearly improves the magnitude and breadth of the cellular immune response induced against the HIV-1 antigens as compared to NYVAC-B. In addition, the replication capability of NYVAC-C7L allows the administration of the vaccine at low and safe doses, thus reducing manufacture burden and making production of NYVAC-C7L vaccines cost effective. Therefore, NYVAC recombinants expressing C7L gene are attractive live viral vectors for the development of vaccines and highlight the use of safe attenuated replication competent VACV vectors as improved platform for vaccine development against HIV/AIDS and other infectious diseases.Overall, the present data demonstrate that both NYVAC-B and NYVAC-B-C7L in combination with DNA-B are highly immunogenic in mice, induced vigorous and broad T cell responses, comprising of both CD42 atmosphere at 37°C. African green monkey kidney cells (BSC40) and human cells (HeLa) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn calf serum (NCS). 3T3 murine cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (CS).Cells were maintained in a humidified air 5% COJ2R) were used to test virus biodistribution in vivo. NYVAC-Luc and WR-Luc recombinants have been previously described C7L gene, this new recombinant vector NYVAC-C7L-Luc encodes the luciferase reporter gene under the transcriptional control of the synthetic early/late (E/L) virus promoter.Recombinant viruses with the luciferase gene inserted in the viral thymidine kinase (TK) locus  of NYVAC-B genome under the transcriptional control of the synthetic early/late (E/L) virus promoter. BSC-40 cells were infected with NYVAC-B recombinant at a multiplicity of 0.01 PFU/cell, and then transfected with 10 µg of pJR101-C7L DNA using lipofectamine reagent according to manufacturer's instructions (Invitrogen). Recombinant NYVAC-B viruses containing the C7L gene were selected by consecutive rounds of plaque purification in BSC-40 cells stained with X-Gluc (5-bromo-4-chloro-3-indoxyl-β-D-glucuronidase acid). In addition to C7L gene, this new recombinant vector NYVAC-B-C7L contains, as for NYVAC-B, the same cassette of HIV-1 genes in the TK locus (J2R) under the transcriptional control of the synthetic early/late virus promoter.Parental NYVAC and recombinant NYVAC-B expressing the HIV-1 All viruses were grown in BSC-40 cells, similarly purified through two 45% (w/v) sucrose cushions, and titrated by plaque assay in BSC-40 cells. Purity of the recombinants was confirmed by PCR analyses. Throughout the manuscript we will refer to parental NYVAC as replication-restricted and NYVAC-C7L as replication-competent, based on their different abilities to produce progeny virus in cultured human cells, with limited virus replication cycle for the former and complete replication cycle for the latter.Monolayers of human (HeLa), mouse (3T3) and monkey (BSC-40) cells grown in 12-well tissue culture plates were infected at 0.01 PFU/cell with NYVAC-B, NYVAC-B-C7L and VACV-WR recombinants, at different times postinfection cells were collected with the media, sonicated and virus yields determined by plaque assay in BSC-40 cells as previously described Bx08gp-120 and IIIBGPN was visualized following western blotting using rabbit polyclonal anti-gp-120 antibody  and rabbit polyclonal anti-Gag p24 serum , respectively. Detection of cellular β-actin protein by specific antibody was used as an internal loading control.HeLa, 3T3 and BSC-40 cells grown in 12 well-plates were infected at 5 PFU/cell with the recombinants NYVAC-B or NYVAC-B-C7L in presence or absence of adenine arabinoside , an inhibitor of viral DNA replication. At 2, 4, 8 and 24 hours post-infection (hpi), cells were collected and centrifuged at 1500 rpm for 10 min. Cellular pellets were lysed in cold buffer  and samples containing equal amounts of protein were run in 10% SDS-PAGE. The expression of HIV-1 6 cells were fixed and permeabilized using cytofix/cytoperm solution (Pharmigen Inc.) according to manufacture's instructions. For detection of viral vaccinia antigen expression, cells were stained using a polyclonal anti-vaccinia antibody (VACV-WR) and then incubated with phycoerythrin conjugated anti-rabbit immunoglobuling (Pharmingen). To test HIV-1 antigen expression we used anti-human immunodeficiency virus (HIV) Gag antibody conjugated to phycoerithrin . A total of 100000 events were acquired on a LSR II FACS (Beckton Dickinson) and analysed using FlowJo software (FlowJo).Fluorescent activated cell sorting (FACS) analysis was used to assay the frequency of antigen expressing cells BX08gp-120 (pCMV-BX08gp-120) and HIV-1 IIIB Gag-Pol-Nef (GPN) fusion protein (pcDNA-IIIBGPN) have been previously reported The two DNA constructs expressing the HIV-1 The HIV-1 peptide pools Gag-1, Gag-2, Env-1, Env-2, GPN-1, GPN-2, GPN-3 and GPN-4 were provided by the EuroVacc Foundation and were previously described 7 PFU/mouse of WR-Luc, NYVAC-Luc or NYVAC-C7L-Luc. At different times post-infection mice were sacrificed and mouse tissues were processed for luciferase analysis and plaque assay titration.BALB/c mice were purchased from Harlan. To analyze the biodistribution of virus recombinants in animals, BALB/c were immunized by intraperitoneal route (i.p.) with a dose of 2×10BX08gp-120 + 50 µg of pcDNA-IIIBGPN) by intramuscular route (i.m.) and two weeks later received an i.p. inoculation dose of 1×107 PFU of NYVAC-B or NYVAC-B-C7L in 200 µl of PBS. Ten days after the last immunization, mice were sacrificed and spleens processed for ELISPOT assay and Intracellular Cytokine Staining (ICS). When long-lived immune response was assayed, the animals (n = 4 per group) were sacrificed at day 53 after the boost. Three independent experiments were performed.To test the immunogeniciy induced by the HIV-1 recombinants, we structured the study around our standard immunization regimen (DNA/Pox combination) which we have previously shown that it elicited strong immune responses to HIV-1 antigens Gene expression of recombinant viruses was monitored by the luciferase assay to quantify heterologous gene expression in tissues from mice inoculated i.p with NYVAC-Luc, NYVAC-C7L-Luc or WR-Luc. Tissues were collected at 24, 48 or 72 h post-infection. Peritoneal cells were harvested by mouse peritoneal cavity lavage with 10 ml of sterile PBS, centrifuged at room temperature for 5 min at 1200 rpm and stored at -70°C. At indicated times post-inoculation, animals were sacrificed, spleen, liver, draining lymph nodes and ovaries were dissected under sterile conditions and stored at −70°C. Tissues from individual mice were homogenized in luciferase extraction buffer   using an Eppendorf-fitted Dounce homogenizer. Luciferase activity was measured in the presence of luciferin and ATP according to the manufacturer's instructions using a Lumat LB 9501 luminometer , and was expressed as Reference Luciferase Units (RLU) per milligram of protein. Protein content in tissue extracts was measured with a bicinchoninic protein assay reagent kit . Tissues from individual mice were collected and homogenized in complete DMEM media to test for the production of infectious virus by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (PFU) per milligram of protein.5–106 splenocytes (depleted of red blood cells) were plated in triplicate in 96-well nitrocellulose-bottomed plates previously coated with 6 µg/ml of anti-mouse IFN-γ mAb R4-6A2 . HIV-1 peptide pools from clade B and negative control (CTRL) pool were resuspended in RPMI 1640 supplemented with 10% FCS and added to the cells at a final concentration of 5 µg/ml for each peptide. Plates were incubated at 37°C, 5% CO2 for 48 h, washed extensively with PBS containing 0.05% of Tween20 (PBS-T) and incubated 2 h at room temperature (RT) with a solution of 2 µg/ml of biotinylated anti-mouse IFN-γ mAb XMG1.2  in PBS-T. Afterwards, plates were washed with PBS-T and 100 µl of peroxidase-labeled avidin  at 1∶800 dilution in PBS-T was added to each well. After 1 h of incubation at RT, wells were washed with PBS-T and PBS. The spots were developed by adding 1 µg/ml of the substrate 3,3′-diaminobenzidine tetrahydrochloride  in 50 mM Tris-HCl, pH 7.5 containing 0.015% hydrogen peroxide. The spots were counted with the aid of a stereomicroscope. The responses were given as the number of spot-forming cells per million of splenocytes. In all the cases the background levels were subtracted to each specific peptide pool.The vaccine- specific cellular immune response in mice was determined using ELISPOT assay measuring the secretion of IFN-γ by splenocytes after stimulation with different peptides pools. Briefly, 106 splenocytes (depleted of red blood cells) were resuspended in RPMI 1640 supplemented with 10% FCS and containing 1 µl/ml Golgiplug (BD Biosciences) to inhibit cytokine secretion. Cells were seeded on M96 plates and stimulated with overlapping peptides that span the entire HIV-1 BX08gp-120 and IIIBGPN proteins  added to the cells at a final concentration of 5 µg/ml for each peptide pool. Cells were incubated at 37°C, 5% CO2, and then analyzed by ICS. After 6 h of stimulation, cells were washed, stained for the surface markers, fixed, permeabilized using the BD Cytofix/Cytoperm™ Kit (Becton Dickinson) and stained intracellularly using the appropriate fluorochromes. To analyze the adaptive immune responses (day 10 post-boost), the following fluorochrome-conjugated antibodies were used: CD3-FITC, CD4-Alexa 700, CD8-PerCP, IL-2-PE, IFN-γ-APC and TNF-α-PECy-7. For analyses at day 53 post-boost, the following antibodies were used: CD4-Alexa 700, CD8-FITC, IFN-γ-PECy-7 and IL-2-Alexa-647. All antibodies were from BD Biosciences. Cells were acquired using an LSRII flow cytometer (Becton Dickinson) equipped with a high throughput system. Sample analysis was performed using FlowJo version 8.5.3 . The number of lymphocyte-gated events ranged between 105 and 106 dead cells were excluded using violet LIVE/DEAD stain kit (Becton Dickinson). Lymphocytes were gated on a forward scatter area versus side scatter area pseudo-color dot plot and dead cells were removed according to violet LIVE/DEAD staining. To analyze the adaptive immune responses, CD4+ and CD8+ events (gate previously on CD3+ cells) were gated versus IFN-γ, TNF-α and IL-2, and then combined together using the boolean operator. For the analyses at day 53 after the boost, CD4+ and CD8+ events were gated and the secretion of IFN-γ and IL-2 was analyzed. Data were analyzed using a one-tailed z test, to test if the antigen-specific response was higher than the negative control response .The phenotypes of responding T cells were analyzed by ICS and fluorescence-activated cell sorting analysis as described elsewhere a posteriori distribution of a priori assumption  is the Beta distribution with parameters a posteriori. Analogously, we can derive the distribution of the proportion of cells responding to RPMI, obtaining the distribution For the statistical analysis, we have developed a novel approach that corrects measurements for the medium response (RPMI) and at the same time it allows the exact calculation of confidence intervals and p-values of hypothesis tests. For ELISPOT and ICS statistical analysis, it was proceeded as follow. Given the total number of cells, Whenever two corrected proportions need to be summed, In the ELISPOT experiment, three replicates were obtained for each kind of antigen. The average response to that antigen was computed using only the corrected proportions significantly larger than the corresponding RPMI. The division implied by the averaging process needed a reinterpolation of the probability density function which we carried out using cubic splines as implemented in MATLAB 2008a.Figure S1Expression of HIV-1 antigens by NYVAC-B and NYVAC-B-C7L vectors in mouse and monkey cells. Western blot showing the kinetics of expression of gp-120 and GPN with time of infection. Murine 3T3 (A) or monkey BSC40 (B) cells were infected with 5 PFU/cell in the absence or presence of 40 µg/ml of cytosine arabinoside, AraC (A), cell extracts collected at various times, analyzed by SDS-PAGE, and Western blots reacted with specific antibodies to Env and GPN. Actin was used as a loading control. M: uninfected mock cells.(8.71 MB TIF)Click here for additional data file.


