Caenorhabditis elegans vulval development leads to the generation of seven distinct vulval cell types , each with its own unique gene expression profile. The mechanisms that establish the precise spatial patterning of these mature cell types are largely unknown. Dissection of the gene regulatory networks involved in vulval patterning and differentiation would help us understand how cells generate a spatially defined pattern of cell fates during organogenesis. We disrupted the activity of 508 transcription factors via RNAi and assayed the expression of ceh-2, a marker for vulB fate during the L4 stage. From this screen, we identified the tailless ortholog nhr-67 as a novel regulator of gene expression in multiple vulval cell types. We find that one way in which nhr-67 maintains cell identity is by restricting inappropriate cell fusion events in specific vulval cells, namely vulE and vulF. nhr-67 exhibits a dynamic expression pattern in the vulval cells and interacts with three other transcriptional regulators cog-1 (Nkx6.1/6.2), lin-11 (LIM), and egl-38 (Pax2/5/8) to generate the composite expression patterns of their downstream targets. We provide evidence that egl-38 regulates gene expression in vulB1, vulC, vulD, vulE, as well as vulF cells. We demonstrate that the pairwise interactions between these regulatory genes are complex and vary among the seven cell types. We also discovered a striking regulatory circuit that affects a subset of the vulval lineages: cog-1 and nhr-67 inhibit both one another and themselves. We postulate that the differential levels and combinatorial patterns of lin-11, cog-1, and nhr-67 expression are a part of a regulatory code for the mature vulval cell types.Regulation of spatio-temporal gene expression in diverse cell and tissue types is a critical aspect of development. Progression through  Caenorhabditis elegans vulva arise. This organ is one of the most intensively studied, and while the signaling network that initiates vulval development and sets the gross pattern of cell differentiation is well understood, the network of transcription factors that specifies the final cell fates is not understood. Here, we identify nhr-67, a new transcription factor that regulates patterning of cell fates in this organ. Transcription factors do not necessarily act alone, and we explore how NHR-67 works with three other regulatory factors (each with human homologs) to specify the different properties of the vulval cells. We also demonstrate that the interconnections of these transcription factors differ between these seven diverse cell types, which may partially account for how these cells acquire a certain pattern of cell fates.During development, in which the single-celled egg generates a whole organism, cells become different from each other and form patterns of types of cells. It is these spatially defined fate patterns that underlie the formation of complex organs. Regulatory molecules called transcription factors influence the fate patterns that cells adopt. Understanding the role of these transcription factors and their interactions with other genes could tell us how cells establish a certain pattern of cell fates. This study focuses on studying how the seven cell types of the  Alleles used in this study: LGI, lin-11(n389); LGII, cog-1(sy275), eff-1(hy21); LGIII, unc-119(ed4); LGIV, unc-31(e169), egl-38(n578), dpy-4(e1166sd), dpy-20(e1282); LGV, him-5(e1490). A complete list of strains is included in -1::GFP] , syIs107-3::GFP] , ayIs4 [17::GFP] , guEx64 26::GFP] , syIs63 -1::GFP] , syIs80 syEx756 was generated by injecting the pNP10 construct [unc-119(ed4); him-5 background using unc-119(+) [http://www.stratagene.com) as coinjection markers.Transgenic lines were generated using standard microinjection protocol that produces high-copy number extrachromosomal arrays . syEx756onstruct  into uncc-119(+)  and pBSKceh-2::YFP transgene. RNAi feeding protocol is similar to that previously described [Escherichia coli strain expressing double-stranded RNA at 20 °C. Animals were scored after 36 h (during the L4 stage) using Nomarski microscopy. We resorted to nhr-67 RNAi feeding for the rest of this study since the nhr-67 deletion allele (ok631) results in L1 lethality and/or arrest . All subsequent nhr-67 RNAi feeding experiments were done as described above. nhr-67 RNAi feeding experiments that entailed the restriction of cell fusion  were conducted at 25 °C.A reverse genetics screen was conducted against 508 transcription factors  from theescribed . Embryosnhr-67::Δpes-10::GFP reporter gene constructs: The pPD97–78 vector, which includes the Δpes-10 basal promoter driving GFP and the unc-54 3′ UTR (gift from Fire lab), was used as a template to generate 2-kb Δpes-10::GFP products. The primers used for amplification are 5′-GCTTGCATGCCTGCAGGCCTTG-3′ and 5′-AAGGGCCCGTACGGCCGACTAGTAGG-3′. All nhr-67 gene fragments were amplified from the C08F8 cosmid and were stitched together with the Δpes-10::GFP fragment via PCR fusion [Δpes-10::GFP. The primers used to amplify this template are 5′-CTGCTCAAAACTTTTGCTCC-3′ (forward) and 5′-CAAGGCCTGCAGGCATGCAAGCTTAAAGAACTACTGTAGTTTTTG-3′ (reverse). Construct (2) spans from the fourth intron to the 3′ noncoding region fused to minimal Δpes-10::GFP. This product was generated using the forward primer 5′-GTTCGATCATGGATCCTCTCC-3′ and the same reverse primer as construct (1). Construct (3) is an nhr-67p::GFP reporter that contains 1 kb of the native promoter stitched in-frame with a 700-bp coding fragment of GFP . The resulting 1.7-kb gene product was subsequently fused to 4.5 kb of nhr-67 regulatory sequences (that span from the fourth intron to the 3′ noncoding region) via PCR. Construct (4) contains 6-kb sequence upstream of the predicted first ATG of nhr-67, appended to minimal Δpes-10::GFP. The following primers were used to amplify this product: 5′-GAACCCGGCGACGTTACGGGGCTTC-3′ and 5′-CAAGGCCTGCAGGCATGCAAGCCATCTGTGAAACCGCAGTCATCAT-3′.R fusion  and wereunc-119(ed4); him-5 worms using unc-119(+) [lin-11(n389); syEx749 doubles were constructed by injecting the 8-kb nhr-67::Δpes-10::GFP construct into lin-11(n389); unc-119(ed4); him-5 background using unc-119(+) as a rescue marker.Reporter constructs were injected into c-119(+)  and pBSKFigure S1nhr-67 RNAi–treated hermaphrodite.A mid-sagittal optical view of an adult (5.3 MB TIF)Click here for additional data file.Figure S2nhr-67 is expressed in the AC in hermaphrodites and (B) in the linker cell in males.(A and B) Nomarski (left), fluorescence (center), and overlaid (right). (A) (3.8 MB TIF)Click here for additional data file.Table S1(51 KB XLS)Click here for additional data file.Table S2(22 KB XLS)Click here for additional data file.ajm-1:WBGene00000100 ; cdh-3:WBGene00000395 (NM_066286); ceh-2:WBGene00000429 (NM_059345); cog-1:WBGene00000584 (NM_182115); eff-1:WBGene00001159 (NM_001026819); egl-17:WBGene00001185( NM_075706); egl-26:WBGene00001193 (NM_061251); egl-38:WBGene00001204 (NM_069435); lin-3:WBGene00002992 ; lin-11:WBGene00003000 (NM_060295); nhr-67: WBGene00003657 (NM_069693); pax-2:WBGene00003938 (NM_068112); unc-53: WBGene00006788 ; and zmp-1:WBGene00006987 (NM_171138).The WormBase Gene IDs (www.wormbase.org) as well as the Refseq accession numbers (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) for the genes described in this study are


By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca++/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.M-phase Promoting Factor  is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the  Cdc20) Cdc20 activation in vitro cell cycle system derived from Xenopus eggs.Rapid MPF activation is granted by an activation loop in which the cdc25C phosphatase removes inhibitory phosphorylations of cdk1 at thr-14 and tyr-15, while MPF stimulates cdc25C activity and lowers activity of wee1, the cdk1 tyr-15 kinase 35S]labelled methionine, were taken at 2 min intervals , refractory to inhibitory phosphorylation, enter mitosis with slower kinetics than control extracts and are delayed in exiting mitosis Cdc20 is required for efficient ubiquitin ligase activity Cdc20 activation in vitro translated wild type human Cdc20 and a mutant Cdc20 version, non-phosphorylatable at 7 cdk1 consensus sites (Cdc207A) Cdc20 activation We asked why interruption of the MPF activation loop was required for timely activation of cyclin degradation. In this system, cyclin degradation tightly relies on the ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) in association with its co-activator Cdc20 and assembly of APC/Cin vitro kinase assays, wee1-ser-549 phosphorylation did not increase wee1 activity and wee1 isolated from interphase was slightly more active than that isolated from M-phase By our analysis it cannot be excluded that the pathways leading to MPF inactivation via inhibitory phosphorylation take the upper hand only after initial degradation of small cyclin B amounts. In addition, the fact that cyclin degradation is activated when cdk1 activity is sustained by non-degradable cyclin B Under our experimental conditions, degradation of full-length cyclin initiated 40–45 min from addition of non-degradable cyclin B . To deteFurther investigation will be required to establish the role for phosphorylations Cdc20 activation. MPF degradation further weakens the MPF activation loop promoting MPF inhibitory phosphorylation. Consequent additional MPF activity loss further helps APC/CCdc20 activation. We cannot exclude that degradation of a small pool of cyclin initiates the loop. However, the observation that MPF binds to wee1 in M-phase even in the absence of cyclin degradation . Histone H1 kinase assays were performed as described Xenopus egg extracts were prepared essentially as described in vitro translated in the presence of [35S]labelled methionine and cysteine  using a T7-TNT coupled system (Promega). Production and purification of GST-human cyclin B1-cdk1wt and GST-human cyclin B1-cdk1AF kinases frombaculovirus-infected Sf9 cells was performed as described 2 addition. Equal amounts to the endogenous level of cyclin B1-cdk1 activity were added of recombinant enzymes.Cdc20 wt and 7A plasmids were a generous gift of A. Hershko. Proteins were Figure S1Cdc25C and cdk1 phosphorylation changes are induced upon CaCl2 addition to CSF-arrested extracts and prevented by CaMKII inhibition. Cdc25C and cdc25C-phospho-ser-287  and cdk1-phospho-tyr15 contents from portions of a CSF-arrested extract, pre-incubated for 20 min with buffer, as control, or with the CaMKII inhbitor peptide CaMKII Ntide (0.5 mM), taken at the indicated time points after CaCl2 addition.(0.16 MB TIF)Click here for additional data file.Figure S2Cyclin B and cyclin A stability in cyclin B1-cdk1AF-treated CSF-arrested extracts. Portions of a CSF-arrested extract were pre-incubated for 20 min at 23 degrees C with buffer or recombinant cyclin B1-cdk1AF. Then, CaCl2 was added and samples taken at the indicated time points. Upper panels, Cyclin B1 and cyclin A were detected by immunoblot. Lower left panel, densitometric quantisation  of the cyclin A content in control  and cyclin B1-AF-treated  extrac portions. Lower right panel, densitometric quantisation  of the cyclin B1 content in control  and cyclin B1-AF-treated  extract portions. Error bars refer to variability within three independent experiments.(0.10 MB TIF)Click here for additional data file.Figure S3Nuclear morphology in cyclin B1-cdk1AF-treated CSF-arrested extracts. Demembranated sperm nuclei (300/µl extract) were allowed to undergo DNA replication by 120 min incubation with interphase extracts. Nuclei were then moved into metaphase by incubating one part of the interphase extract (+nuclei) with two parts of a CSF-arrested extract for 50 min. After incubation, portions were treated either with buffer (control) or cyclin B1-AF and further incubated at 23 degrees C for 20 min. Samples were fixed at the indicated time points after CaCl2 addition and visualised by Hoechst staining. A portion of the cyclin B1-cdk1AF-treated extract also received roscovitine (2 µM in DMSO) 1 min after CaCl2 addition (Cyclin B1-AF+Rosco). DMSO addition alone did not affect morphological changes in cyclin B1-AF-treated extracts (not shown).(0.08 MB TIF)Click here for additional data file.Figure S4Cdc20-Cdc27 interaction in CSF-arrested extracts. Cdc27 was immunoprecipitated (Cdc27 Ip) and bound Cdc20 visualised by immunoblot to from untreated CSF-arrested samples, pre-incubated for 20 min at 23 degrees C before CaCl2 addition to maintain similar conditions to the experiments with recombinant cyclin B1-cdk1 complexes. Samples were taken at the indicated time points after CaCl2 addition. Mock precipitations (MkIp) were performed with non-immune Ig from time 0 samples(0.05 MB TIF)Click here for additional data file.


