Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet β cells. To determine whether specific lysosomal proteases might influence the outcome of a T cell–mediated autoimmune response, we examined the functional significance of cathepsin inhibition on autoimmune T1D-prone non-obese diabetic (NOD) mice.+ T cell infiltration via inhibiting granzyme activity. It was discovered that cathepsin L inhibition prevents cytotoxic activity of CD8+ T cells in the pancreatic islets through controlling dipeptidyl peptidase I activity. Moreover, the gene targeting for cathepsin L with application of in vivo siRNA administration successfully prevented CY-induced diabetes of NOD mice. Finally, cathepsin L mRNA expression of peripheral CD8+ T cells from NOD mice developing spontaneous T1D was significantly increased compared with that from control mice.Here it was found that specific inhibition of cathepsin L affords strong protection from cyclophosphamide (CY)-induced insulitis and diabetes of NOD mice at the advanced stage of CD8+ T cell-mediated autoimmune diabetes, and inhibition of cathepsin L as a powerful therapeutic strategy for autoimmune diabetes.Our results identified a novel function of cathepsin L as an enzyme whose activity is essential for the progression of CD8 The MHC class II expression on PLN B cells from CY-treated NOD mice was increased compared with that from control NOD mice -specific T cell receptor (TCR)-transgenic mice (OT-II) was conducted. In brief, purified CD4+ T cells from the spleen of OT-II mice were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and co-cultured with T cell-depleted spleen cells as APCs from B6 mice in the presence of OVA protein with or without CatL-inh or CatS-inh for three days. A small inhibitory effect of CatL-inh  on OT-II T cell response to OVA was observed although inhibitory effect of CatL-inh at high concentration (5 µM) on the T cell response was found . By contrast, the expression levels of CD4+, CD25+CD4+, B220+ B, and CD11c+ cells from NOD mice were similar to the levels of B6 mice  of cathepsin L was performed. A significantly enhanced cathepsin L activity of pancreatic CD8+ T cells from CY-treated NOD mice was observed compared with that from control NOD mice  and ionomycin (IM), the increased level of enzymatic activity of cathepsin L was confirmed by confocal analysis using Magic Red™  RNA for cathepsin L gene silencing provides long-term suppression of diabetes development in CY-treated NOD mice. Evidence was obtained demonstrating that application of siRNA targeting cathepsin L (CatL siRNA) intraperitoneally (i.p.) caused a marked decrease in the development of CY-induced diabetes in NOD mice showing a high level of blood glucose . To confted mice . Histoloted mice . Furtherted mice . There wted mice . In addited mice . These r+ T cells from peripheral blood lymphocytes (PBLs) of non-diabetic and diabetic NOD mice from three to nine months of age. As shown in + T cells from diabetic NOD mice was significantly higher than that from non-diabetic NOD mice. However, there was a range of cathepsin L expressions even in non-diabetic mice. Some differences in the disease development or onset in NOD littermates may be associated with expression levels of cathepsin L. By contrast, enhanced expression of granzyme B in diabetic NOD mice compared with that in non-diabetic NOD mice could not be found (data not shown). On the other hand, when we compared the granzyme B expressions of infiltrating CD8+ T cells in the pancreas tissues between non-diabetic and diabetic NOD mice by flow cytometry, significantly higher granzyme B expression of diabetic pancreatic CD8+ T cells was observed in diabetic NOD mice  and the antigen presentation in cortical thymic epithelial cells + T cells in the development of T1D other than the maintenance of Treg cells in the periphery and the thymic selection. Two reports using cathepsin L-deficient mice focused on Treg cells which control autoimmune diabetes