In the present study, we intended to investigate the expression of c-FLIP in human HCC tissues, and its relation with drug-induced cell apoptosis through the specific inhibition of c-FLIP expression by siRNA in 7721 cells.in vitro with cells treated with doxorubicin.c-FLIP expression was quantified immunohistochemically in HCC tissues(eighty-six cases), and corresponding noncancerous tissues (fifty-seven cases). Patients with HCC were followed up for cancer recurrence. Then, the c-FLIP gene was silenced with specific siRNA in 7721 HCC cells. c-FLIP expression was detected by RT-PCR, Western Blot and immunocytochemical staining. The cellular viability and cell apoptosis were assayed Positive immunostaining was detected for c-FLIP in 83.72% (72/86) human HCC tissues, 14.81% (4/27) hepatic cirrhosis, 11.11% (2/18) hepatic hemangioma tissues, and absent in normal hepatic tissues. The overexpression(more than 50%) of c-FLIP in HCC adversely affected the recurrence-free survival. Through c-FLIP gene silencing with siRNA, the expressions of c-FLIP mRNA and protein were remarkably down-regulated in 7721 HCC cells. And doxorubicin showed apparent inhibition on cell proliferations, and induced more apoptosis.These results indicate that c-FLIP is frequently expressed in human HCCs, and its overexpression implied a lesser probability of recurrence-free survival. The specific silencing of c-FLIP gene can apparently up-regulate drug-induced HCC cell apoptosis, and may have therapeutic potential for the treatment of human HCC. Human HCC (hepatocellular carcinomas) is the common hepatic highly malignant tumor. Most patients, especially in China, present at diagnosis with a high stage. The etiopathogenisis and developments of HCC are not well known. Deregulation of cell proliferation and cell apoptosis underlies neoplastic initiation and development, which involves multiple gene alterations, and is regulated by complicated signal transduction pathways. It has become clear that deregulated apoptosis plays a pivotal role in tumorigenesis, malignancy and metastatic potential .Accumulating evidence suggests that multiple intrinsic and extrinsic signaling molecules contribute to the resistance to death ligands- and chemotherapeutics-induced apoptosis in cancer cells. c-FLIP(cellular FLICE-inhibitory protein) is a novel member of IAP(inhibitor of apoptosis protein) family, which inhibits the apoptosis signaling mediated by the death receptors Fas, DR4, and DR5,3. c-FLI2 acid residues at the active site[L(long form) and 25 kDa c-FLIPS(short form), have the capacity to block DR-mediated apoptosis. c-FLIPL and c-FLIPS have been described to be recruited to the DISC and prevent caspase-8 activation in slightly different ways. Although the precise physiological role of c-FLIP is still debated, it is generally accepted that c-FLIPS interferes with the initial cleavage between the p20 and the p10 subunits of caspase-8, while c-FLIPL blocks the final cleavage step between the prodomain and the p20 subunit of the p43/41 intermediate unit. In contrast to c-FLIPS, c-FLIPL can interact with both FADD and caspase-8, and it has the more potent inhibitory activity and prevents caspase-8 activation by acting as a substrate trap [c-FLIP has homology to caspase-8 and caspase-10, but lacks their protease activity due to the absence of key NHtive site. c-FLIP In addition, c-FLIP is a target for the major survival pathways involved in carcinogenesis, namely the NF-κB, Akt/PKB and MAPK pathways . MoreoveL in many cells by siRNA sensitizes the cells to ligands- and chemotherapeutics-induced apoptosis [RNA interference (RNAi) represents a phenomenon of double-stranded RNA (dsRNA)-mediated post-transcriptional gene silencing (PTGS). RNAi can highly induce specific target gene silencing in mammalian cells using small interfering RNA (siRNA) . It has poptosis .In this study, the expression of c-FLIP in human HCC tissues and corresponding noncancerous tissues was analyzed by immunohistochemical staining. And then, the plasmids, which could encode siRNA against c-FLIP, were constructed and transfected into 7721 cells, a typical human HCC cell line, to inhibit the c-FLIP expression for the further study on its biological activity.Eighty-six patients with HCC presenting at Tangdu and Xijing Hospital of FMMU between 1999 and 2006, for whom sufficient paraffin embedded tissue was available, were enrolled in the present investigation. All the patients were not given the adjuvant radio- and/or chemo-therapy before the resection. Of the patients, seventy were male and sixteen were female with median age 65 years (range 31 to 76). The mean size of tumor was 5.5 ± 2.1 cm (mean ± SD) in diameter with a range from 2.5 to 11.0 cm. Tumor staging was in accordance to the AJCC staging system. 27 cases of hepatic cirrhosis, eighteen cases of hepatic hemangiomas, and twelve cases of normal hepatic tissues were used as the control.All tissues were scored by two pathologists blinded to disease status. Grading of HCC was based on Edmondson methods . Histopa2O2 for 10 min and microwaved at 750 W in citrate buffer (pH 6.0) for 15 min. Tissue sections were then blocked for 20 min with normal rabbit serum and incubated overnight at 4°C with rabbit anti-human c-FLIP polyclonal antibodies, diluted 1:200. Incubation with PBS instead of the primary antibody served as a negative control. After washing twice with PBS for 2 min each, immunostaining was performed using the standard S-P technique  and visualized with diaminobenzidine tetrahydrochloride solution.Sections (4 μm) were deparaffinized, rehydrated, immersed in 3% HStaining was assessed blindly by one observer. A minimum of five randomly selected fields (200×) were examined, with a mean of 1500 cells counted throughout the whole section. The labeling index was defined as the percentage of neoplastic cells with clear cytoplasmic immunoreactivity of the total number of neoplastic cells counted. The threshold for c-FLIP positivity was 10%. The intensity of staining was scored as 0: achromatic, 1: light yellow, 2: yellow, 3: brown.Bgl II and Hind III sites were respectively generated at the 5' and 3' ends of the templates (as shown below). si-526:According the sequence of the c-FLIP mRNA, the siRNA oligonucleotides were designed and synthesized to the targeted RNAi regions at 526~544, 1164~1182, 1305~1323 nt. GGAGCAGGGACAAGTTACATTCAAGAGATGTAACTTGTCCCTGCTCCTTTTTGGAAA-3' (Forward);5'-CCCGGAGCAGGGACAAGTTACATCTCTTGAATGTAACTTGTCCCTGCTCCGGG-3' (Back).5'-TTTCCAAAAAsi-1164:GCGAGGGCTGTGCACAGTTTTCAAGAGAAACTGTGCACAGCCCTCGCTTTTTGGAAA-3' (Forward);5'-CCCGCGAGGGCTGTGCACAGTTTCTCTTGAAAACTGTGCACAGCCCTCGCGGG-3' (Back).5'-TTTCCAAAAAsi-1305:ACGCCCACTCCTGGATCTTTTCAAGAGAAAGATCCAGGAGTGGGCGTTTTTTGGAAA-3' (Forward);5'-CCCACGCCCACTCCTGGATCTTTCTCTTGAAAAGATCCAGGAGTGGGCGTGGG-3' (Back).5'-TTTCCAAAAABgl II- and Hind III-compatible overhangs, and then ligated into pSuper (linked overnight at 16°C). After E. Coli, DH5α, transfected by the recombinant vectors, the positive clones were selected. With positive plasmids, the sequences were checked by sequencing a PCR-amplified region containing the oligonucleotides. The recombinant plasmids were named as pSuper-Si1, pSuper-Si2, pSuper-Si3 and pSuper-Neg(no target segment inserted), respectively.These above siRNA-encoding complementary single-stranded oligonucleotides were hybridized to give 2 by routine passage every 3 days or as needed.HCC cell line, 7721, showed stronger staining intensity (results not shown), and was used as the target cell for the following study. 7721 cells were cultured in RPMI1640 containing 10% fetal bovine serum(FBS), and maintained in a humidified 37°C incubator with 5% CO5 cells/well, corresponding to a density of 80% at the time of transfection. 4 μg plasmids and 8 μL LipofectamineTM 2000 were mixed respectively with RPMI1640 without FBS. These reagents were combined and incubated for 20 min before adding the cells in the mixed liquor. Cells were incubated at 37°C for 8 hr, then fresh RPMI1640 with 10% FBS was added. After another 48 hr cultivation, 400 μg/mL G418  was added in. When the cell clones formed after 14 days' growth, cells were screened out to be kept on cultivating. At last, the stable transfection 7721 cell clones were collected and given extended culture.Plasmids were transfected into cells according to the manufacturer's protocol of LipofectamineTM 2000 . In brief, 24 hr prior to transfection, cells were seeded without antibiotics in 6-well plate at 3 × 106 cells using TRIzol reagent . The first strand cDNA was prepared using the Superscript Amplification System kit  according to the manufacturer's instructions. For PCR, the primer sequences and expected product sizes were as follows: c-FLIP (512 bp), Forward: 5'-ATGTCTGCTGAAGTCAT CC-3', Back: 5'-ATCCTCACCAATCTCCTGCC-3'; β-actin (475 bp), Forward: 5'-TGACGGGGTCACCCACACTGTGCC-3', Back: 5'-CTGCATCCTGTCGGCAATGCCAG-3. Amplification was performed for 25 cycles  in a PERKIN ELMER Thermal Cycler PE2400.Total cellular RNA was extracted from 1 × 10The PCR products were analyzed on 2% agarose gels and visualized by ethidium bromide staining. Quantitation of expression levels was achieved after adjustment for the expression levels of the housekeeping gene β-actin by densitometry . The relative level of expression was then represented as the ratio of c-FLIP/β-actin.g for 10 min at 4°C. Cell lysates were washed three times in cold lysis buffer. 