One difference between these settings is the availability of immunoglobulin Fc receptors. The question therefore arises whether a herpesvirus exposed to apparently neutralizing antibody can still infect Fc receptorImmune sera blocked murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts, but failed to block and even enhanced its infection of macrophages and dendritic cells. Viral glycoprotein-specific monoclonal antibodies also enhanced infection. MHV-68 appeared to be predominantly latent in macrophages regardless of whether Fc receptors were engaged, but the infection was not abortive and new virus production soon overwhelmed infected cultures. Lytically infected macrophages down-regulated MHC class I-restricted antigen presentation, endocytosis and their response to LPS.IgG Fc receptors limit the neutralization of gamma-herpesviruses such as MHV-68. Thus, they differ fundamentally from epidemic viruses, whose replication and transmission are blocked by established immunity. The chief defence against epidemic viruses is neutralizing antibody in vitro, making related gamma-herpesviruses an important source of information. One of the most experimentally accessible is murine gamma-herpesvirus-68 (MHV-68), a natural, B cell-tropic parasite of yellow-necked mice + T cells + T cells Our understanding of gamma-herpesvirus neutralization has been limited by the narrow species tropisms of Epstein-Barr virus (EBV) and the Kaposi's Sarcoma-associated Herpesvirus (KSHV). Both fail to reproduce their basic pathogenesis in experimental animals and propagate poorly Our aim with MHV-68 is to understand why established antibody responses do not prevent gamma-herpesvirus transmission. Standard MHV-68 pathogenesis studies have a limited capacity to answer this question as they focus on acute infection; by the time MHV-68-specific antibody is made, virus titers are low. And no MHV-68 transmission model yet exists. Immune sera protect antibody-deficient mice against disease in vivo phenomena but to identify the mechanisms behind them. Fortunately, MHV-68 offers several advantages for in vitro analysis: it is readily propagated and modified, cells with relevant deficiencies can be derived from knockout mice, and monoclonal antibodies can be generated from virus carriers. Our approach has therefore been first to understand as much as possible about neutralization in vitro. Immune sera appear to block MHV-68 infection of fibroblasts + and FcR− cells. Our data suggest that FcRs allow otherwise neutralized virions to remain infectious and are therefore likely to compromise infection control.It is essential with MHV-68 not only to record + cells, we incubated MHV-68 virions with sera from immune mice and then added the antibody-coated virions to either BHK-21 fibroblasts or RAW264.7 macrophages (+) expresses eGFP from a human cytomegalovirus (HCMV) IE1 promoter, so eGFP expression provides a convenient marker of infection + MHV-68 infection of wild-type, bone marrow-derived macrophages, but inhibited the infection of IgG FcR-deficient macrophages .Our second question was whether BAC eGFPomponent . Perinuc+ cells were not necessarily lytic, and neither lytic nor latent cells were necessarily eGFP+. In contrast, the expression of eGFP-tagged gM from the endogenous gM promoter correlated closely with ORF65 expression  infection phenotype. Flow cytometry established that approximately 50% of lytically infected RAW264.7 cells had down-regulated cell surface MHC class I expression . Immune sera were collected at 3–6 months post-infection, by which time the viral load and serum antibody have reached steady state +F4/80+) harvested after 7–14 d. Peritoneal macrophages were obtained by peritoneal lavage of naive mice with Dulbecco's modified Eagle medium plus 5% fetal calf serum. Cells not adherent to tissue culture plates  were discarded. The adherent cells were 80–85% CD11b+CD11c−F4/80+CD19−. In flow cytometry-based assays, FSC/SSC gating increased this to >95%. Dendritic cells were grown from bone marrow progenitors in RPMI with 10% fetal calf serum, 50 µM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin and 7.5ng/ml GM-CSF. Bone marrow cells were first put on tissue culture plates  and the adherent (macrophage-rich) cells discarded. The culture medium was changed every 2d. After 3d, non-adherent (granulocyte-rich) cells were discarded. After 7d, the non-adherent cells (90% CD11c+MHC class II+Gr1−) were harvested. BHK-21 cells, RAW264.7 cells and 293T cells were grown in Dulbecco's modified Eagle medium with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum. Murine embryo fibroblasts were cultured in the same medium plus 50 µM 2-mercaptoethanol.BALB/c and C57BL/6J mice were purchased from Harlan U.K. Ltd. , housed in the Cambridge University Department of Pathology and infected intranasally with 2×10+ virus), the BAC cassette was removed by passaging the virus through NIH-3T3-CRE cells g, 3 min). Virions were then concentrated from supernatants by high speed centrifugation . Virus titers were determined by plaque assay on BHK-21 cells Infectious MHV-68 was derived from a genomic BAC, which contains eGFP with an HCMV IE-1 promoter as part of a loxP-flanked BAC cassette + viruses were washed in PBS and analysed directly for green fluorescence. To assay endocytosis, macrophages were incubated with Alexa 647-conjugated 10kDa dextran (Invitrogen) at 100 µg/ml or 0.2 µm Crimson (625/645) Fluospheres (Invitrogen) at 2×109 beads/ml, washed in PBS and analysed for red fluorescence. For surface staining, cells were incubated  with MHV-68 glycoprotein-specific mAbs followed by fluorescein-conjugated rabbit anti-mouse IgG pAb (Dako Cytomation) or Alexa 633-conjugated or Alexa 488-conjugated goat anti-mouse IgG pAb (Invitrogen). Fluorescent conjugates of mAbs against CD86, CD11c, CD11b, F4/80, MHC class II or MHC class I were from BD Biosciences. For intracellular cytokine staining, cells were fixed in 1% paraformaldehyde (30 min), permeabilized with 0.1% saponin, and incubated with phycoerythrin-conjugated rat anti-mouse IL-6 or anti-mouse TNF-α mAbs (BD Biociences). For ORF65 staining, cells were permeabilized in 0.1% Tween-20 after fixation and incubated with mAb 12B8 followed by Alexa 633-conjugated goat anti-mouse IgG pAb. Cells were washed with PBS after antibody staining and analysed on a FACS Calibur using Cellquest software (BD Biosciences).Cells exposed to eGFPAdherent cells were washed in PBS, fixed in 2% paraformaldehyde and permeabilized with 0.1% Tween-20. EGFP fluorescence was visualized directly. The MHV-68 ORF65 capsid component was stained with mAb 12B8 MHV-68 glycoprotein-specific hybridomas were all derived from MHV-68-infected mice by fusing spleen cells with NS0 cells DNA was extracted from infected cells and a portion of the MK3 ORF (genomic co-ordinates 24832-25071) amplified by real-time PCR b) mice were infected with wild-type or K3-deficient b-restricted MHV-68 epitope from ORF6 b [59]. After a further 18h, the cells were washed ×1 in PBS and lysed in PBS/5 mM MgCl2/1% NP-40/0.15 µM chlorophenol-red-beta-D-galactoside (Merck Biosciences) to assay beta-galactosidase activity. The absorbance at 595nm was read on a Biorad Benchmark Microplate Reader.Peritoneal macrophages from C57BL/6 (H2


Malignant carcinoid tumours, islet cell tumours and medullary carcinomas of the thyroid are tumours with similar clinical features. In patients with unresectable or metastatic tumours leukocyte interferon (IFN) and recombinant human (rh) IFN have demonstrated efficacy. Twenty-four evaluable patients with progressive tumours were treated with 2.5 megaunits rh IFN alpha-2b, administered once daily subcutaneously, for a median duration of 7 months (range 0.5-37+). Two carcinoid patients demonstrated a response in tumour size, 80% showed stable disease (SD). Sixty percent of the carcinoid patients with elevated urinary 5-hydroxyindoleacetic (5-HIAA) levels reached a biochemical partial response of the urinary 5-HIAA levels (median duration 13.5 months). In the patients with an islet cell or medullary tumour and an elevated tumour marker, the marker did not further increase. Of the 12 carcinoid patients evaluable for a symptomatic response, ten (83%) experienced a relieve of symptoms. IFN alpha-2b dose reduction or discontinuation due to toxicity was necessary in three and ten patients, respectively. No neutralising IFN alpha-2b antibodies developed despite prolonged treatment. In conclusion, IFN alpha-2b had a beneficial effect in patients with progressive tumours, while long-term IFN alpha-2b treatment did not augment neutralising antibodies. In view of the IFN alpha-2b-related toxicity, administration of IFN alpha-2b on alternating days may be preferable.


Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.11–20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8+ T cells in 5/5 healthy donors.In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E7in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response  Cervical cancer contributes to approximately 12% of the global cancer burden in women and represents the second most frequent gynecological malignancy in the world . In a moOne of the major risk factors for the development of cervical cancer is infection with human papillomavirus (HPV). More than 20 oncogenic HPV genotypes have been characterized, while HPV type 16 (HPV-16) and type 18 (HPV-18) are the most prevalent in cervical cancer  and is rStimulation of the immune system against specific tumor antigens might become a suitable secondary (or even primary) therapy to treat cancer. Because tumor cells do not efficiently function as antigen-presenting cells (APC) for the activation of CTL, a vigorous immune response is generally absent or defective. To overcome this problem, transfer of tumor antigens from tumor cells to professional APC, like DC, might be a valuable strategy .DC are the most efficient antigen-capturing and -presenting cells of the immune system and are potent inducers of (primary) immune responses directed against tumors and viral antigens . In theiin vitro cultured mature DC loaded with an HLA-A*0201-restricted HPV-16 E7 peptide for the in vitro activation of antigen-specific IFN-γ-producing CD8+ T cells. After several rounds of in vitro stimulation of CD8+ T cells using peptide-pulsed mature DC, the presence of HPV-16 E7 peptide-specific T cells was monitored using an IFN-γ enzyme-linked immunospot (ELISPOT) assay.In this study, we used Mature DC were differentiated starting from adherent monocytes as described in the Methods section . Culture+ T cells in a MHC class I-dependent way. For this, E7 peptide-pulsed mature DC (2.104 cells) as well as non-pulsed mature DC (2.104 cells) were co-cultured with an HLA-A*0201+ HPV-16 E7 YMLDLQPETT peptide-specific T cell clone (2.105 cells) for 6 hours in 96-round bottom wells. As showed by the production of IFN-γ by the T cell clone, E7-peptide-pulsed mature DC were able to stimulate these antigen-specific T cells  with HPV-16 E7 peptide-pulsed mature DC, we were not able to detect an HPV-16 E7-specific immune response (data not shown). We therefore added to the protocol 4 additional weekly stimulations with HPV-16 E7-peptide-pulsed mature DC and implemented an IFN-γ ELISPOT technique for detection of antigen-specific IFN-γ-secreting T cells.Previously, we reported a DC-based T cell activation protocol for influenza matrix protein M1 peptide-specific T cells . We want2 [3 HPV-16 E7-peptide-pulsed DC were cocultured together with 15.104 autologous CD8+ T cells, isolated using magnetic bead selection from cryopreserved autologous PBMC  in response to HPV-16 E7-peptide-pulsed target cells as compared to control targets  tested per blood donor, a significant difference was detected between the number of spot-forming CD88 tested Adequate immune monitoring techniques are of utmost importance to validate phase I/II clinical trials designed to test T cell-based vaccines for HPV-related malignancies. Such endpoints include the measurement of HPV-specific CTL- and T helper-mediated immune responses. Routinely used techniques such as ELISA and RT-PCR, do not provide information regarding the frequencies of cytokine-producing cells, in contrast to sensitive methods such as ELISPOT.5 cells [in vitro stimulations with autologous antigen-loaded mature DC [+ T cells. Also, we excluded aspecific IFN-γ production by CD4+ T helper cells or NK cells by subtracting control values for our estimation of effector CD8+ T cell frequencies.In this study, we show efficient detection of HPV-16 E7 peptide-specific IFN-γ-producing autologous T cells after multiple stimulations with HPV-16 E7-peptide-pulsed mature DC using an IFN-γ ELISPOT assay  . Cells w+ D4 T cell clone recognizing the immunodominant HLA-A*0201-restricted HPV-16 E7 epitope (YMLDLQPETT) was a kind gift of Dr. S. Youde . Cloned T cells (1.106) were expanded to large numbers in tissue culture flasks containing 50 mL IMDM supplemented with 10% human (h)AB serum  with 20.106 fresh irradiated (30 Gy) allogeneic peripheral blood mononuclear cells (PBMC) from 2 donors, phytohemagglutin A (PHA)  and interleukin (IL)-2 . Fresh medium and IL-2 were added on day 5. On day 7, the T cells were transferred to a 24-well plate at 2.106 cells/well and cultured in the presence of 100 U/mL IL-2 for up to 14 days.The CD8Peripheral blood mononuclear cells (PBMC) were obtained from hemochromatosis patients after informed consent or from buffy coats provided by the Antwerp Blood Transfusion Center (Red Cross). Mononuclear cells were isolated by Ficoll-Hypaque gradient separation . Monocytes were directly isolated and used for DC culture, as described below.et al. [-7 M prostaglandin E2 (PGE2) , as previously described by Jonuleit et al [Immature monocyte-derived DC were cultured from monocytes as described by Romani et al.  with minit et al . Mature it et al . BrieflyScreening of blood donors for HLA-A*0201-positivity was performed on whole blood by incubation with the supernatant of the BB7-2 hybridoma , followed by staining with fluorescein (FITC)-conjugated rabbit anti-mouse immunoglobulins . For phenotyping of cultured DC and T cell populations, the following murine anti-human monoclonal antibodies were used for direct immunofluorescence staining: CD80-FITC, CD83-phycoerythrin (PE) , CD86-PE, CD3-peridinin chlorophyll-a protein (PerCP), CD14-PE, CD19-FITC, HLA-DR-PE, CD4-FITC, CD16-FITC, CD56-FITC, CD8-PE and CD8β-PE. Non-reactive isotype-matched antibodies were used as controls. All antibodies were purchased from Becton Dickinson , unless stated otherwise. Cells were stained with antibody for 15 min at 4°C, washed and surface labelling of living cells was analyzed by flow cytometry on a FACScan (Becton Dickinson).6 cells/mL) for 2 h at room temperature in 5-mL polystyrene tubes with 20 μg/mL peptide in serum-free IMDM supplemented with 2.5 μg/mL β2-microglobulin (Sigma). Peptide-pulsed DC and K562-HLA-A*0201 cells were used, respectively, as stimulators for T cells in DC/T cell co-cultures and as antigen-specific stimulator cells in an IFN-γ ELISPOT assay.An HPV-16 HLA-A*0201-restricted E7 protein-specific peptide  11–20, YMLDLQPETT) was used for activation or for detection of HPV-16 E7 peptide-specific T cells, when pulsed on DC or on K562-HLA-A*0201 cells respectively. The peptides (>95% pure) were purchased from Eurogentec  and were dissolved in 100% DMSO to 10 mg/mL, further diluted to 1 mg/mL in serum-free IMDM and stored in aliquots at -80°C. The peptides were used at a final concentration of 20 μg/mL. DC or K562-HLA-A*0201 cells were washed once with IMDM and subsequently incubated  according to manufacturer's instructions. Routinely, 10–20.106 CD8+ T cells were obtained starting from 100.106 cryopreserved non-adherent cells with purity levels ≥ 85%.For DC/T cell co-culture experiments, frozen non-adherent PBMC were thawed and CD8+ T cells. Multiple co-cultures of 15.103 peptide-pulsed DC with 15.104 purified CD8+ T cells were initiated in AIM-V medium supplemented with 5% hAB serum in 96-round bottom wells . After 7 days of co-culture, T cells were restimulated with 15.103 cryopreserved peptide-pulsed mature DC. At this point, additional cytokines were added to the co-cultures: 5 ng/mL IL-7 (Biosource) and 10 U/mL IL-2 (Biosource). Restimulation of cultured T cells was done weekly. After 5 stimulations, T cells were analyzed for antigen specificity in an IFN-γ ELISPOT assay.HPV-16 E7 peptide-pulsed mature monocyte-derived DC were used for stimulation of autologous CD85 cells) were co-incubated with stimulator cells (2.104 cells) in 96-round bottom plates for 6 hours at 37°C in a total volume of 200 μL IMDM supplemented with 5% hAB serum. Triplicate supernatant samples from these co-cultures were tested for IFN-γ secretion by an IFN-γ enzyme-linked immunosorbant assay (ELISA) (Biosource). Measurements are presented as pg/mL released IFN-γ by 2.105 CTL per 6 hours. Results are expressed as the mean ± standard deviation. Comparisons were validated using Student's t test.HPV-16 E7 peptide-pulsed mature DC were used as stimulator cells for the E7 peptide-specific D4 CTL, while non-pulsed mature DC were used as control cells. CTL  were co-incubated with stimulator cells (1.104 cells) overnight at 37°C in anti-IFN-γ antibody coated 96-well PVDF-plates  in a total volume of 100 μL. These plates were further analyzed for IFN-γ secretion by IFN-γ ELISPOT staining technique  according to manufacturer's instructions. Frequencies of antigen-specific IFN-γ-secreting cells were calculated based on the number of responder cells and the number of spots per well after subtraction of background. Responses were considered positive if a minimum of 15 spot-forming cells (SFC) were counted after the control had been subtracted and if the ratio of SFC between positive experiments and their respective control was larger than 3.K562-HLA-A*0201 cells, pulsed with HPV-16 E7 protein-specific peptide, were used as stimulators of cultured CD8The author(s) declare that they have no competing interests.NC carried out the DC culture, the T cell activation experiments and the ELISPOT assays. She also drafted the manuscript. PP carried out the T cell clone experiment and helped drafting the manuscript. ML participated in acquiring and analyzing flow cytometry data. GN kept cell lines into culture and performed the IFN-γ ELISA. DRVB and ML participated in coordination and analysis of flow cytometry. VFIVT participated in design and coordination of the study and critically revised the manuscript. ZNB reviewed and gave final approval of this version of the article to be submitted for publication.