in NOD mice Cat-L−/− splenocytes-transferred recipients + T cells in addition to the Treg cells. Moreover, although significantly increased Treg cells in cathepsin L-deficient NOD mice were detected, there was no difference in the Treg cell number of PLNs between CY and CY+CatL-inh-treated NOD mice in our experiment. As for the discrepancy, the cathepsin L inhibitor might be effective for peripheral T cells, but not T cell differentiation including Treg cells in the thymus. The effectiveness may depend on the dose, time span, or timing of CatL-inh administration, while the weakness of the treatment with specific CatL-inh is that the metabolic time is considerably short in vivo. Within approximately 24 hours, the effectiveness had disappeared in the animal's bodies. In addition, it was reported that there was an effect of cathepsin L inhibition using CatL-inh on antigen processing in the peripheral lymphoid tissues + T cells, possible roles in other immune cells besides CD8+ T cells should be researched.Cathepsin L functions under the acidified conditions in the endosome, and plays an important role in the DPPI signaling events in CD8+ T cells in PLN from cathepsin L siRNA-treated NOD compared with that from control NOD mice .As for the adverse effects of injection of cahtepsin L inhibitor, when body weight, health condition, and a histopahtological examination of all organs from CatL-inh-administerred mice was checked, there was no significant change. This suggests that the CatL-inh has no toxicity or side effects, and the application may be clinically useful. Also the administration of siRNA targeting cathepsin L genes using athelocollagen may have a temporary effect on the peripheral T cells. It is possible that the siRNA might not have any effect on the gene-knockdown in the thymus. When atelocollagen, which is positively charged, interacts with the negatively charged siRNA duplex, a siRNA/atelocollagen complex is formed. This is a nanosize particle with a diameter of 100–300 nm. The siRNA/atelocollagen complex was highly stable against nucleases and a prolonged release of genes and oligonucleotides without the digestion of RNase + T cells in the thymus and periphery of cathepsin L deficient mice was observed, and that CD8+ T cells in the deficient mice were relatively increased + T cell differentiation + T cells from NOD mice might play a critical role in the pathogenesis of T1D. It is known that CD8+ T cells as well as CD4+ T cells are pivotal effector cells for rapid destruction of pancreatic b-cells in CY-induced T1D + cells from spleen against enriched islet cells from NOD pancreas was significantly reduced by the addition of CatL-inh. Splenic CD8+ T cells including a very low number of b cell antigen-specific CD8+ T cells in NOD mice were stimulated with PMA and ionomycin, and used for in vitro CTL assay. The antigen-specificic CTL activity of CD8+ T cells in development of autoimmune diabetes of NOD mice remains unsolved.A previous report showed that a reduced number of CD4+ T cells in peri-pancreatic LN was significantly enhanced compared with that of the other immune cells including CD4+T cells, Treg cell, B cells, and dendritic cells. Those findings suggested that cathepsin L in CD8+ T cells might be activated at an advanced stage of the disease in NOD mice. However, it is still unclear why cytotoxic activity of CD8+ from NOD mice could be upregulated via the axis of cathepsin L, DPPI, and granzyme B. In addition, we should pay attention for using the mRNA level of cathepsin L in the peripheral CD8+ T cells as the level dose not strictly reflect the onset or development of autoimmune diabetes in NOD mice. Further investigation would be required for the clinical application in human patients.On the other hand, it would be helpful for diagnosis of spontaneous diabetes in NOD mice to measure cathepsin L expression in the peripheral T cells. Under three months of age, there was no change in the cathepsin L expression. In