100 μg of total protein was loaded on SDS-polyacrylamide gels, separated by electrophoresis, and transferred to nitrocellulose membranes  using standard procedures. The blots were stripped. Blocking of membranes and incubation with the primary  and appropriate secondary Abs were performed. Bands were visualized with an ECL detection kit .The transfected 7721 cells were incubated for 30 min at 4°C in lysis buffer . Lysates2O2 for 15 min to remove endogenous peroxidase activity. Slides were incubated overnight at 4°C with rabbit anti-human c-FLIP polyclonal antibodies. Incubation with PBS instead of the primary antibody served as a negative control. After washing twice with PBS for 2 min each, immunostaining was performed using the standard S-P technique and visualized with diaminobenzidine tetrahydrochloride solution.Cells were fixed in situ in paraformaldehyde (4% in PBS), and smeared onto slides precoated with 0.01% poly-L-lysine and air dried for 48 hr. Slides were washed in PBS and put into 3% H4 cell/well cell suspension was plated in 96-well microplates. After treated with doxorubicin for 0–8 days, the number of cells per well is obtained by using counting chamber.For cell viability determination, 2 × 107/mL) were fixed in 4% formaldehyde in PBS for 25 min at 4°C. Each glass slide was added 50–100 μL of cell suspension. After air-dry slides at room temperature for 5 min, slides were then washed with PBS for two times. The slides were put into 2% H2O2 for 5 minutes to remove endogenous peroxidase activity. After removing excess liquid carefully, 50 μL of incubation buffer  were added to each sample. For negative controls: Prepare a control incubation buffer without TdT Enzyme by combining 45 μL of Equilibration Buffer, 5 μL of Nucleotide Mix and 1 μL of autoclaved, deionized water. They were covered with chambered coverslip caps and placed in an incubator under a humidified atmosphere at 37°C for 60 min. Slides were then dipped in stop solution, and incubated 30 min at 37°C. After being washed with PBS at room temperature, the slides were observed under a fluorescence microscope. Apoptosis was indicated by the presence of green or yellow-green fluorescence within the nucleus of cells as confirmation of fluorescein-12-dUTP incorporation at 3'-OH ends of fragmented DNA.Cells were treated with the indicated doses of doxorubicin for 48 hr, and then carefully harvested by centrifugation and reattached to gelatin-covered glass slides before labeling. In brief, cells . But no correlation was found between the expression of c-FLIP and the tumor stage and size.The positive rate in human HCC tissues was related to HCC grade. The rate of c-FLIP positive expression increased with Edmondson standard. The positive expression of c-FLIP displayed in 13/18 (72.22%) samples of Grade I HCC, 20/25 (80.00%) of Grade II, 18/21 (85.71%) of Grade III, and 21/22(95.45%) of Grade IV class (P = 0.204). But c-FLIP overexpression  implied a lesser probability of survival , using a semi-quantitative RT-PCR assay. The comparable amplification efficiencies were validated by the uniformity of control β-actin RT-PCR product yields. RT-PCR results showed that the expression levels of c-FLIP mRNA were inhibited in the transfected cells Figure. , but theThen we examined the effect of siRNA on the expression of c-FLIP protein with Western Blot and immunocytochemical staining. First, c-FLIP protein expression was analyzed by Western blot analysis Figure. . pSuper-Cells were treated with doxorubicin for 0–8 days, and then the growth curves were obtained. Cell proliferation was inhibited obviously when c-FLIP expression was knocked down by siRNA. Our data showed that si-526-siRNA significantly decreased the growth rate of 7721 cells, with a >50% decrease after 3 days repeatedly in three separate experiments Figure. .Then, the cells were assayed by the TUNEL method to assess the drug-induced apoptosis. Positive TUNEL staining would be indicative of the DNA fragmentation that was characteristic of apoptosis. Without c-FLIP RNAi, the fewer 7721 cells were TUNEL positive. In contrast, in cells transfected with the specific siRNA vector, pSuper-Si1, the apoptosis induced by treatment with doxorubicin was significantly elevated Figure. .Tumor cells have developed different ways to escape apoptosis induced by DR-triggering such as surface DR down-regulation, loss or mutation. Other mechanisms elaborated by tumor cells to develop cell death resistance include aberrant expression of anti-apoptotic molecules such as c-FLIP, Bcl-2, Bcl-xL, survivin and Livin. The current belief holds that perturbations in apoptotic death regulation constitute a vital step in cancer evolution .Each step in DR-mediated apoptosis is well regulated. c-FLIP is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs). Furthermore, c-FLIP over-expression can activate nuclear factor (NF)-κB activation induced by TNF-α or TRAIL. c-FLIP has a more central role in the antiapoptotic NF-kB response than the TRAF/IAP complex. On the other hand, c-FLIP expression is regulated by NF-κB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. So, c-FLIP plays an important role in cell survival not simply by inhibiting DR-mediated apoptosis but also by regulating NF-κB activation in human HCCs ,18. MoreThere is increasing evidence that in regard to its anti-apoptotic functions, c-FLIP can be considered as a tumor-progression factor. At present, the role of c-FLIP, as an anti-apoptotic protein involved in the regulation of the DR extrinsic apoptotic pathway, remains unclear. Therefore, the identification of the precise role of c-FLIP in cancer cells represents an essential aim to further target and restore deregulated death pathways.c-FLIP is generally expressed in embryonic tissues, but is not expressed in most normal adult tissues, whereas is over-expressed in the majority of human cancers. It indicates that c-FLIP may associate with the tumorigenesis and progress of most human cancers. Published information regarding the significance of c-FLIP over-expression in human tumors has only recently begun to accumulate -24.Human HCCs show resistance to apoptosis mediated by several death receptors. c-FLIP is constitutively expressed in human HCC cell lines, and is expressed with a higher positive rate in human HCC tissues than in noncancerous liver tissues. In the present study, positive immunostaining was detected for c-FLIP in 83.72% of human HCC samples, but was absent from normal hepatic tissues. The other authors' and our studies suggest that c-FLIP may play an important role in human HCCs. For the patients with c-FLIP overexpression, they may have a shorter recurrence-free survival time.Now, RNAi, that can induce highly specific target gene silencing in mammalian cells using siRNA, has been a powerful tool in studying the cell function of any gene. c-FLIP expression can be inhibited by RNA interference using siRNAs, evidence from reduced levels of c-FLIP mRNA and c-FLIP protein.In this study, the c-FLIP-targeted siRNA vectors were designed to specifically silence c-FLIP. Then, the plasmids transcript containing c-FLIP-targeted siRNA and negative siRNA were constructed and transfected into 7721 cells. We found that there were significant differences between 7721/pSuper-Si1 and 7721/pSuper-Neg in c-FLIP expression at both mRNA and protein levels Figure. . The pheL was sufficient to sensitize MDA435 cells to doxorubicin. Our study showed that c-FLIP gene silencing enhanced doxorubicin-induced HCC cell apoptosis (Figure. Some studies reported that siRNA-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant, and null colorectal cancer cell lines . And theThe results of the present investigation demonstrated that c-FLIP is frequently expressed in human HCCs, correlated with Edmondson standard. The HCC patients with c-FLIP overexpression may have a shorter recurrence-free survival time. The specific silencing of c-FLIP can apparently inhibit its expression on both protein and mRNA levels. The inhibition of c-FLIP expression can down-regulate HCC cell viability and up-regulate drug-induced cell apoptosis. Our data suggest that targeting c-FLIP in conjunction with anticancer therapies may have therapeutic potential by enhancing HCC cell death.The authors declare that they have no competing interests.XD and GB participated in the preparation of the tissue sections and the construction of the siRNA vectors, and helped in coordinating the work. GB also participated in data analysis and interpretation and in manuscript preparation. XH and QQ have been involved in western blot analysis, PCR assays and IHC and ICC assays of c-FLIP expression. HZ and FY participated in cell culture and cellular work. JL participated in study design and critical revision of the manuscript. QM participated in the study design and coordination and helped to revise the manuscript. All authors read and approved the final manuscript.