Caenorhabditis elegans, two of the earliest events following fertilization are secretion of the chitinous eggshell and completion of meiosis, and in this report we demonstrate that the eggshell is essential for multiple developmental events at the one-cell stage.Fertilization restores the diploid state and begins the process by which the single-cell oocyte is converted into a polarized, multicellular organism. In the nematode, chs-1(RNAi) or combined RNAi with the chitin-binding proteins, CEJ-1 and B0280.5, does not interfere with normal meiotic timing, lagging chromosomes are observed at meiosis, and polar-body extrusion fails. We also demonstrate that chitin, and either CEJ-1 or B0280.5, are essential for the osmotic/permeability barrier and for movement of the sperm pronucleus/centrosome complex to the cortex, which is associated with the initiation of polarization.We show that the GLD -1-regulated hexosamine pathway enzyme, glucosamine-6-phosphate N-acetyltransferase (GNA)-2, is required for synthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the substrate for eggshell chitin synthesis by chitin synthase-1 (CHS-1). Furthermore, while C. elegans development, playing an essential role in multiple actin-dependent early events. Furthermore, the earliest meiotic roles precede osmotic barrier formation, indicating that the role of the eggshell is not limited to generation of the osmotic barrier.Our results indicate that the eggshell is required in single-cell  Caenorhabditis elegans, this relief is provided by major sperm protein (MSP), budded off from sperm stored in the spermatheca ) is polymerized from the sugar nucleotide donor, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), synthesized by the hexosamine pathway -4GlcNAcnocephala . The C. rulation . This harulation -20. Furtrulation ,22. Take1) with distinct developmental potentials III), XA782 (gld-1(q485)/gna-2(qa705) unc55(e1170)I; unc-119(ed3) III; ruIs57 [unc-119(+) pie-1::GFP::tubulin]), XA785 (gna-2(qa705) unc-55(e1170)I; qaEx747 ), XA787 (gna-2(qa705) unc-55(e1170)I; qaEx749 [gna-2::GFP pRF 4 pBS KS-]), XA792 (sup-46(qa707)I), XA794 (gna-2(qa705) unc55 (e1170) sup-46(qa707)I), XA796 (gna-2(qa705) unc55 (e1170) sup-46(qa708)I), XA797 (sup-46(qa708)I) and DE2 (dnEx2 [pRF 4 pBS KS-]). We also obtained the following CGC strains: N2, CB4856, AZ212 (unc-119(ed3); ruIs32 [unc-119(+) pie-1::GFP::H2B]III), AZ244 (unc-119(ed3) III; ruIs57 [unc-119(+) pie-1::GFP::tubulin], DR1 (unc-101(m1)I), DR101 (dpy-5(e61)unc-55(e1170) I), DS97 (mat-2(ax76)II), JK1466(gld-1(q485)/dpy-5(e61)unc-13(e51)I), JH1576(unc-119(ed3) III; axIs1140 [pie-1::gfp-mbk-2]), JH1580(unc-24(e1172)mbk-2(pk1427)IV/nT1 [let-?(m435)] and KK866 .The following additional strains were also constructed: XA781 (Sma1 site of pPD129.36 (a gift of A. Fire): pAK57-2 (B0280.5 primers 5'-TTCCTTCAAGGTGCGAGTTT-3' and 5'-AGTGCAAGCAGTGAATGACG-3'), pAK56-2 (cej-1 primers 5'-GAGACCACCACAGAAGCATATGCTCCAG-3' and 5'-GTGGTTTCAACGACTTCTGACTCGATC-3'), pAK55-1 (chs-1 primers 5'-TTCGTCCATCGGAAGTTTTC-3' and 5'CCAAGTACATGTCGATTGCG-3'). The gna-2 RNAi feeding vector, pAK38-2, was a PCR-generated cDNA fragment (primers 5'-CGGGATCCGATGCTATGAAAAAGGC-3' and 5'-GCTCTAGACTACAAGAGTGGCAGCACC-3') cloned into the BamH1 and XbaI sites of pPD129.36. Double-stranded RNA production was induced in liquid culture, and bacteria were collected by centrifugation and spread on NGM plates supplemented with ampicillin, tetracycline and isopropyl-beta-D-thiogalactopyranoside (IPTG). Escherichia coli strain OP50 on unsupplemented NGM was used as a control, unless otherwise stated. For combined RNAi (co-RNAi), approximately equal amounts of each bacterial preparation were mixed before spreading on the plates.RNAi was performed by feeding . The folo) were transferred to individual plates and allowed to lay eggs. Total hatchling number/hermaphrodite was determined by aspirating F1 larvae prior to adulthood. For experiments quantifying % Him, the Po was transferred to a new plate each day until no new hatchlings were observed, and the sex of the hatchlings on each progeny plate was determined at the L4 or adult stage. Results were analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Hatchling number was determined, rather than brood size or % of brood that hatched, because brood-size measurements would have been inaccurate for the following reasons: (i) embryos with a defective eggshell are fragile and sometimes break as they are laid; these embryos would have be excluded from the brood count; (ii) embryos with a defective eggshell are often laid as part of a large cohesive mass, which precludes accurate quantification of the number of embryos in the mass; and (iii) gna-2(qa705) and chs-1(RNAi) embryos are pale brown and non-refractile, making them easily confused with (unfertilized) oocytes.To determine hatchling number, single L1/L2 larvae (Pgna-2(qa705) Mel phenotype, young adult Unc-55 hermaphrodites were picked from gld-1(q485)/gna-2(qa705) unc55 (e1170)I plates. UDP-GlcNAc was dissolved in water  and injected into the rachis of a single gonad arm as described previously [To test biochemical rescue of the eviously . The numgna-2(qa705) were collected in M9 buffer, washed three times in M9, incubated for 30 min at room temperature (RT), washed three times in H2O, resuspended in 50 μl H2O, frozen in dry ice/ethanol and stored at -80°C for no longer than 1 week. Samples were thawed rapidly, sonicated for 20 seconds, centrifuged, and the supernatant filtered and injected onto an anion exchange column . Detection was by ultraviolet absorption at 254 nm. UDP-GlcNAc, UDP GalNAc, UDP-Glc and UDP-Gal were quantified and UDP-GlcNAc and UDP-GalNAc were expressed as a percentage of UDP-Glc + UDP-GalFor HPLC analysis 1000 adult hermaphrodite wild type or 2O, 150 or 300 mM KCl, transferred to the appropriate  2% agarose pad on a glass slide, and viewed by DIC. Compression by the glass coverslip was prevented by depositing embryos into the fluid-filled space that was created by removing a central disc from the pad. To test whether osmotic support could rescue PAR-2 polarization wild-type worms expressing PAR-2::GFP were fed gna-2 RNAi, chs-1 RNAi or cej-1+B0280.5 coRNAi, and embryos were dissected quickly in 150 mM KCl followed by freeze-fracture (control), or were dissected in minimal EGM [gna-2(RNAi), chs-1(RNAi) or cej-1+B0280.5(coRNAi) embryos dissected in M9 are very fragile and usually break, whereas the KCl provides some osmotic support. However, dissection and freezing was completed in <5 min, which should not allow for reversal of polarity defects.For fixed embryos, young adult hermaphrodites were washed in 150 mM KCl and transferred to 0.1% poly-lysine-coated slides. Embryos were released by cutting at the gonad, or gonads were released by cutting just behind the pharynx, followed by freeze-fracture . Slides imal EGM  and incugna-2(qa705) hermaphrodites expressing tubulin::GFP or histone H2B::GFP were transferred to agarose pads, and embryos were examined in utero, using a Leica DMLSFA confocal microscope. The effect on meiotic DNA segregation, polar-body extrusion, SPCC-cortical association, pseudocleavage furrow formation and pronuclear migration of chs-1(RNAi) or cej-1+B0280.5(coRNAi) was also determined in worms expressing histone H2B::GFP. For meiosis duration (determined with tubulin::GFP), timing was defined as follows:To examine meiotic spindles, meiotic DNA segregation, polar-body extrusion, SPCC-cortical association, pseudocleavage furrow formation and pronuclear migration, wild-type or • Meiosis I: time from entry into the spermatheca until tubulin::GFP waned to a small spot . Time from entry into the spermatheca was used instead of nuclear envelope breakdown (NEBD) because it is easily and objectively assessed without DIC optics, and because it excludes any oocytes that undergo NEBD but fail to be ovulated (and fertilized) in a timely manner.• Meiosis II: time from the end of meiosis I (defined above) until the meiosis II spindle waned to a very pale structure.