addition, we have confirmed that the expression of cathepsin L mRNA of CD8+ thymus-derived naturally occurring Treg cells play an indispensable role in the maintenance of self-tolerance and immune homeostasis + T cells against pancreatic b-cells through unknown molecular mechanisms.Foxp3-expressing CD4+ T cells. Targeting these CD8+ T cells using cathepsin L inhibitor could be a strategy for the prevention and cure of diabetes in the near future.Our previous report indicated that cathepsin S inhibitor can prevent Sjogren' syndrome which is one of the organ-specific autoimmune diseases found in salivary and lacrimal glands + T cell response in autoimmune diabetes NOD mice via inhibiting granzyme B activation. These data point to cathepsin L as a novel regulatory mechanism of cytotoxicity, suggesting a potential use of cathepsin L inhibition including siRNAs delivery as a new therapeutic target in autoimmune diabetes.Taken together, the results of the experiments presented in this report show that treatment with cathepsin L inhibition prevents the cytotoxic CD8This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of Tokushima (toku09021).NOD mice were reared in our specific pathogen-free mouse colony and given food and water ad libitum. Experiments were humanely conducted under the regulation and permission of the Animal Care and Use Committee of the University of Tokushima, Tokushima, Japan. Prediabetic (7 to 8-weeks-old) female NOD mice were intraperitoneally injected twice with 4 mg cyclophosphamide (CY).Specific inhibitors for cathepsin B (CA074), cathepsin L (Clik148 and Clik195), and cathepsin S (Clik60) were developed with the help of computer-graphic modeling based on the stereo-structure The blood glucose levels and the urine glucose were monitored weekly with a Glucometer  using 50 µl blood from the tail vein, and with Keto-Diastix , respectively.All organs were removed from the mice, fixed with 4% phosphate-buffered formaldehyde (pH 7.2) and prepared for histological examination. The sections were stained with hematoxylin and eosin (H&E). For semiquantitative evaluation of infiltration, histological analysis using sections containing five or more islets was performed as previously described Surface markers on lymphocytes in pancreatic lymph nodes (PLNs) and pancreas were identified by mAbs with BD FACSCant flow cytometer . Rat mAbs to FITC-, PE-conjugated anti-CD8 and CD4 mAbs  were used. For detection of T cell activation markers, PE-Cy5-conjugated anti-CD44 mAbs (eBioscience) were used. Intracellular Foxp3 and granzyme B (eBioscience) expressions with an intracellular Foxp3 detection kit (eBioscience) were analyzed according to the manufacturer's instructions. Enzymatic activities of cathepsin L were assessed using Magic Red™ cathepsin detection kit for cathepsin L . Briefly, lymphocytes were incubated with Magic Red™ fluorogenic substrates of cathepsin L according to the manufacturer's instructions. The red fluorescence was detectable as a result of intracellular enzymatic cleavage. The data were analyzed with FlowJo FACS analysis software (Tree Star).Cathepsin L activity was assayed in the thymus and PLNs from CY-treated and control NOD mice using a cathepsin L activity assay kit . Briefly, 10 µg cytoplasmic lysates of thymus and PLNs were incubated with cathepsin L substrates labeled with amino-4-trifluoromethyl coumarin (AFC) at 37°C for an hour. The AFC cleaved by cathepsin L was read with a fluorometric microplate reader  at excitation and emission wavelengths of 400 nm and 505 nm.+ cells, CD4+ cells, CD4+CD25+ cells, B220+ cells and CD11c+ cells were prepared from PLNs by positive selection using anti-CD8, CD4, CD25, B220 (eBioscience) and CD11c  antibodies, and magnetic beads . Total RNA was extracted using TRIzol reagent  and reverse transcribed. Transcript levels of cathepsin L, DPP1 and β-actin were detected using PTC-200 DNA Engine Cycler (BioRad) with SYBR Premix Ex Taq . Primer sequences were as follows: cathepsin L: forward, 5′-GTGGACTGTTCTCACGCTCA-3′ and reverse, 5′-TATCCACGAACCCTGTGTCA-3′, DPPI: forward, 5′-TCTGTCAATGAGTGAGCTGTGTCAA-3′ and reverse, 5′-TGCGCTCATGTGTGTATGGAAG-3′ and b-actin: forward, 5′- AAATCTGGCACCACACCTTC -3′ and reverse, 5′- AGAGGCGTACAGGGATAGCA -3′.CD8The frozen sections of pancreas tissues from control and CY-treated NOD mice were fixed with cold acetone, blocked with M.O.M.