P ≤ 0.001). ER status (P = 0.026) and histological grade (P = 0.031). Expression of E-CD was not found to be related to recurrence, distant metastases, lymph node stage, vascular invasion, primary tumour size, prognostic group or survival. Thus E-CD expression in human breast cancer appears to have minimal prognostic value, but may have a role as a phenotypic marker. © 2001 Cancer Research Campaign http://www.bjcancer.comEpithelial cadherin (E-CD) is a member of the cadherin family of cell adhesion molecules and has been implicated as an invasion suppressor molecule in vitro and in vivo. We analysed 174 breast tumours from the Nottingham/Tenovus Breast Cancer Series immunohistochemically for E-CD expression using the mouse monoclonal antibody HECD-1 (Zymed Laboratories Inc.). In normal epithelial cells E-CD was strongly expressed at cell–cell boundaries. 66% of the breast cancers examined had reduced intensity of E-CD expression with 74% having significant reductions in the proportion of E-CD-positive tumour cells. Using a combined intensity/proportion score, significant associations were found between E-CD expression and tumour type (


We found that major stability determinants are located in the N-terminus. Moreover, testing of all 20 amino acids in the position after the initiator methionine revealed strong differences in protein stability and indicated an important role of the penultimate N-terminal amino acid residue in determining the protein half life. We propose that the stability of plastid proteins is largely determined by three factors: (i) the action of methionine aminopeptidase , (ii) an N-end rule-like protein degradation pathway, and (iii) additional sequence determinants in the N-terminal region.Although chloroplast protein stability has long been recognised as a major level of post-translational regulation in photosynthesis and gene expression, the factors determining protein stability in plastids are largely unknown. Here, we have identified stability determinants  Plastids arose through endosymbiosis of a formerly free-living cyanbacterium, and have therefore retained numerous prokaryotic features in their gene expression machinery, for example organization of genes into operons and translation on 70S-type ribosomes. However, over the course of evolution, plastids have also acquired numerous novel features that are clearly not of eubacterial origin and make the regulation of plastid gene expression quite complex. These include the presence of multiple RNA polymerases and promoter types , the preBiochemical and genetic studies have unravelled several plastid proteolytic activities  is technically demanding and involves time-consuming and laborious techniques , PetD (subunit IV of the cytochrome b6f complex), Rps12  and PGK . PsbE, PetB, PetD and Rps12 are encoded by the plastid genome, but PGK is encoded by the nuclear genome and its N-terminus is generated by post-import cleavage of the transit peptide that directs the protein to the chloroplast. We used the sequences of the N-terminal eight amino acids from PsbE, PetB, PetD and Rps12 to generate N-terminal fusions with GFP in vector pWA-N. Due to codon constraints caused by requirement for an NcoI-compatible overhang for cloning, nine amino acids of the mature N-terminus were used for PGK. Likewise, the sequences of the C-terminal eight amino acids were used to produce C-terminal fusions with GFP in vector pWA-C (Whether the major (in)stability determinants of plastid proteins reside in the N-terminus, the C-terminus or both is currently unknown. In bacteria, it is known that C-terminal sequence motifs can also trigger protein degradation  was consor pWA-C . The resEscherichia coli, and hence perform a side-by-side comparison of protein stability determinants between bacteria and plastids.All 30 cassettes were cloned into a plastid transformation vector suitable for targeting the transgenes to a defined location in the plastid genome by homologous recombination . As plasE. coli cells. As any sequence modification of the gfp transcript can potentially affect mRNA stability, we wished to exclude the possibility that differences in gfp mRNA accumulation interfere with the assessment of protein stability as determined by GFP accumulation. We therefore compared gfp mRNA accumulation for all constructs  for mRNA stability. Quantification of hybridisation signal intensities and normalisation of the gfp signal to the bla signal revealed similar gfp mRNA accumulation levels for all constructs  were selected for polysome analysis. When the distribution of the analysed . This ingfp mRNA accumulation levels and polysome association are comparable in the transplastomic lines and not influenced by the sequence stretches fused to the 5′ or 3′ end of the gfp coding region made it clear that any difference observed in GFP accumulation is probably due to a difference in protein turnover. Consequently, GFP accumulation levels serve as a reporter of protein stability in plastids. Chloroplast-localised GFP fluorescence was detectable in the transplastomic lines, indicating proper protein folding of GFP in planta. GFP accumulation was not high enough to be directly detectable by Coomassie staining. We therefore measured protein accumulation in the 30 transplastomic lines using an anti-GFP antibody  but the others remain unprocessed  or undergo more extensive N-terminal processing. Although N-terminal Met excision had been confirmed (for the native chloroplast proteins) by protein sequencing (http://www.uniprot.org/) for all plastid N-termini used in this study, it is likely that some of the amino acid exchanges in the 20 constructs with mutations of the penultimate N-terminal amino acid residue result in inefficient N-terminal Met removal. This could be the case in constructs where the initiator Met is followed by a bulky amino acid that is known to impair Met removal   , confirmhttp://www.isv.cnrs-gif.fr/tm/maturation/images/chloro.html).Having established that the penultimate N-terminal amino acid residue represents an important determinant of chloroplast protein (in)stability, we wished to identify putative substrates of the underlying protein degradation pathway in plastids. When analysing the penultimate N-terminal amino acids of all plastid genome-encoded proteins in tobacco, we found that the two strongest instability-conferring amino acids, Cys and His  and 7a, In the absence of plastid-encoded proteins with destabilising Cys or His residues, we next considered the possibility that Cys and His serve as protein degradation signals for nuclear-encoded proteins targeted to plastids. The N-termini of imported proteins are generated by post-import proteolytic processing, which removes the N-terminal transit peptide from the protein. Previous work has established that the cleavage site is defined by the loosely conserved sequence motif -X-(Ala/Cys)↓Ala . ConsequIn this study, we have identified key determinants of protein stability in chloroplasts. Our data obtained with fusions of N- and C-terminal sequences from five plastid proteins to GFP provide strong evidence that the N-terminus harbours important information affecting the half life of the protein. Although the identity of the penultimate N-terminal amino acid residue is part of this stability information, additional, and perhaps more significant, stability determinants reside downstream, as revealed by the strong differences in stability conferred by N-termini with identical or similar penultimate N-terminal residues  and 6. WE. coli has mainly involved cleavage of reporter proteins, in which the N-terminus of the test protein was generated by post-translational proteolytic processing  revealed that we used only one codon that is rare in E. coli (i.e. used at a frequency of less than 10%): AGA for arginine (used for only 2% of the Arg codons in E. coli K12). However, as Arg resulted in high GFP accumulation in E. coli , in the fully sequenced genome of the model plant E. coli  plants were obtained from surface-sterilised seeds germinated on agar-solidified MS medium .Sterile tobacco  harbouring the various plasmid constructs were grown in YT medium  with ampicillin (100 μg/ml) at 37°C under continuous shaking (180 rpm).rrn promoter, the gene 10 leader from phage T7 (G10L), the gfp coding region and the rps16 3′ UTR was assembled from previously described elements . The synthetic fragments have NcoI overhangs on both sides and contain an internal AgeI restriction site (underlined). The basic vector pWA-Gly contains glycine as the first amino acid after the initiator Met. To change the second codon of the psbE–gfp fusion gene, pairs of annealed synthetic oligonucleotides were inserted into the NcoI/AgeI-digested vector pWA-Gly. The oligonucleotide pairs had the sequences 5′-CATGNNNGGATCTA-3′ and 5′-CCGGTAGATCCNNN-3′, where NNN indicates the codon to be changed. Annealing of the oligonucleotides generated NcoI and AgeI overhangs for cloning into pWA-Gly. For each amino acid, the most preferred codon was chosen according to the codon usage table for the tobacco plastid genome (http://www.kazusa.or.jp/codon/). To test larger N-terminal sequences for their impact on protein stability, the sequences for the first eight amino acids of the PetD, PetB and Rps12 proteins and the first nine amino acids of the PGK protein were inserted into the NcoI-digested pWA-1 vector as annealed synthetic oligonucleotide pairs with NcoI overhangs. The oligonucleotide pairs used were 5′-CATGGGAGTAACTAAAAAACCTGA-3′ and 5′-CATGTCAGGTTTTTTAGTTACTCC-3′ for PetD, 5′-CATGTCTAAAGTATATGATTGGTT-3′ and 5′-CATGAACCAATCATATACTTTAGA-3′ for PetB, 5′-CATGCCTACTATTAAACAATAAAT-3′ and 5′-CATGATTTATTGTTTAATAGTAGG-3′ for Rps12, and 5′-CATGGCTAAAAAATCTGTAGGAGATCT-3′ and 5′-CATGAGATCTCCTACAGATTTTTTAGC-3′ for PGK. C-terminal sequences from the same