• Meiosis I + II: time from entry into the spermatheca until the meiosis II spindle waned to a very pale structure.As an additional way to determine meiotic timing, we also examined chromosome segregation using histone H2B::GFP. Timing was defined as follows:• Meiosis I: time from entry into the spermatheca until chromatin masses were separated (anaphase I).• Meiosis II: time from entry into the spermatheca until chromatin masses were separated a second time (anaphase II).• Pronuclear meeting: time from entry into the spermatheca until maternal and paternal pronuclei met.t test (tubulin::GFP experiment) or one-way ANOVA followed by Tukey's multiple comparison test (histone H2B::GFP experiment). Lipophilic dye permeability was tested by DIC/UV microscopy of living embryos dissected in 150 mM KCl containing 30 μM FM 4–64 (Molecular Probes).Failure of the SPCC to associate closely with the cortex was scored as any embryo in which the sperm pronucleus  failed to come within half of the sperm pronuclear diameter of the cortex. Results were analysed by GLD Germline differentiation abnormalAPC/C Anaphase-promoting complex/cyclosomeCBD Chitin-binding domainCDC Cell division cycle relatedCEJ Cell junction proteinCHS Chitin synthasecoA Coenzyme ACUL CullinCYK Cytokinesis defectDIC Differential interference contrastDYRK Dual specificity tyrosine phosphorylation regulated kinaseEMB Embryogenesis abnormalEMS Ethyl methanesulphonateFITC Fluorescein isothiocyanateGalNac N-acetyl-D-galactosamineGAM Goat anti-mouseGAR Goat anti-rabbitGFP Green fluorescent proteinGlcNac N-acetylglucosamineGLN Glutamine synthetaseGNA Glucosamine-6-phosphate N-acetyltransferaseGTPase Guanosine triphosphataseLEA Lycopersicon esculentum agglutininMAPK Mitogen-activated protein kinaseMAT Metaphase to anaphase transition defectMBK Minibrain kinaseMEI Meiosis defectiveMEKK Mitogen-activated or extracellular signal-regulated protein kinase kinaseMEL Maternal effect lethalMEX Muscle excessMLC Myosin light chainMSP Major sperm proteinNADPH Nicotinamide adenine dinucleotide phosphate (reduced form)NMY Non-muscle myosinOMA Oocyte maturation defectivePAR Embryonic partitioning abnormalPDZ PSD-95/DLG/ZO-1PFN ProfilinPIE Pharynx and intestine in excessPOD Polarity-osmotic defectivePVF Perivitelline fluidSBA Soybean agglutininSPCC Sperm pronucleus/centrosome complexSQV Squashed vulvaSTA Solanum tuberosum agglutininTRITC Tetramethylrhodamine-5-(and-6)-isothiocyanateUDP Uridine diphosphateVAB Variable abnormal morphologyWGA Wheat germ agglutininZIF Zinc finger-interacting proteinZYG Zygote defective embryonic lethalgna-2(qa705), and performed microscopy and RNAi experiments. AK performed microinjections and conducted the suppressor screen. Plasmids and C. elegans strains were generated by WLJ and AK, and HPLC was conducted by JWD. WLJ, AK and JWD conceived of the study, and the manuscript was drafted by WLJ, with AK and JWD revising it critically. All authors read and approved the final manuscript.WLJ isolated and characterized


Glycosaminoglycans (GAGs) commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces. Glycosaminoglycans (GAGs) are ubiquitous components of mammalian epithelial surfaces GAGs have received relatively little attention in gamma-herpesvirus infections because the archetypal gamma-herpesvirus, Epstein-Barr virus (EBV), appears to ignore them MHV-68 infection of fibroblasts is highly sensitive to inhibition by soluble heparin Our aim was to understand the GAG dependence of MHV-68 in the context of its GAG-binding glycoproteins. Our starting hypothesis was that gp150, ORF4 and gB could all have functionally important GAG interactions. We have previously characterized monoclonal antibodies (mAbs) for the identification of gp150 MAb 9C7 recognized transfected ORF4 much like T3B8 and T2B11 in We used GPI-linked ORF4 truncations to map the domains recognized by each mAb . The preAn uncharacterized 40-45 kDa ORF4 product has been detected in 50×concentrated supernatants of MHV-68-infected cells MHV-68-infected NMuMG cells showed a slightly different pattern, releasing ORF4 products of both 45 kDa and 55 kDa . The 55 The gp70 ST domain is predicted to be heavily O-glycosylated. We analyzed the contribution of glycans to the ORF4 apparent size by treating virus lysates with PNGase F to remove N-linked glycans or with sialidase plus O-glycanase to remove common O-linked glycans . O-glycaSince heparin inhibits MHV-68 infection of GAG-expressing cells The immune rabbit serum used strongly recognizes gp150 (as can be seen in the lysate lane of We identified the precipitated 150 kDa band by mass spectrometry. Its recovered peptides gave 67% coverage of ORF75c and 14% coverage of ORF75b. These ORFs are predicted to encode tegument proteins that would not be accessible to heparin in intact virions. The 20 kDa band was identified as the ORF65 capsid component (67% coverage). The association of the ORF75c, ORF75b and ORF65 gene products with heparin presumably reflected that they normally interact with another negatively charged polymer such as DNA. The only glycoprotein that bound convincingly to heparin-agarose was gp70.1 Fc domain for each GPI anchor  as a fusion with IgG1-Fc . The fus1-Fc . Cell bi1-Fc failed to bind to cell surfaces . We therefore focussed on the region 21-151. This contains many of the cationic residues in gp150, most of which is predicted to be highly acidic and heavily O-glycosylated. There are 3 cationic residues in the region 41–81 and 4 in 81–151.Gp150 amino acid residues 1-151 fused to IgGsurfaces , as did surfaces . However+ but not GAG− CHO cells (Each GST fusion protein was recognized in ELISAs by at least 5 different gp150-specific mAbs (data not shown). Thus, the native conformation of each gp150 fragment appeared to be preserved. GST-M7:21-151 and GST-M7:41-151 both bound to BHK-21 and L929 cells, whereas GST-M7:81-151 and GST-M7:108-151 did not . ConvincHO cells , whereas+ cells  in accordance with Home Office Project Licence 80/1579. A 107 PFU intraperitoneal boost was given 3–4 months later. Spleens were harvested after a further 3 days. Splenocytes were fused with NS0 cells and selected with azaserine (1 µg/ml)/hypoxanthine (100 µM) Female BALB/c mice were were infected intranasally with MHV-68  so as to place the gp150 fragments downstream of and in frame with GST.The full-length ORF4 coding sequence (genomic co-ordinates 9873-11039) E.coli. Expression was induced for 4 h in log phase growth with 400 µM IPTG. The E.coli were lysed in 1% Triton X-100/50 mM TrisCl pH 7.4/150 mM NaCl/5 mM EDTA/1 mM PMSF/5 mM N-ethyl-maleimide/Complete protease inhibitors (Roche Diagnostics). The GST fusion proteins were precipitated with glutathione-sepharose beads (APBiotech), washed×3 in lysis buffer and×2 in Tris-saline, eluted with 50 mM glutathione, and dialysed×3 against PBS.Plasmids derived from pGEX-4T1 were transformed into BL-21 Baby Hamster Kidney cells (BHK-21), L929 cells, NMuMG cells, CHO-K1 cells, the GAG-deficient CHO-K1 mutant pgs745, and 293T cells were all grown in Dulbecco's modified Eagle medium  supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum . CHO-K1 cells expressing ORF4 were generated by transfection with pMSCV-ORF4-IRES-ZEO using Fugene-6  and selected in 500 µg/ml Zeocin . 293T cells were also transfected using Fugene-6.35S-cysteine/methionine  1-Fc fusion proteins were detected with a horseradish peroxidase-conjugated goat anti-human IgG-Fc pAb . Where indicated, proteins were deglycosylated before immunoblotting with PNGase F or sialidase+O-glycanase .BHK-21 cells were infected with MHV-68 , starved for 45 min in cysteine/methionine-free RPMI , then labelled for 1 h with Cells were incubated with glycoprotein-specific mAbs , washed×2 in PBS, incubated with fluorescein-conjugated rabbit anti-mouse IgG pAb , washed×2, and analysed on a FACS Calibur . The binding of Fc fusion proteins was detected with a phycoerythrin-conjugated goat anti-human IgG-Fc pAb . The binding of GST fusion proteins was detected with a biotinylated goat anti-GST pAb  followed by phycoerythrin-conjugated streptavidin (BD Biosciences). Graphs were plotted with FCSPress v1.3.