™ blocking reagent , and then stained with the antibodies FITC-conjugated antibody to CD8 (eBioscience) and biotinylated antibody to CD4  for over night. After washes in PBS, the sections were stained with Alexa Fluor 488 goat anti-FITC IgG and Alexa Fluor 568-conjugated streptavidin  for 40 minutes. The nuclear DNA was stained with 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) (Invitrogen). The sections were visualized with a laser scanning confocal microscope (Carl Zeiss) at a magnification of 400× or 630×. Quick Operation Version 3.2 (Carl Zweiss) for imaging acquisition and Adobe Photoshop CS2 (Adobe System) for image processing was used.+ subset, lymphocytes in pancreas and PLNs were treated for 30 min at 4°C with mAbs specific for CD4, CD25, CD11c, NK1.1, B220, and MHC class II (eBioscience), and then CD8+ T cells were isolated by negative selection with magnetic beads .For purification of CD8Cells were cultured in RPMI 1640 containing 10% FCS, L-glutamine, penicillin/streptomycin and 2-mercaptoethanol in 96-well round-bottom microtiter plates stimulated with (0.5∼5 ng/ml) PMA and 500 ng/ml ionomycin .+ T cells stimulated with PMA and ionomycin (IM) was assessed by confocal microscopic analysis using a Magic Red™ cathepsin detection kit. In brief, CD8+ T cells were stimulated with PMA and IM for 24 hours. Cells were stained with Magic Red™ according to the manufacture's instructions. After staining, cells were deposited onto poly-L-lysine-coated glass slides and were fixed with 0.25 to 1% paraformaldehyde. Subsequently, cells were stained with anti-CD8 mAb and visualized with a laser scanning confocal microscope. A 63×1.4 oil DIC objective lens was used.Cathepsin L activity of CD8+ T cells were stimulated with PMA/IM in the presence of cathepsin inhibitor (10−6 M) for 40 hours. Cell extracts were added to H-Gly-Arg-AMC in assay buffer  and incubated for 1 hour at 37°C. After incubation, substrate cleavage was measured by fluorophotometer at excitation and emission wavelengths of 380 nm and 460 nm.DPPI activity was assayed by the liberation of the fluorescent leaving group, 7-amino-4-ethyl coumarin (AMC), and measured as increase in fluorescence using a fluorometer. Briefly, purified CD8+ T cells as effector cells in spleen from NOD mice were stimulated with PMA/IM in the presence of cathepsin L inhibitor  for 24 hours. Maximum cytotoxicity was induced by 0.1% Triton X. 51Cr -labeled target cells were incubated with the effector cells for 6 hours at the indicated E:T ratios. 51Cr release of the supernatants was measured with a g-scintillation counter . Corrected % lysis was calculated as: corrected % lysis = 100× (test 51Cr released – spontaneous 51Cr released)/(maximum 51Cr released - spontaneous 51Cr released).For target cells, the enriched islets were obtained from NOD mice at 4-weeks-old as previously described in vivo therapeutic effects on the T1D in CY-treated NOD mice. Sequences of the oligonucleotide were as follows: cathepsin L: 5′-GAGCGAUAUGGGAGAAGAATT-3′ and negative control: 5′-ATCCGCGCGATAGTACGTA-3′. Briefly, equal volumes of atelocollagen Small interfering RNA (siRNA) of cathepsin L and negative control  were used for analysis of t test was used for statistical analysis. Values of p<0.05 were considered as significant.The Student' Figure S1(A) Effect of Cat-L and S inhibitors on antigen processing and presentation. Splenic CD4+ T cells from OVA-specific T cell receptor transgenic mice (OT-II) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and co-cultured with T cell-depleted spleen cells as APCs from B6 mice in the presence of OVA protein with or without CatL-inh or CatS-inh for 3 days. (B) Proliferation to OVA was evaluated by divided cells (%). Data are shown as means ± s.d. of triplicate wells *, P<0.05, **, P<0.01. Results are representative of two independent experiments.