plastid proteins were integrated at the 3′ end of the gfp coding region as BstbI/XbaI-digested PCR products generated using the forward primer 5′-GAGTACAACTATAACTCACAC-3′ and one of the following gene-specific oligonucleotide primers: 5′-AAAATCTAGATTAAAATGATCGTGAAAATTCATCTAA-TTTGTATAGTTCATCCATGCC-3′ for PsbE, 5′-AAAATCTAGATTA-AAATAATCCTAAAGTTAAAGATTTTTTGTATAGTTCATCCATGCC-3′ for PetD, 5′-AAAATCTAGATTATAAAGGTCCAGAAATTCCAGGTTTTTTGTATAGTTCATCCATGCC-3′ for PetB, 5′-AAAATCTAGATTATTTAGGTTTTTTTACTCCATATTTTTTGTATAGTTCATCCATGCC-3′ for Rps12, and 5′-AAAATCTAGATTATACAGCTACAGGAGCATCAGC-TTCTTTGTATAGTTCATCCATGCC-3′ for PGK .All vectors are based on the previously described plastid transformation vector pRB95  with a Hepta adaptor. Spectinomycin-resistant lines were selected on a modified MS medium containing 500 mg/L spectinomycin. Plastid transformants were identified by double resistance tests on medium containing both spectinomycin and streptomycin (500 mg/L each) (Plastid transformation was performed using the biolistic protocol . Briefly/L each) . For eachttp://www.peqlab.de). RNA from E. coli was isolated using a NucleoSpin® Extract II kit . RNA samples were purified by treatment with RNase-free DNase I  and tested for the absence of residual DNA by PCR. For Southern blot analysis, DNA samples  were digested using BglII or XcmI, separated by gel electrophoresis in 1% agarose gels, and transferred onto Hybond nylon membranes  by capillary blotting using standard protocols. RNA samples were electrophoresed in formaldehyde-containing 1% agarose gels and blotted onto Hybond nylon membranes or transferred directly onto membranes using a slot-blot apparatus . For slot blotting, RNA samples were diluted in 200 μl in DNase I buffer and heat-denatured. Hybridisations were performed at 65°C (psaB coding region (psbZ coding region [obtained using primers psbZ-F (5′-GCTGATAGAGGGATCAAAT-3′) and psbZ-R (5′-GGGTCATTTTGGTTTTGGG-3′)] were used as RFLP probes to verify plastid transformation and assess homoplasmy. An E. coli 16S rRNA-specific probe and a gfp-specific probe were produced as described previously  and P16Srrn-R (5′-GGCGGTGTGTACAAGGCCC-3′). Probes were purified by agarose gel electrophoresis after extraction of the DNA fragments of interest from excised gel slices using a GFX™ PCR kit , and then radiolabelled by random priming using [α-32P]dCTP.Total plant DNA was isolated from fresh leaf tissue by a cetyltrimethylammoniumbromide-based method . Total c at 65°C . A 550 bPolysome isolation was performed as described previously . Each grE. coli cells (harvested at an OD600 of approximately 1.0) was performed as described previously  were also analysed. Protein samples were separated by electrophoresis on 15% SDS-containing polyacrylamide gels and blotted onto polyvinylidene difluoride membranes . Membranes were treated with blocking buffer  for 1 h, and then incubated for 1 h with polyclonal GFP antibody  diluted 1:10 000 in buffer . Detection was performed using the ECL Plus detection system  and an anti-mouse secondary antibody . To exclude technical or biological variation, all experiments were repeated at least three times. Equal loading was confirmed by Coomassie staining of the high-molecular-weight region of the gel using standard protocols and quantitatively assessing the amount of large subunit of Rubisco (RbcL) in all plant samples. Image quantification was performed using the Java software package imagej (http://rsb.info.nih.gov/ij/). The recombinant GFP signals were used for ECL exposure adjustment and normalisation between replicates.Extraction of total soluble protein from plant material and from http://www.miltenyibiotec.com) with affinity chromatography columns from the same supplier. Total soluble protein extract  was used for the chromatography. The eluted GFP fusion protein was further purified by gel electrophoresis and prepared for mass spectrometry by tryptic digestion as described previously (http://msquant.sourceforge.net), and searched for the N-terminal peptides using the Mascot algorithm (http://www.matrixscience.com). The search parameters included: 50 ppm precursor mass accuracy, 0.8 Da fragment mass tolerance, carbamidomethyl cysteine as a fixed modification, and oxidation of methionine as a variable modification. One missed cleavage was allowed.GFP fusion proteins were purified from leaf material using the μMACS GFP isolation kit (Miltenyi Biotech GmbH,


Porphyromonas gingivalis is an obligate anaerobe that requires heme for growth. To understand its heme acquisition mechanism, we focused on a hemin-binding protein (HBP35 protein), possessing one thioredoxin-like motif and a conserved C-terminal domain, which are proposed to be involved in redox regulation and cell surface attachment, respectively.The periodontal pathogen hbp35 gene was transcribed as a 1.1-kb mRNA with subsequent translation resulting in three proteins with molecular masses of 40, 29 and 27 kDa in the cytoplasm, and one modified form of the 40-kDa protein on the cell surface. A recombinant 40-kDa HBP35 exhibited thioredoxin activity in vitro and mutation of the two putative active site cysteine residues abolished this activity. Both recombinant 40- and 27-kDa proteins had the ability to bind hemin, and growth of an hbp35 deletion mutant was substantially retarded under hemin-depleted conditions compared with growth of the wild type under the same conditions.We observed that the P. gingivalis HBP35 exhibits thioredoxin and hemin-binding activities and is essential for growth in hemin-depleted conditions suggesting that the protein plays a significant role in hemin acquisition. Porphyromonas gingivalis has been implicated as a major pathogen associated with chronic periodontitis. The establishment of P. gingivalis at a periodontal site and progression of disease is dependent on the ability of the bacterium to utilize essential nutrients, of which iron (preferably in the form of heme) plays a crucial role. P. gingivalis lacks the majority of enzymes in the biosynthetic pathway for the porphyrin ring, hence it is unable to synthesize protoporphyrin IX, the precursor of heme  insertion mutant (KDP168), suggesting that M115 and M135 are the N-terminal amino acid residues of the 29-and 27-kDa proteins, respectively. The use of M115 and M135 as alternative translation initiation sites was supported by the finding that no HBP35 translational product was detected in the hbp35 [M115A and M135A] insertion mutant (KDP170). Moreover, recombinant HBP35 proteins with a C-terminal histidine-tag were produced in an E. coli strain expressing the hbp35 gene and purified by a histidine-tag purification system. Immunoblot analysis revealed that the purified products contained 40-, 29-, and 27-kDa proteins immunoreactive to the anti-HBP35 anitibody. Edman sequencing revealed that the N-terminal amino acid residue of the recombinant 27-kDa protein was M135  could bind hemin and that HBP35 was suggested to possess a putative heme binding sequence (Y50CPGGK55). To determine the hemin binding region of HBP35, we constructed and purified rHBP35 (Q22-P344), rHBP35 (Q22-P344 with C48S and C51S) and truncated rHBP35 (M135-P344) proteins with N-terminal histidine-tags using a histidine-tag purification system and carried out hemin binding assays using a hemoprotein peroxidase assay. As shown in Figure 22-P344), rHBP35 (Q22-P344 with C48S and C51S) and truncated rHBP35 (M135-P344) proteins were found to have hemin binding ability, implying that the hemin binding site is located in M135-P344 of HBP35 protein.Shibata et al.  found thhbp35 insertion mutant, the full length deletion mutant, the complemented strain which was constructed by replacing the intact hbp35 gene into the hbp35 full length deletion mutant, and the wild-type strain were hemin-starved after being grown in enriched BHI broth containing hemin .Since HBP35 protein is a hemin-binding protein, we determined the contribution of HBP35 proteins to acquisition or intracellular storage of heme. The et al.  and [GenBank: AP009380], respectively. The DNA primers used in this study are shown in Additional file P. gingivalis hbp35 insertion mutant was constructed as follows. A DNA fragment corresponding to a region (0.80 kb) containing the C-terminal lower portion of PG0615 and the N-terminal upper portion of the PG0616 gene was generated by PCR using P. gingivalis W83 chromosomal DNA as the template with a forward primer, MS1, containing a KpnI site (underlined) and a backward primer, MS2, containing an EcoRI site (underlined). The resulting fragment was cloned into the pGEM-T Easy vector (Promega) to yield pKD732. A DNA fragment corresponding to a region (0.70 kb) containing the C-terminal portion of the PG0616 gene was generated by PCR using P. gingivalis W83 chromosomal DNA as the template with a forward primer, MS3, containing a BglII site (underlined) and a backward primer, MS4, containing a NotI site (underlined). The resulting fragment was cloned into the pGEM-T Easy vector to yield pKD733. The BglII-NotI region of pKD733 containing the 0.70-kb fragment was swapped with both equivalent sites of pKD704 ) was verified by PCR analysis.f pKD704 , resultiporation . The celg pMD125  as the tof pAL30  which cohbp35 mutant lacking the last five amino acid residues (K340-P344), a DNA fragment corresponding to a region (1.5 kb) containing the C-terminal lower portion of PG0615 and PG0616 lacking K340-P344 was generated by PCR using pMD125 as the template with a forward primer, MS9, containing a KpnI site (underlined) and a backward primer, MS10, containing a BamHI site (underlined). The resulting fragment was cloned into the pCR4 vector to yield pKD741. A DNA fragment corresponding to a region (0.47 kb) containing the PG0617 gene and PG0618 gene upper region was generated by PCR using pMD125 as the template