In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated.Phosphatidylinositol 4,5-bisphosphate (PIP3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3.Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP DAG and Ca2+ activate protein kinase C (PKC), which stimulates cell growth amino]hexyl]-1H-pyrrole-2,5-dione; Calbiochem) or U73343 -trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione; Calbiochem), 0.03 M ET-18-OCHDrosophila spermatocytes were imaged using an upright Zeiss Axioplan 2 microscope with a Zeiss Apochromat 100× oil immersion objective lens (NA 1.4). Phase-contrast and fluorescent images were recorded with an Axiocam CCD camera using AxioVision software for image acquisition. Live crane-fly spermatocytes were followed using a phase-contrast inverted Nikon microscope with a Nikon 100× oil-immersion objective (NA 1.3). Real-time images were recorded on videotape or on DVD.Live Drosophila spermatocytes were performed essentially as described -1H-hexahydro-l,4-diazepine·HCl (MLCK inhibitor)MLCK myosin light chain kinaseNEM N-ethylmaleimidePI phosphatidylinositolPIP phosphatidylinositol 4-phosphate2 phosphatidylinositol 4,5-bisphosphatePIPPLC phospholipase CDrosophila myosin regulatory light chain)Sqh Spaghetti squash -trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (PLC inhibitor)U73343 1-amino]hexyl]-2,5-pyrrolidine-dione (inactive isomer of U73122)Drosophila experiments and drafted the manuscript. LF designed and carried out the crane-fly experiments and commented on the manuscript. AF participated in the design of the study, analysis of the data, and helped edit the manuscript. JAB conceived the study, participated in its design and edited the manuscript. All authors read and approved the final manuscript.RW designed and carried out the Meiosis in a crane-fly spermatocyte. Time-lapse phase-contrast micrographs of a dividing crane-fly primary spermatocyte. Times are hr:min:s. Movie corresponds to Figure Click here for fileDrosophila spermatocyte. Time-lapse phase-contrast micrographs of a dividing Drosophila primary spermatocyte. Mitochondria and parafusorial membranes appear dark and outline the spindle during meiosis. Times are hr:min:s:ms. Movie corresponds to Figure Meiosis in a Click here for fileInhibition of PLC with U73122 causes cleavage furrow regression in a dividing crane-fly spermatocyte. Dividing crane-fly spermatocyte treated with 4.3 μM U73122 at 1 min 36 sec (14:51:00). Note that this cell was followed for only a short time after treatment. Times are hr:min:s. Movie corresponds to Figure Click here for fileDrosophila spermatocyte. Dividing Drosophila spermatocyte treated with 10 μM U73122 at 3 min 20 sec and washed at 18 min 20 sec. Times are hr:min:s:ms. Movie corresponds to Figure Inhibition of PLC with U73122 causes cleavage furrow regression in a dividing Click here for file3 causes cleavage furrow regression in a dividing crane-fly spermatocyte. Dividing crane-fly spermatocyte treated with 35 μM ET-18-OCH3 at 13:32:00. Times are hr:min:s. Movie corresponds to Figure Inhibition of PLC with ET-18-OCHClick here for file3 causes cleavage furrow regression in a dividing Drosophila spermatocyte. Dividing Drosophila spermatocyte treated with 35 μM ET-18-OCH3 at 5 min. 35 sec. Times are hr:min:s:ms. Movie corresponds to Figure Inhibition of PLC with ET-18-OCHClick here for fileThe alkylating agent NEM inhibits cytokinesis in a dividing crane-fly spermatocyte. Dividing crane-fly spermatocyte treated with 1 mM NEM at 15:02:00. Times are hr:min:s. Movie corresponds to Figure Click here for fileDrosophila spermatocyte. Dividing Drosophila spermatocyte treated with 1 μM NEM at 2 min 21 sec. Times are hr:min:s:ms. Cell treated similarly is shown in Figure The alkylating agent NEM inhibits cytokinesis in a dividing Click here for fileDrosophila spermatocyte. Dividing Drosophila spermatocyte treated with 80 μM ML-7 at 3 min 30 sec. After the cell is washed at 4 min 55 sec, furrowing resumes. Times are hr:min:s:ms. Cell treated similarly is shown in Figure Inhibition of MLCK with ML-7 causes cleavage furrow regression in a dividing Click here for file


Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues.Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in  Caenorhabditis elegans, the DAF-2 insulin-like pathway regulates development, metabolism, and longevity. All known components of DAF-2 insulin-like signaling are structurally and functionally conserved in mammals, suggesting that insights gained from studying this pathway in C. elegans may shed light on pathogenetic mechanisms underlying cancer and diabetes. In this study, the authors describe a genetic screen designed to identify novel components of DAF-2 insulin-like signaling in C. elegans. They have characterized three genes that may encode parts of a novel multimolecular membrane-associated complex that potentiates DAF-2 insulin-like signaling in two neuroendocrine cells, the XXX cells. Two of these genes encode proteins similar to mammalian protein tyrosine phosphatases. These results suggest that protein tyrosine phosphatase–like molecules may transduce IGF signals in mammalian endocrine cells and highlight the role of endocrine circuits in the pathogenesis of cancer and diabetes.Insulin and insulin-like growth factor (IGF) signaling regulates critical physiological processes in a wide variety of multicellular organisms. In humans, dysregulation of IGF signaling underlies the pathogenesis of cancer and diabetes. In the nematode  Drosophila ; sdf-9p: nucleotides 10417 to 16170 of YAC Y44A6D; eak-6p: nucleotides 4381 to 5426 of cosmid F10G8. PCR primers were tailed with BglII linkers. Promoter fragments were purified using the Qiaquick PCR Purification Kit (Qiagen), digested with BglII, and repurified. eak-4 and eak-6 promoter fragments were subcloned into BamHI-digested GFP reporter plasmid pPD95.67 (a gift from Andrew Fire) to generate eak-4p::GFP and eak-6p::GFP, respectively; the sdf-9 promoter fragment was subcloned into BamHI-digested RFP ) animals from three independent lines were assayed for rescue of dauer formation at 25 °C. Rescue phenotypes were compared with those of nontransgenic (GFP[−]) siblings.A construct containing the sion PCR ,98. An eFigure S1daf-2(e1370), akt-1(mg306), eak-6(mg329), and eak-4(mg348);akt-1(mg306) animals were grown at 25 °C  or 27 °C (akt-1 and eak-6), and dauers were examined under Nomarski optics for pharyngeal morphology and the presence of alae. The isthmus and terminal bulb of the pharynx are denoted by arrows, and the alae are denoted by arrowheads. All dauers examined had alae; however, constriction of the isthmus and terminal bulb seen in daf-2 dauers (top panel) is not observed in eak-6 or eak-4;akt-1 dauers . akt-1 dauers exhibited an intermediate degree of pharyngeal constriction (second panel). eak-4, sdf-9, akt-1 sdf-9, and eak-6;akt-1 dauers had alae and exhibited pharyngeal morphology comparable to eak-6 and eak-4;akt-1 dauers.(2.9 MB PDF)Click here for additional data file.Figure S2eak mutants enhance the dauer arrest phenotype of age-1/PI3K and pdk-1 loss-of-function mutants.(A) eak mutants do not enhance the dauer arrest phenotype of an akt-2 loss-of-function mutant.(B) All error bars indicate standard deviation. All experiments were performed twice. Refer to (826 KB PDF)Click here for additional data file.Figure S3eak;akt-1 25 °C dauer arrest phenotypes are suppressed by a mutation in daf-16/FoxO.(A) eak 27 °C dauer phenotypes are suppressed by a weak loss-of-function mutation in daf-18/PTEN.(B) eak 27 °C dauer phenotypes are suppressed by a gain-of-function mutation in akt-1. All error bars indicate standard deviation.(C) All experiments were performed twice. Refer to (781 KB PDF)Click here for additional data file.Figure S4This experiment was performed twice. Refer to (553 KB PDF)Click here for additional data file.Figure S5eak-4(mg348) males were mated with BC1216  hermaphrodites, and F1 cross-progeny were scored for dauer arrest at 27 °C. sDf21 is a deficiency on chromosome IV that deletes eak-4.1 cross-progeny from eak-4(mg348) X BC1216 do not show enhanced dauer arrest at 27 °C compared to eak-4(mg348).(A) F(B) Comparison of observed to expected embryonic lethality among progeny confirms the identity of the BC1216 strain.(630 KB PDF)Click here for additional data file.Figure S6daf-2 wild-type and daf-2(e1370) L1 animals harboring an SDF-9::GFP translational fusion construct were grown at 25 °C and analyzed using fluorescence microscopy.(558 KB PDF)Click here for additional data file.Table S1(15 KB XLS)Click here for additional data file.