(0.39 MB TIF)Click here for additional data file.Figure S2The mRNA expressions of cathepsin L in PLN cells from C57BL/6 mice. The mRNA expressions of cathepsin L in purified CD8+ T cells, CD4+ T cells, CD25+CD4+ T cells, B220+ B cells and CD11c+ dendritic cells of PLNs from C57BL/6 mice were detected by real-time PCR. Data are shown as means ± s.d. of triplicate wells, and representative of three mice.(0.20 MB TIF)Click here for additional data file.Figure S3Evaluation of toxic effect of cathepsin L inhibitor on CD8+ T cells. CD8+ T cells from NOD mice were incubated with CatL-inh (10-5 M) for 24 hours. Apoptotic or necrotic cells were detected with annexin-V and propidium iodide (PI) staining by flow cytometry. Results are representative of three independent experiments.(0.63 MB TIF)Click here for additional data file.Figure S4The toxic effect of CatL-inh on enriched pancreatic islet cells. (A) The islet cells were enriched from the pancreas of NOD mice, and then stained with anti-insulin mAb  and Alexa568-conjugated mouse IgG (H+L) as the secondary antibody. The nuclei were stained with DAPI. Insulin-producing cells (red) were detected by confocal microscopic analysis. Results are representative of two independent experiments. (B) The islet cells were incubated with CatL-inh  for 24 hours. Apoptotic or necrotic cells were detected with annexin-V and propidium iodide staining by flow cytometry. Results are representative of three independent experiments.(0.78 MB TIF)Click here for additional data file.Figure S5The population of PLN cells from the mice administered with control siRNA or CatL siRNA. The graph shows the mean frequency of CD8+ T cells, CD4+ T cells, B220+ B cells and CD11c+ dendritic cells. Data are shown as means ± s.d. of 3 mice.(0.21 MB TIF)Click here for additional data file.Figure S6Granzyme B expression of pancreatic CD8+ T cells from non-diabetic and diabetic NOD mice was analyzed by flow cytometric analysis. The result is representative of two independent experiments.(0.47 MB TIF)Click here for additional data file.


Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required.Due to the global occurrence of multi-drug-resistant malarial parasites (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 μg mL-1 in shake-flask cultures and 1 g L-1 in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast.Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast. Terpenoids (or isoprenoids) are a large and diverse class of natural products derived from five-carbon building unit, isopentenyl diphosphate (IPP) ,2. The cIn primary metabolism, terpenoids are indispensable components for various physiological processes, such as respiration (ubiquinone), photosynthesis (plastoquinone), membrane fluidity (cholesterol), and intracellular signaling cascades (protein prenylation). Terpenoid metabolism is also responsible for creating a wide array of related, yet chemically distinct natural products, which play important roles in interactions among organisms and defense mechanisms against biotic stresses ,5. Many The pharmaceutical, chemical, and food industries that supply terpenoid commodities face two critical issues. First, the chemical complexities of terpenoids hinder economic chemical synthesis of terpenoids. To date, the supply of many terpenoid compounds still depends on the isolation of natural terpenoids or the pathway intermediates from plant sources. Second, chemical intermediates and solvents required for the organic chemical synthesis of terpenoids are often petroleum-derived chemicals whose availability is finite. To circumvent these problems, current biotechnological efforts have been focused on devising novel