with a forward primer, MS11, containing a BamHI site (underlined) and a backward primer, MS12, containing a NotI site (underlined). The resulting fragment was cloned into the pGEM-T Easy Vector to yield pKD742. The BamHI-NotI site of pKD742 was inserted into the same sites of pKD741 to yield pKD743. To create a BglII site located 8 bp upstream of PG0617 in pKD743, the two-stage PCR-based overlap extension method , hbp35[M135A] or hbp35[M115A M135A] insertion mutants which had an insertion with the ermF-ermAM DNA cassette that was located just upstream of F110, pKD746, pKD747 and pKD748 were linearlized with NotI and introduced into P. gingivalis 33277, giving KDP168, KDP169 and KDP170, respectively.To create 1-P344) with a C-terminal histidine-tag overexpression system, a 1.0-kb PCR fragment was amplified using forward primer MS19, containing an NcoI site (underlined) and backward primer MS20, containing an XhoI site (underlined), and then cloned into the pCR4 to yield pKD749. The EcoRI-XhoI sites of pKD749 were inserted into the same sites of pET21d(+), resulting in pKD750. To create a recombinant HBP35 protein (Q22-P344) with an N-terminal histidine-tag overexpression system, a 0.97-kb PCR fragments were amplified using forward primer MS21 and backward primer MS22 and then cloned into the pET30 Ek/LIC vector (Novagen), resulting in pKD751. Site-directed mutagenesis of the thioredoxin active site in HBP35 was performed using a QuickChange II Site-Directed Mutagenesis kit. A double amino acid substitution mutant (rHBP35 Q22-P344 with C48S C51S) was created with the oligonucleotide sense primer MS23, containing C48S and C51S substitutions (underlined), and antisense primer MS24, containing C48S and C51S substitutions (underlined), using the recombinant plasmid pKD751 as the template to yield pKD752. Proper mutation was confirmed by DNA sequencing. To create a recombinant truncated HBP35 protein (M135-P344) with an N-terminal histidine-tag overexpression system, a 0.66-kb PCR fragments were amplified using forward primer MS25 and backward primer MS22, and then cloned into pET30Ek/LIC vector, resulting in pKD753.To create a recombinant HBP35 protein (AE. coli BL21(DE3)pLysS harboring pKD750, pKD751, pKD752 or pKD753 was cultured in LB medium containing 100 μg/ml of Ap at 37°C to OD600 of 0.4-0.6, and then IPTG was added to the culture at 1 mM, followed by an additional 3-h incubation. The cells were harvested, suspended in buffer A  and then disrupted with a French Press. The mixture was centrifuged at 3,000 × g for 15 min to separate the inclusion body fraction (pellet) from the soluble fraction (supernatant). The supernatant was loaded onto a pre-equilibrated Ni2+-NTA agarose column (Invitrogen) of 2 ml in bed volume and incubated at 4°C for 30 min. The column was washed three times with buffer B  and the bound protein was eluted with 10 ml of elution buffer  as 1-ml fractions. The fractions were analyzed by SDS-PAGE. The pure fractions were pooled and then dialyzed against milliQ water and stored at -20°C until further use. N-terminal amino acid sequencing (Edman sequencing) of the purified rHBP35 protein with the C-terminal histidine-tag was carried out using the service facility in CSIRO .SDS-PAGE was performed according to the method of Laemmli . ProteasP. gingivalis cells were harvested from 400 ml of fully-grown culture by centrifugation at 10,000 × g for 30 min at 4°C, washed twice with 10 mM HEPES-NaOH (pH 7.4) containing 0.15 M NaCl, and resuspended in 20 ml of HEPES containing 0.1 mM TLCK, 0.1 mM leupeptin and 0.2 mM PMSF. The cells were disrupted with a French Press by three passes at 100 MPa in the presence of 25 μg/ml each of RNase and DNase. Unbroken cells were removed by centrifugation at 1,000 × g for 10 min and the supernatant was subjected to ultracentrifugation at 100,000 × g for 60 min. The precipitates were treated with 1% Triton-X100 in HEPES containing 20 mM MgCl2 for 30 min at 20°C. The inner and outer membrane fractions were recovered as a supernatant and a pellet, respectively, by ultracentrifugation at 100,000 × g for 60 min at 4°C  website [http://www.tigr.org]) using an in-house Mascot search engine  and BioTools 2.2 software  and by comparison with tryptic peptide mass lists generated by using General Protein Mass Analysis for Windows software .To identify the 27-kDa protein, eviously . Gel pie22 to P344 of HBP35 and the 0.80-kb DNA fragment coding for M1 to S266 of ErmF were obtained by PCR that were used as the radiolabelled hbp35 and ermF probes, respectively. To label the DNA probes, [α-32P]dCTP and the ready-to-go DNA labeling beads kit  was used. The radiolabelled products were analyzed with a fluoro-image analyzer FLA-5100 (Fujifilm).Total RNA extraction and Northern blot analysis of mRNA were carried out as described previously  with som2O2, by the method of Shibata et al.  insertion mutant), KDP169 (hbp35 [M135A] insertion mutant) and KDP170 (hbp35 [M115A M135A] insertion mutant) were depicted.Click here for fileE. coli expressing the hbp35 geneN-terminal amino acid sequencing of the recombinant 27-kDa protein produced in an . rHBP35 products, which were partially purified using a C-terminal histidine-tag, were analyzed by SDS-PAGE followed by staining with CBB (left) or immunoblot analysis with anti-HBP35 antibody (right). The N-terminal amino acid sequence of the recombinant 27-kDa protein was determined by Edman sequencing, resulting in M135 as an N-terminal residue.Click here for fileBacterial strains and plasmids used in this study.Click here for fileOligonucleotides used in this study.Click here for file


The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression.L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold.We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF).Our findings suggest that NFI-A, in particular its brain-specific isoform, represses  Moreopression ,6. More pression , and KLFpression . Nuclearpression ,10). In L1 gene, exon 1, intron 1, exon 2 with the luciferase cDNA inserted to replace the L1 start codon by the luciferase start codon, intron 2 including the neural restrictive silencer element, exon 3, intron 3 and exon 4. The NFI-A expression plasmid pCHNFI-A has been described previously [BstXI and KpnI. 3' overhangs were removed by incubation with Platinum Pfx DNA Polymerase  at 68°C for 15 min, and the resulting blunt ends were ligated using the Rapid DNA Ligation Kit . For ChIP experiments, Myc-tagged expression constructs for the standard and brain-specific NFI-A isoforms were made by cutting out the HA tag with NotI and SfiI from pCHNFI-A and pCHBNFI-A, respectively. Two oligonucleotides, NFI-Myc 1 (GGCCGCTATGGAACAAAAACTCATCTCAGAAGAGGATCTGCAC) and NFI-Myc 2 (CAGATCCTCTTCTGAGATGAGTTTTTGTTCCATAGC) , which contain the coding sequence of the Myc epitope combined with Kozak box, start codon and the compatible nucleotide overhangs, were annealed. The resulting double-stranded oligonucleotide was added to a 5-10 fold excess of the respective NotI/SfiI-digested NFI-A expression vector, and ligated using the Rapid DNA Ligation Kit.The L1 reporter plasmid L1-11 , a kind eviously  murine oeviously , which, eviously  in an HAThe CMX expression plasmid coding for β-galactosidase  was kind2 atmosphere.Mouse neuroblastoma cells (N2A) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM L-glutamine, and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). For reporter gene assays, N2A cells were grown in Opti-MEM without phenol red  supplemented with 5% fetal calf serum, 200 units/ml of penicillin G, and 200 μg/ml streptomycin. Chinese hamster ovary cells (CHO) were grown in Glasgow minimum essential medium (GMEM), which contained 10% fetal calf serum, 4 mM L-glutamine, and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). All cell types were grown at 37°C under a 5% CO32P-dCTP using Klenow polymerase (Roche). Binding reactions were performed in a total volume of 10 μl consisting of 20 mM HEPES pH 7.8, 100 mM NaCl, 2 mM MgCl2, 7 mM DTT, 0.5 μg Cot-1 DNA, and 3 μl of cell lysate. Complete protease inhibitor was added according to the manufacturer's specifications (Roche). Binding reactions were incubated for 20 min followed by the addition of 2 μl of the labeled oligonucleotides, and incubated further for 20 min at room temperature. For the supershift, 1 μl of undiluted anti-HA  was added before loading, and incubated for another 20 min. In the case of competition experiments, radioactively labeled oligonucleotides were mixed with a 5-100 fold molar excess of the respective unlabeled oligonucleotides before being added to the binding reaction. Complexes were resolved by nondenaturing PAGE, and dried gels were exposed to BioMax MR film .For EMSAs, HA-NFI-A constructs were expressed in CHO cells. Cells were plated onto 90-mm dishes in CHO culture medium. After reaching confluence, cells were washed once with CHO culture medium without serum and antibiotics. Transfections were performed with Lipofectamine (Invitrogen) according to the manufacturer's instructions. Per dish, 13 μg of the respective plasmid were transfected with 26 μl Lipofectamine and 39 μl Plus Reagent (Invitrogen). Three h later, the transfection reaction was stopped by addition of 6.5 ml CHO culture medium. 