We hypothesize that over-expression of transporters associated with antigen processing (TAP1 and TAP2), components of the major histocompatibility complex (MHC) class I antigen-processing pathway, enhances antigen-specific cytotoxic activity in response to viral infection. An expression system using recombinant vaccinia virus (VV) was used to over-express human TAP1 and TAP2  in normal mice. Mice coinfected with either vesicular stomatitis virus plus VV-hTAP1,2 or Sendai virus plus VV-hTAP1,2 increased cytotoxic lymphocyte (CTL) activity by at least 4-fold when compared to coinfections with a control vector, VV encoding the plasmid PJS-5. Coinfections with VV-hTAP1,2 increased virus-specific CTL precursors compared to control infections without VV-hTAP1,2. In an animal model of lethal viral challenge after vaccination, VV-hTAP1,2 provided protection against a lethal challenge of VV at doses 100-fold lower than control vector alone. Mechanistically, the total MHC class I antigen surface expression and the cross-presentation mechanism in spleen-derived dendritic cells was augmented by over-expression of TAP. Furthermore, VV-hTAP1,2 increases splenic TAP transport activity and endogenous antigen processing, thus rendering infected targets more susceptible to CTL recognition and subsequent killing. This is the first demonstration that over-expression of a component of the antigen-processing machinery increases endogenous antigen presentation and dendritic cell cross-presentation of exogenous antigens and may provide a novel and general approach for increasing immune responses against pathogens at low doses of vaccine inocula. The development of protective vaccines against infectious diseases such as AIDS, SARS, and West Nile virus has become a societal priority but remains a scientific challenge. In recent years, the threat of bioterrorism agents such as anthrax and smallpox has heightened the need for the rapid development of effective new vaccines. One of the major stumbling blocks to the implementation of any vaccine is the toxic side effects on the vaccine candidate. For example, a significant number of doses of a new vaccine against smallpox have been commissioned, but approximately 20% of the individuals targeted to be inoculated will suffer toxicity due to vaccination. Furthermore, an additional difficulty in the production of vaccines is the creation of sufficient doses to vaccinate a large population. The authors have identified a novel approach that appears to address these issues. They demonstrate that the inclusion, in low doses of vaccines, of a normal component of the antigen-processing pathway, the transporter associated with antigen processing (TAP), confers protective immunity against lethal viral loads during viral challenges. This new paradigm is shown to be applicable to many viruses, including poxviruses, and could significantly advance the creation of new vaccines and improve those that already exist. Therefore, these cells lack the capacity to direct the priming of antigen-specific T cells  or high-dose [1.5e7 TCID50]), SV (low-dose [1.6e5 CEID50] or high-dose [1.6e7 CEID50]), VV-PJS-5 (low-dose [3e4 PFU] or high-dose [5e6 PFU]), VV-hTAP1,2 (3e4 PFU), and VV-NP-VSV (3e6 PFU). Titrations determined the infectious doses of VSV and SV that would generate a minimum (low dose) and maximum (high dose) CTL response against peptide-pulsed RMA targets. Splenocytes were harvested 6 d after infection and cultured (RPMI-1640/10% FBS complete media) for 2–3 d to eliminate nonspecific natural killer cell killing. For VSV infection alone or via coinfection with VV-hTAP1,2 or VV-PJS-5, splenocytes were incubated without peptide. For infections with SV or VV-NP-VSV, splenocytes were stimulated with SV peptide  or VSV-NP peptide . VV-hTAP1,2 or VV-PJS-5–infected mouse splenocytes were restimulated in vitro for 3 d with irradiated syngeneic splenocytes infected with VV-PJS-5 (3.4e7 PFU/1e8 syngeneic splenocytes).H-2Kb cells, RMA cells, or naïve splenocytes as targets. T2-Kb targets were infected with VV-NP-VSV alone or in combination with VV-hTAP1,2 or VV-PJS-5 (multiplicity of infection [MOI] = 10) for 6 h. The RMA target cells were pulsed with VSV-NP52–59 peptide (5–25 μM) or SV-NP324–332 peptide (5–25 μM) for the relevant CTL assay. For VV antigen-specific killing, the RMA targets were infected overnight with VV-PJS-5 (MOI = 0.34). For the measurement of endogenous antigen processing, targets were generated by the in vitro stimulation of naïve splenocytes (2e7 cells) for 2 d with lipopolysaccharide (LPS) , followed by overnight infection with either VV-PJS-5 or VV-hTAP1,2 (5e6 PFU).Cytotoxic activity was measured in standard 4-h 51Cr-release assays using T2-K+/VSV-NP52–59–specific splenocytes was measured in two separate experiments using a H-2Kb–VSV-NP52–59 epitope-specific tetramer conjugated to phycoerythrin  and a fluorescein-conjugated rat anti-mouse CD8a (Ly-2) monoclonal antibody (clone 53–6.7) after Fc receptor block , . Mice were coinfected with VV-PJS-5 (3e4 PFU) plus low-dose VSV, or VV-hTAP1,2 (3e4 PFU) plus low-dose VSV, or infected with low-dose VSV, or high-dose VSV. Six days after infection, CD8+/VSV-NP52–59–specific fluorescence as a percentage of total CD8+ fluorescence was determined by flow cytometry.The frequency of CD8Human TAP1 expression in splenocytes from the mice infected with VV-hTAP1,2 was determined by SDS-PAGE and Western blot. The blots were probed for human TAP1 with rabbit anti-human TAP1 antiserum  and visualized by enhanced chemiluminescence .RT-PCR was used to detect human TAP1 and TAP2, and mouse TAP1 and TAP2, in spleens 24 h after infection. In addition, a time-course quantification of human TAP1 and mouse TAP1 was performed using quantitative real-time PCR (QRT-PCR). Total RNA was extracted  from mouse spleens 4 h, 8 h, and 24 h after i.p. infection with VV-hTAP1,2 (3e4 PFU). QRT-PCR reactions  were performed in duplicate using a Light Cycler  for mouse TAP1 and for human TAP1, plus a ribosomal small subunit S15. The sequences of the primer pairs used in the RT-PCR and the QRT-PCR are listed in The threshold cycle (CT) above background was determined for mouse TAP1 and human TAP1 and normalized to the lowest S15 CT value among the reactions. The differences in CT were used to calculate the abundance of human TAP1 relative to mouse TAP1. Relative abundance = 2 (CTmTAP1–CThTAP1). The CT values represent the average of three mice per time point.Visualization of human TAP expression in spleen-derived antigen presentation cells from mice infected with VV-hTAP1,2 or VV-PJS-5 was performed using confocal fluorescence microscopy. Splenocytes were double labeled with rabbit anti-human TAP1 antiserum (Stressgen Biotechnologies) and one of the following cell-surface markers: rat anti-mouse B220 , rat anti-mouse MAC-1 , or rat anti-mouse NLDC-145 antibodies (DC marker) . The presence of human TAP1 was determined in approximately 300 cells per surface marker in 20 randomly chosen fields. Splenocytes from VV-PJS-5–infected mice were used as negative controls.−/− mice were used as controls.TAP heterodimer activity, in the presence or absence of adenosine triphosphate (ATP), was detected by a streptolysin-O–mediated peptide-transport assays in splenocytes harvested 24 h after mice were infected with VV-hTAP1,2 or VV-PJS-5 using a radio-iodinated peptide library containing a glycosylation site (NXT)  ,42. Spleb antibody (clone AF6–88.5) (BD Biosciences Pharmingen) or 25.D1.16 antibody specific for Kb/SIINFEKL  [b and H-2Kb–SIINFEKL complexes.Splenic DCs were isolated using CD11c+ magnetic beads run through an AutoMacs  after enrichment by centrifugation . DCs were infected with VV-PJS-5, VV-mTAP1, or VV-hTAP1,2 (3e6 cells/1e4 PFU) for 2 h, followed by incubation with GM-CSF (10 ng/ml complete RPMI 1640) and OVA (10 mg/ml)  or PBS (18 h at 37 °C). DCs stained with phycoerythrin–conjugated rat anti-mouse CD11c antibody, Fc blocker (Fcγ III/II receptor), and either FITC-conjugated anti-H-2KH/NIAID) . Flow cyVV-hTAP1,2 and VV-PJS-5 viruses were demonstrated to replicate equally in cell culture. Forty-two mice were randomized into seven groups (six mice per cage) and were vaccinated with three different doses of VV-hTAP1,2 or VV-PJS-5  or PBS. Fourteen days later, mice were weighed and challenged with a lethal dose of VV-WR  under isoflurane anesthesia. Weight was recorded daily for 14 d, and any mouse falling below 25% of pre-challenge weight was euthanized. Mean weight going forward was calculated from the remaining survivors.b, VSV-NP, and CD8+ cells generated by the vaccination protocols. A two-way ANOVA, after square-root transformation of the data, was used to analyze the main effects of dose and the recombinant VV on mouse weight 5 d after VV-WR challenge. Bonferroni procedure corrected p-values for multiple comparisons. A chi-square test  compared flow-cytometry histograms for differences in total H-2Kb or H-2Kb–SIINFEKL complexes. p-Value of < 0.01  was considered significant, and T(X) > 10 was empirically determined as a cut-off value.The z-statistic was calculated to determine the statistical significance of the differences in the proportions of H-2Khttp://www.ebi.ac.uk/swissprot) accession numbers for the proteins discussed in this paper are calnexin (BC040244), calreticulin (NM 007591), LMP2 (U22919), LMP7 (U22031), LMP10 (U77784), MHC class I heavy chain (V00747) and β2-microglobulin (NM 009735), TAP1 (NM 013683), TAP2 (NM 011530), and tapasin (A7316613). The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the strains discussed in this paper are mouse strain C57BL/6 (V00746), recombinant VV carrying human TAP1 (NM 000593) and TAP2 (NM 018833) genes, SV (X00087), VSV, Indiana Strain (J02428), and VV Western Reserve strain (AY243312).The Swiss-Prot (


Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzedC. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses attom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response intom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles intom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defectiveunc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at theC. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses intom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects ofunc-13 mutants are partially suppressed intom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming. C. elegans neuromuscular junction. Tomosyn inhibits vesicle priming by regulating the size of the readily releasable vesicle pool.This paper examines the in vivo role of the syntaxin binding protein tomosyn in synaptic transmission at the To test this possibility, we measured the evoked responses oftom-1 mutants in 0.5 mM Ca2+. In lower calcium, the evoked response amplitudes oftom-1 mutants were still comparable to the WT , p > 0.1; and 1,533 ± 202 pA,n = 8, fortom-1(nu468),p > 0.05); however, the response duration remained significantly prolonged , p = 0.016; and 4.2 ± .37 ms fortom-1(nu468), p = 0.0014), again resulting in increased charge integrals (6.9 ± 0.82 pC for WT vs. 10.7 ± 1.2 pC fortom-1(ok285), p = 0.018; and 13.3 ± 2 pC fortom-1(nu468), p = 0.006). These data suggest that the enhanced release observed intom-1 mutants is due primarily to a prolongation of the evoked response.To directly test whether mutations in= 0.37) A and2B.0.0001) B, was gr0.0001) B. The entom-1 mutants could be rescued by expressing TOM-1 protein specifically in cholinergic neurons. Recordings fromtom-1(nu468) mutants possessing an integrated TOM-1A cDNA array (jaIs1052) reduced the excitatory postsynaptic current amplitude when compared to both WT ortom-1(nu468) ) and reversed the increased response duration to WT levels ), resulting in a significant decrease in total charge integral  relative to both WT (p = 0.0001) andtom-1(nu468) (p < 0.0001)  times that of the WT. Together, these data suggest that TOM-1A is required presynaptically to regulate synaptic transmission. This observation is consistent with the neuron-specific expression pattern of TOM-1 based on atom-1 promoter::GFP fusion construct  methyl-2-aminoethane-sulfonic acid), 0.25 mM CaCl2, 4 mM Na2ATP, 36 mM sucrose, and 5 mM EGTA . Hyperosmotic data were recorded using an 800 mOsm extracellular solution achieved through addition of sucrose. Data were acquired using Pulse software  run on a Dell computer. Subsequent analysis and graphing was performed using Pulsefit (HEKA), Mini analysis  and Igor Pro .Electrophysiological methods were as previously described . Brieflytom-1(ok285), tom-1(nu468), tom-1(ok285);unc-13(s69), unc-13(s69), and TOM-1A rescue young-adult hermaphrodites were prepared for high-pressure freezing as previously described [E. coli and immobilized by high-pressure freezing at ˜180 °C under high pressure in a Bal-Tec HPM010 and moved to liquid nitrogen.N2,scribed . BrieflyFreeze substitution was performed in a Reichart AFS machine  as previously described  using taSerial sections were cut at a thickness of 40–50 nm, collected on formvar-covered carbon coated copper grids , and counterstained in 2% or 2.5% aqueous uranyl acetate for 4 min, followed by Reynolds lead citrate for 2 min. Images were obtained on a JEOL JEM-1220 transmission electron microscope  operating at 80 kV. Micrographs were collected using a Gatan digital camera .tom-1(ok285), tom-1(nu468), tom-1(ok285)unc-13(s69), unc-13(s69), and (jaIs1052) TOM-1A rescued animals were performed from ventral nerve cord serial sections. The analysis was performed blind. Images were quantified using NIH Image software. A synapse was defined as a set of serial sections containing a presynaptic specialization and two flanking sections from both sides without presynaptic specialization. Several morphometric measurements were obtained: the number of vesicles per profile, the distance from each vesicle membrane perpendicular to the plasma membrane, and the distance to the proximal edge of the presynaptic specialization.Morphometric analysis of WT,Figure S1C. elegans His6-tagged SNAREs and rat His6-tagged SNAREs. Purified syntaxin, SNAP-25, and synaptobrevin were mixed, incubated to allow complex formation, and separated on a PAGE gel to visualize complexes. One aliquot of each sample was loaded without heating (rt), and the other was heated to 95 °C for 5 min (b) prior to loading. The position of complexes, syntaxin, SNAP-25, andC. elegans synaptobrevin (which migrates just above the dye front) are labeled on the gel. Unfortunately, vertebrate (v) synaptobrevin migrates in the dye front and is not identifiable on the gel. Various combinations ofC. elegans (c) and rat protein (v) mixes were tried. All reactions containing purifiedC. elegans SNAP-25 failed to form complexes efficiently, while all other combinations form complexes. The positions of molecular weight markers are on the left.Complex assays were performed usingC. elegans His6-tagged SNAREs and rat SNAP-25 were purified as described in the main text. Rat His6-tagged synaptobrevin and syntaxin were a gift from Michael Crowder (Washington University School of Medicine) and purified as described in [Methods:ibed in . To form(292 KB JPG)Click here for additional data file.Figure S2C. elegans GST syntaxin or GST alone was incubated with either rat SNAP-25 (vSNAP-25) orC. elegans tomosyn C-terminal domain (TOM-1Ct). Glutathione agarose beads were added to collect complexes. The supernatant was removed, and the beads were washed extensively. Aliquots of the supernatant (sup) and the protein eluted from the glutathione agarose (beads) were separated on SDS-PAGE and stained for protein with Coomassie Blue. Although syntaxin bound to SNAP-25 in the absence of TOM-1Ct or synaptobrevin, TOM-1Ct did not interact with syntaxin in the absence of SNAP-25. Molecular weights of markers are labeled on the left.A Coomassie-stained gel shows GST pull-down assays performed to eliminate the possibility that TOM-1Ct forms a complex with syntaxin in the absence of SNAP-25.Methods: proteins and GST pulled-down assays were performed as described in the main text.(543 KB JPG)Click here for additional data file.Figure S3tom-1(nu468) cholinergic synapse. (D) Cholinergic synapse injaIs1052  mutant background). (E)tom-1(nu468) GABAergic synapse. (F) GABAergic synapse injaIs1052  mutant background). (G)unc-13(s69). (H)tom-1(ok285) unc-13(s69) double mutant. Arrows point to membrane-contacting synaptic vesicles.(A) WT. (B) tom-1(ok285). (C)(2.8 MB JPG)Click here for additional data file.http://www.ebi.ac.uk/swissprot;http://www.ebi.ac.uk/embl) accession numbers for the following genes and gene products are syntaxin-1a (O35526), SNAP-25 (P60879), synaptobrevin-2 (a.k.a. VAMP-2) (P63044), tomosyn (Q8K400), synaptotagmin (P46096), Munc13 (Q9Z1N9), ROCK (P70335), Munc18 (Q548T0_MOUSE),tom-1 (Q49HI2_CAEEL), TOM-1B (P91392_CAEEL), TOM-1A (P91389_CAEEL), TOM-1C (Q8T3B2_CAEEL), synaptobrevin (SNB-1) (O02495_CAEEL), syntaxin (UNC-64) (O16000_CAEEL), SNAP-25 (RIC-4) (O62414_CAEEL), andunc-13 (P27715).The Swiss-Prot/TrEMBL (


Haloquadratum walsbyi dominates NaCl-saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. The genome sequence was analyzed and physiological and physical experiments were carried out in order to reveal how H. walsbyi has specialized into its narrow and hostile ecological niche and found ways to cope with the desiccation stress.The square halophilic archaeon A rich repertoire of proteins involved in phosphate metabolism, phototrophic growth and extracellular protective polymers, including the largest archaeal protein (9159 amino acids), a homolog to eukaryotic mucins, are amongst the most outstanding features. A relatively low GC content (47.9%), 15–20% less than in other halophilic archaea, and one of the lowest coding densities (76.5%) known for prokaryotes might be an indication for the specialization in its unique environmentAlthough no direct genetic indication was found that can explain how this peculiar organism retains its square shape, the genome revealed several unique adaptive traits that allow this organism to thrive in its specific and extreme niche. The two unique features of these cells are the wafer like rectangular shape and a cell thickness of not more than 0.1 μm. Already known since the early 1980s as Walsby's square bacterium  for the genome and plasmid PL47 respectively.The genome can be accessed via HaloLex . General2O residual DNA was digested using the "DNA-free" kit (Ambion) following the manufacturers instructions. The quality of the RNA was checked using the 2100 Bioanalyzer (Agilent) and the RNA Nano LabChip (Agilent).The RNA was extracted with peqGold RNAPure extraction solution (Peqlab Biotechnology) following the manufacturers instructions. After dissolving RNA in DEPC-HTotal RNA was reverse transcribed into cDNA using SuperScript II (Invitrogen) following the manufacturer's instructions with 2 μg total RNA per reaction as template and the gene specific primers pcr4-rev and pcr7-rev, respectively.The PCR reactions were performed using HotStarTaq (Qiagen) (50 μl per reaction) and 0.5 μl of the cDNA samples as template. The following temperature profile was applied on a Thermocycler T3 (Biometra): 95°C 15 min; 40×  or 90 sec (1 kbp-PCRs). Subsequently the PCR reactions were analyzed by standard agarose gel electrophoresis.pcr1-for: 5'-CAT TGG ATC GGT GTC TGC ACA GCA AC-3'pcr1-rev: 5'-GCG CCG CTT GAA GGA GTT ATT TGC G-3'pcr2-for: 5'-GAT CAC GCT CGA CGA CCT CG-3'pcr2-rev: 5'-CGT TGA TGA CGC CAG CCT GC-3'pcr3-for: 5'-CCA CTG GTC AGG TGA ATG CCT C-3'pcr3-rev: 5'-CTT CCT GTC GCA TCC GAC TGG-3'pcr4-for: 5'-GAC GCT ACT GCC ACC GGC GAT G-3'pcr4-rev: 5'-GCA GAC CCG TGT TCG AAC CGT CC-3'pcr5-for: 5'-GGA CTT GCT GGC ACG ATC GAC-3'pcr5-rev: 5'-CTC CAG ATG TGC CAA CCT CGC-3'pcr6-for: 5'-GCG GTT GAG TGG TAT CTT CAC C-3'pcr6-rev: 5'-GCT ATC GGT GGC GGT GTC G-3'pcr7-for: 5'-CTC CCC ATC CAG TAG TCG GTC ATT GG-3'pcr7-rev: 5'-GAT TGT ATC CTC TCA AAT GCC CCG CTA AG-3'Shotgun library preparation and sequencing was performed by PP. MF and FP provided genome assembly, initial ORF prediction and bioinformatics tools integrated into the HaloLex system and performed the genome statistics. AW performed the mRNA detection study of Halomucin. MR programmed and supported the genome analysis system MIGENAS and supported genome annotation. HB supervised the study, performed the manual annotation and wrote the manuscript jointly with FRV, FP and DO.H. walsbyi proteins. List of all Haloquadratum proteins ordered by function category and function class.complete list of Click here for file