24 h after transfection, cell monolayers were washed once with PBS and harvested by scraping into PBS supplemented with Complete protease inhibitor (Roche). Cells were pelleted by spinning for 10 min at 4°C (300 × g), the supernatant was removed and the pellet resuspended in low salt lysis buffer ). The cell suspension underwent three freeze-thaw cycles in dry ice/ethanol. Cell debris were removed by centrifugation , and the supernatant was used for DNA binding analysis. The electrophoretic mobility shift experiments were essentially performed as described . In brieCGCTTGGTGCCAAgcatc-3'; N, 5'-aggtCTGGCTTTGGGCCAAgagccgc-3'; Nc, aggtCTCGCTTTGGGCCAAgagccgc-3'; SIS, 5'-agcttaccagaaggtcaaggtcaaatgaagctagct-3' Sequences corresponding to the NFI binding site are capitalized; mutations in the negative control oligonucleotides are shown in boldface. The sequence of one strand is shown after the fill-in reaction.Oligonucleotides used were as follows: L, 5'-gctatTTGGCTTGGTGCCAAgcatc-3'; Lc, 5'-gctatTTet al. 2003). Anti-HA  was used as primary antibody ). Horseradish peroxidase-conjugated goat anti-mouse secondary antibody  was applied at a 1:10,000 dilution in 4% milk powder in Tris-HCl-buffered saline (pH 7.3). Detection of Myc-NFI-A st and Myc-NFI-A bs expression in N2A cells was performed following the same protocol, using anti-Myc  as a primary antibody. Transfection of the respective plasmids was performed as described under "Chromatin immunoprecipitation assay". However, 6-well plates were used, with DNA amounts and volumes of media and reagents adjusted accordingly. To check for the amount of protein loaded per lane, an anti-GAPDH antibody was applied .In order to check HA-NFI-A expression by Western Blot, transfection of CHO cells was performed as described for EMSAs. However, 6-well plates were used, with DNA amounts and volumes of media and reagents adjusted accordingly. 24 h after transfection, cells were lysed by incubating in Ripa buffer  NP-40, 1 × Complete protease inhibitor (Roche)) for 1 h at 4°C. Cell debris were removed by centrifugation . The supernatant was mixed with sample buffer and boiled for 5 min. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunologically detected as described  per dish according to the manufacturer's instructions. 48 h later, ChIP analysis was essentially performed as described . For red4 mouse neuroblastoma (N2A) cells were seeded in 96-well tissue culture plates  and transfected the next day with 2.5 μl Lipofectamine 2000 (Invitrogen) per microgram of DNA. 80 ng reporter plasmid (L1-11), 60 ng of the effector plasmids (pCHBNFI-A or pCHBNFI-Am) or control CMX plasmid coding for β-galactosidase were applied per well. EGFP fluorescence generated by cotransfection of 60 ng pEGFP-C3  was determined after 48 hours with a Wallac 1420 Multilabel Counter . Luciferase activity was measured with the Bright-Glo Luciferase Assay System according to the manufacturer's instructions . Light emission was normalized to the level of EGFP fluorescence activity from the EGFP control plasmid. Transfections were generally performed in septuplicate. Average relative luciferase light units and standard deviations were calculated using the Prism-4 program .For transient transfections, 1 × 10L1 gene regulatory region, we analyzed the nucleotide sequence of this region [5GCCAA [L1 gene regulatory region also contains a previously identified DNA element recognized by Pax-6 and by additional homeodomain proteins [To ask whether NFI-A binds to the murine s region ,5,6 for  [5GCCAA , in intrL1 gene regulatory region in vitro, electrophoretic mobility shift assays (EMSAs) were performed, using both the ubiquitous "standard" isoform of NFI-A (NFI-A st) and a brain-specific isoform (NFI-A bs) [L1 gene (L), or an idealized NFI binding site  . Their uFI-A bs)    analysis. Mouse neuroblastoma (N2A) cells, which express endogenous L1 [L1 gene flanking the NFI full binding site  shows the outcome of a representative experiment: whereas binding of the brain-specific NFI-A isoform (NFI-A bs) to the L1 regulatory region could be confirmed, no signal was obtained for the ubiquitous isoform (NFI-A st). The same result was observed in two further ChIP assays, and only in one experiment, weak binding of NFI-A st was detected. Only weak PCR signals were observed for mock-transfected cells. As an additional negative control, segments of the mouse Chst8 and Chst11 genes were amplified by PCR. No signals were observed when ChIP precipitates were used as a template  to this genomic region in vivo, although the in vitro interaction of NFI-A st with the full binding site in L1 was at least as strong as the one of NFI-A bs. This difference might be caused by the different binding reaction conditions in the two assays. Alternatively, post-translational modifications could be a reason for the different behavior of NFI-A st and NFI-A bs in the two assays, in particular phosphorylation, which has been implied in regulating NFI activity by several studies [L1 gene regulatory region. Differences in protein phosphorylation or other modifications might also explain why the apparent molecular weights of NFI-A bs and NFI-A st differed slightly more than one would expect from their amino acid composition. Finally, it should be noted that eight half binding sites for NFI proteins can be found in the 2400 bp immediately upstream of the full site. NFI-A binds to such pentanucleotide sequences with a reduced affinity [ChIP analysis confirmed that the brain-specific isoform of NFI-A (NFI-A bs) binds to the regulatory region of the mouse  studies -23. Wheraffinity .L1 regulatory region influences L1 expression, we performed reporter gene assays in mouse neuroblastoma (N2A) cells. NFI-A bs caused a reduction in L1 gene activity to approx. 30% of control level. In this context, it is noteworthy that only the brain-specific isoform significantly interacted with the endogenous L1 gene regulatory region. Moreover, we could nearly abolish repression of L1 transcription by NFI-A bs by deleting NFI-A's transregulatory domain. To our knowledge, these results are the first experimental evidence for a specific role of NFI-A bs in the regulation of a neuronal gene.In order to understand how binding of NFI-A to the L1 gene regulatory region be regulated in a physiological context? Several studies suggest that NFI activity is modulated by NFI phosphorylation [How could NFI-A activity at the rylation -23. In oL1 gene as the full NFI binding site identified in our study [L1 gene is also mediated by the HPD element. This might implicate a functional interaction between NFI transcription factors and Pax-6, Barx-2, or Hoxa-1 in regulating L1 expression.Interactions with other site-specific transcription factors might also regulate transactivation/transrepression by NFI-A. NFI proteins physically interact with TTF-1 , Oct-1 , ski 2626, and Cur study  to the LL1 gene activity. A role for NFI-A in regulating L1 expression during development is plausible, as expression of both genes starts around embryonic day 9 in the developing nervous system of the mouse [et al. [In summary, our data suggest that NFI-A is a repressor of mouse he mouse ,11. In a [et al.  have pro [et al. -31. As b [et al. , furtherTS designed and performed experiments shown in Fig.


Sc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrPSc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood.Prion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein , we found that the amount of the full-length form of collapsin response mediator protein-2  decreased in the late stages of the disease, while the amount of its truncated form (56 kDa) increased to comparable levels observed for the full-length form. Detailed analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry showed that the 56-kDa form (named CRMP-2-ΔC) lacked the sequence from serineWe identified the presence of CRMP-2-ΔC in the brain of a murine model of prion disease. Of note, C-terminal truncations of CRMP-2 have been recently observed in models for neurodegenerative disorders such as ischemia, traumatic brain injury, and Wallerian degeneration. While the structural identity of CRMP-2-ΔC in those models remains unknown, the present study should provide clues to the molecular pathology of degenerating neurons in prion diseases in connection with other neurodegenerative disorders. Sc) in the central nervous system and neuronal loss and vacuolation, although these features are not evident in some cases + = 778.5)  , indicated that CRMP-2 in spot b1 was the full-length form. Conversely, the digest derived from spot a did not yield these C-terminal peptides   and 594.4 ([M+2H]2+) were derived from the peptide Val506-Ser517  was ascribed to a b-type ion of Val506-Thr514  being observed in tandem MS [a. In support of this, the values of Mrobs and pIobs of spot a were in good accord with the corresponding theoretical values for non-phosphorylated CRMP-21-517  Figure . Full-le8 Figure . Subsequ7 Figure , while t4 Figure . This b-andem MS . BecauseCompletion of the structural analysis of CRMP-2-ΔC enabled us to use the N3E antibody to evaluate the relative amounts of CRMP-2-ΔC (56 κDa) and the full-length form 61 κDa). Since the full-length CRMP-2 and CRMP-2-ΔC both contain the epitope for the N3E antibody which recognizes the amino acid sequence 142-194 of CRMP-2 , the ant κDa. SinTwo variant forms of CRMP-2, named CRMP-2A and -2B, were previously identified that had different amino terminal regions due to the alternative usage of two first coding exons -33. Ther514 and Thr555 [in vitro (0-DIV). As the transfection efficacy of the neurons in primary culture was rather low, we co-transfected a plasmid encoding green fluorescent protein (GFP) to identify the transfected cells. The cells positive for GFP-fluorescence were examined at 4-DIV for neurites longer than 10 μm, and we found that the cells transfected with CRMP-2-ΔC developed 1.2-fold more neurite tips than the cells transfected with the empty vector  which is predominantly expressed in the adult brain [4 = Thr, P3 = Pro, P1' = Ser, and P2' = Ala) was in modest agreement with the amino acid sequences preferred for cleavage by calpain [Not only in the physiological neuronal processes, the importance of CRMP-2 in the adult brain under nonphysiological conditions has also been implied recently in animal models of neurodegenerative disorders including traumatic brain injury and cerebral ischemia (see below). Here, we performed a proteomic analysis of the brains of mice infected with scrapie prion, and found a C-terminally truncated form of CRMP-2 (CRMP-2-ΔC) in the late pathological stages of the disease. To date, truncated forms of CRMP-2 have been identified in cultured neurons exposed to N-methyl-D-aspartic acid ,35 or delt brain ,33. We s calpain ,41. In t calpain . It shou calpain . We have calpain .522, Thr509, Thr514 and Ser 518 by Cdk5 and GSK-3β is essential for the regulation of CRMP-2 activity [3PO4 (Δm/z = -98) in the MS analysis of CRMP-2-ΔC (data not shown), strongly indicated that it was not phosphorylated. Thus, the augmentation in the development of neurite branch tips of cultured neurons by the overexpression of CRMP-2-ΔC may be explained by the lack of the regulatory phosphorylation sites. Alternatively, it is recognized that CRMPs are assembled into homo- or hetero-tetramers. Interestingly, while the structure of the recombinant CRMP-213-490  was indeed in a tetrameric form, as determined using crystallographic analysis, it behaved like a trimer when analyzed using gel filtration [13-490 [Lines of evidence to date indicate that the sequential phosphorylation of CRMP-2 at Seractivity ,29. Thesltration . This wa [13-490 . If so,  [13-490 . ConsideTo explore the molecular neuropathology of prion diseases, we conducted a proteomic analysis of a murine model of prion diseases. In addition to the quantitative changes of GFAP, glutathione S-transferase-μ1 and peroxiredoxins during the progression of the disease in the brain of ICR and C57BL/6J mice, we identified a unique truncation of CRMP-2. Detailing the relevance of CRMP-2-ΔC to the morphological abnormalities of degenerating axon terminals/dendrites observed in models of scrapie  awaits fg for 1 h at 4°C with a TLA-55 rotor  to obtain the supernatant (soluble fraction) and the pellet (membranous fraction). Protein concentrations were determined using a BCA protein assay kit .Female C57BL/6J and ICR mice (6 weeks old) were purchased from Charles River Laboratories  and CLEA Inc. , respectively. The mice were inoculated with 25 μL of a 0.25% (w/v) brain homogenate in phosphate-buffered saline  prepared from terminally ill ICR mice infected with mouse-adapted scrapie prion (Obihiro-I strain) . As mockThe samples were prepared by adjusting the soluble fraction of the brain homogenate to a total of 400 μg protein/mL in 9 M Urea, 4% CHAPS, 65 mM DTT, and 0.5% IPG buffer 3-10 . Immobiline DryStrips for isoelectric focusing  were rehydrated at 20°C overnight by soaking in 250 μL of the sample solution , then IEF was carried out using a linear gradient from 0-1000 V in 2 h, followed by 8000 V for 6 h. After IEF, the strips were equilibrated with 50 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS, 20 mM DTT, 30% glycerol, and 0.03% bromophenol blue for 15 min, and overlaid on 12.5% polyacrylamide slab gels for SDS-PAGE. Proteins were stained with CBB R-250 or SYPRO Ruby , and gel images were captured using a LAS-1000 plus lumino-image analyzer . Software PDQuest, version 7.3  was used for the image analysis. At least one duplicate set of mice was analyzed for each specified dai. Mr and pI were calibrated using the spots of the following proteins: calmodulin , γ-enolase , serum albumin , peroxiredoxin 6 , carbonic anhydorase II , and aconitase . The signal intensity of each spot was normalized to that of β-actin for statistical analysis. In the dephosphorylation analysis, the samples were incubated with λ-PPase  at 30°C for 15 min prior to 2-DE.480-528) and N3E  [2 . The ECL Plus reagent  and a LAS-3000mini lumino-image analyzer (Fuji Photo Film) were used for detection. Signal intensity was quantified using Image Gauge software (Fuji Photo Film). For the analysis of PrPSc, the membranous fraction  in 30 μL of 0.5% sarkosyl, 25 mM Tris-HCl, and 50 mM NaCl (pH 7.5) was incubated with PK  at 37°C for 120 min. The digestion was stopped by the addition of 4-volumes of 10 mM phenylmethylsulfonyl fluoride in methanol, and the mixture was centrifuged at 20,000 × g for 30 min at 4°C to recover PrPSc as a precipitate. The anti-PrP monoclonal antibody 44B-1 [The anti-CRMP-2 antibodies C4G   were pur2O/acetonitrile/formic acid = 98/2/0.1 (v/v/v) and (B) H2O/acetonitrile/formic acid = 10/90/0.1 (v/v/v), with a gradient from A/B = 95/5 (v/v) to 45/55 (v/v) at a flow rate of 400 nL/min. The eluate was introduced into an LCQ Deca XP spectrometer  using a nano-ESI interface  and a MonoSpray FS monolithic emitter (GL Sciences). The data were collected at an interval of three tandem spectra per spectrum in the data-dependent-scan mode, and analyzed using BioWorks software  and by manual inspection. The theoretical Mr and pI values were calculated using Compute pI/MW [Pieces of the 2-DE gel were rinsed with 100 mM ammonium bicarbonate, and incubated with 10 mM DTT at 56°C for 45 min. Then, protein in the gel was reacted with 55 mM iodoacetamide at room temperature for 45 min, and subjected to in-gel digestion  with tryte pI/MW  and Scante pI/MW  softwaresecure reagent (Applied Biosystems) until use. Electrophoresis was carried out with 1 μg of RNA using 1.0% agarose-formaldehyde gel in 40 mM MOPS-NaOH, 10 mM sodium acetate, and 1 mM EDTA (pH 7.0). After the transfer of the RNA to a positively charged-nylon membrane (Roche Applied Science), the membrane was incubated with digoxigenin-labeled antisense RNA probes for CRMP-2 (complementary to the open reading frame (ORF) from +340 to +1551; corresponding to Thr114-Ser517) or GAPDH (the ORF of from +330 to +878) at 68°C overnight in Ambion ULTRAhyb hybridization buffer (Applied Biosystems). The bound probes were detected using an alkaline phosphatase-labeled anti-digoxigenin antibody (Roche Applied Science) and CDP-Star chemiluminescence reagent , with X-ray films (Fuji Photo Film) and a LAS-3000mini lumino-image analyzer (Fuji Photo Film). Signal intensity was quantified using Image Gauge software (Fuji Photo Film).RNA was prepared from the whole brains of C57BL/6J mice sacrificed at 160 dai using the SV Total RNA Isolation System (Promega), and stored at -80°C in RNAGGAATTCGAGATGTCTTATCAGGGGAAGAAAAATATTCCA-3' and 5'- ATAAGAATGCGGCCGCTTTAGCCCAGGCTGGTGATGTTGG-3'	(the	underlined sequences are for the generation of EcoRI and NotI sites). CRMP-2-ΔC cDNA (CRMP-21-517) was generated using the reverse primer 5'- ATAAGAATGCGGCCGCTTATGAGGCTGGAGTCACC-3' (the underlined sequence is for the introduction of a NotI site). The cDNAs were ligated into the pCIneo mammalian expression vector (Promega) between the EcoRI and NotI sites. Site-directed mutagenesis was carried out using the following primers: 5'- GTGACGCCCAAGACGGTGGCGCCAGCCTCATCAGCTAAG-3' and 5'- CTTAGCTGATGAGGCTGGCGCCACCGTCTTGGGCGTCAC-3' for T514A; 5'- GTGACGCCCAAGACGGTGGATCCAGCCTCATCAGCTAAG-3' and 5'- CTTAGCTGATGAGGCTGGATCCACCGTCTTGGGCGTCAC-3' for T514D; 5'- CATTCCCCGCCGCACCGCCCAGCGCATCGTGG-3' and 5'- CCACGATGCGCTGGGCGGTGCGGCGGGGAATG-3' for T555A; and 5'- CATTCCCCGCCGCACTGATCAGCGCATCGTGG-3' and 5'- CCACGATGCGCTGATCAGTGCGGCGGGGAATG-3' for T555D. The constructs were verified using DNA sequencing.Full-length cDNA of mouse CRMP-2-wt was obtained from a C57BL/6J mouse brain cDNA library (Invitrogen) by PCR using the following primers: 5'- Cerebral cortical neurons from E15 ICR mice  were plated on poly-L-lysine-coated plastic dishes , and maintained in Neurobasal medium (Invitrogen-GIBCO) with B27 (Invitrogen-GIBCO) and 0.5 mM glutamine according to the manufacturer's instructions. Arabinocytidine (2.5 μM) was added to the medium to suppress the growth of glia. The pCIneo vectors encoding CRMP-2 or its mutants (1 μg) were mixed with pEGFP-N1 encoding GFP , and the mixture was introduced into cells using the NeuroPORTER transfection reagent  at the time of plating (0-DIV). The neurons were fixed at 4-DIV using BD Cytofix  for 15 min at room temperature, and observed under a BIOZERO BZ-8000 microscope  with a software BZ analyzer (Keyence) for counting the number of neurite tips and measuring the length of neurites.CRMP-2: collapsin response mediator protein-2; 2-DE: two-dimensional electrophoresis; dai: days after inoculation; DIV: day of culture in vitro; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; Glu-C: endoproteinase Glu-C; IEF: isoelectric focusing; λ-PPase: lambda phosphatase; LC-ESI-MS/MS: liquid chromatography-electrospray ionization-tandem mass spectrometry; MALDI: matrix-assisted laser desorption/ionization; Mr: molecular mass; obsMr: observed Mr; MS: mass spectrometry; ORF: open reading frame; PBS: phosphate-buffered saline; obspI: observed pI; PK: proteinase K; PrP: prion protein; CPrP: cellular prion protein; ScPrP: disease-associated prion proteinThe authors declare that they have no competing interests.FSO performed the experiments, data analysis and protein identification, and wrote the manuscript. YY took care of the mice, collected tissue samples, provided experimental devices, and took part in the experimental design. HH took care of the mice and collected tissue samples. MT helped perform the cell culture experiments. MN and KH took part in the experimental design. KH performed a Northern blotting analysis, participated in the experimental design, data analysis, and wrote the manuscript. All authors read and approved the final manuscript.CRMP-2-ΔC in the brains of C57BL/6J mice in the late stages of the disease. A supplementary data of Western blot analysis for detection of CRMP-2-ΔC in additional individuals of prion-infected and mock control mice.Click here for fileRelative levels of CRMP-2 mRNA determined by quantitative RT-PCR. The mRNA levels of CRMP-2 did not show significant difference between the prion-infected and mock control mice in quantitative RT-PCR analysis.Click here for file