biological processes to manufacture complex terpenoids using enzymes and engineered microbial platforms. One example of biological manufacturing of terpenoids is the production of the anti-malarial drug artemisinin precursor, artemisinic acid, using recombinant enzymes in microbial platforms ,8.Artemisia annua). Artemisinin is a potent anti-malarial drug whose mode of action in curing malaria is proposed to include inhibition of the SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) activity of Plasmodium falciparum  as well as genes known to respond to oxidative and osmotic stresses. The studies presented here provide the basis for future strain optimization to improve artemisinic acid production.ADS plasmid, the 5'-end of Leu2-coding sequence from pRS425-Leu2::ADS was amplified by PCR using a pair of primers: forward primer, 5'-ACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTG-3', and a reverse primer, 5'-ATGTCTGCCCCTAAGAAGATCGTCGTTTTGCCAGGTGACCACGTTGG-3'. This PCR-product was used as a template for the second PCR using a pair of primers: a forward primer, 5'-ATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACC-3' and a reverse primer, 5'-CGGGGCGCCTTTTTTTATATATATTTCAAGGATATACCATTGTAATGTCTGCCCCTAAGAAGATCG-3'. The deleted promoter was introduced using the reverse primer. The PCR-product was digested using EcoRV and NarI and cloned into the EcoRV- and NarI-digested pRS425-Leu2::ADS.The Leu2d allele codes for an identical Leu2 protein sequence, but a significant portion of its promoter was deleted, leaving only a 29-bp promoter region. In order to generate pRS425-Leu2d::EcoRV and DraIII, and the EcoRV- and DraIII-digested fragment from pRS425-Leu2d::ADS was ligated into the pESC-Leu plasmid. The ADS expression cassette in pRS425-Leu2d::ADS was amplified by using primers 5'-GTCAATCACTACGTGTAGTACGGATTAGAAGCCGCCGAGC-3' and 5'-GTCAATGCCGGCCAAATTAAAGCCTTCGAGCGTCCCA-3'. The PCR-product was digested using DraIII and NaeI and ligated into the corresponding sites in pESC-Leu2d or pESC-Leu. The coding sequence for AMO from pESC-URA::AMO/CPR was digested using PacI and NotI and ligated into the PacI and NotI of pESC-Leu2d::ADS and pESC-Leu::ADS. The coding sequence for CPR from pESC-URA::AMO/CPR was digested by NotI and NheI and ligated into the NotI and NheI sites of pESC-Leu2d::ADS/AMO and pESC-Leu::ADS/AMO, resulting in the plasmids, pESC-Leu2d::ADS/AMO/CPR and pESC-Leu::ADS/AMO/CPR, respectively.In order to generate pESC-Leu2d vector, the pESC-Leu plasmid was digested using MATαα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) [GAL1-tHMGR PGAL1-upc2-1 erg9::PMET3-ERG9 PGAL1-tHMGR PGAL1-ERG20 plus pRS425-Leu::ADS and pESC-Ura::AMO/CPR) have been previously described [ura3Δ0 and lys2Δ0 in EPY300 was accomplished by transformation with a URA3 PCR amplicon , selecting for uracil autotrophy, then transforming the resulting uracil autotroph with a LYS2 PCR amplicon  and selecting for lysine autotrophy. All OD600  measurements were taken using a Beckman DU-640 spectrophotometer. Culture tubes containing 5 mL of CSM medium with 2% glucose omitting Leu, His, and Met were inoculated with the strain of interest. These inocula were grown at 30°C to OD600 between 1 and 2. Unbaffled culture flasks (250 mL) containing 50 mL CSM medium omitting Leu and His or YPG medium  supplemented with 1 mM Met were inoculated to an OD600 of 0.03–0.05 with the initial seed cultures for induction. For plasmid stability determination, dilutions of the cultures were plated on CSM or CSM without Leu (2% glucose) and grown for 3–4 days at 30°C before counting colonies.Strains and their genotypes are given in Table  ura3Δ0) . The chrescribed . StrainsGas chromatography (GC) and GC-mass spectrometry were used to identify and quantify amorphadiene and artemisinic acid as described previously .ACT1 (5'-GCAAACCGCTGCTCAATCTTCT-3' and 5'-CAAAGCGGTGATTTCCTTTTGC-3'); PDR1 (5'-ACTTACCGCAGCCCTTTGTGAG-3' and 5'-TGTCGCATGTGAAGTCCACGTA-3'); PDR3 (5'-TTCTCCTTGCCCCATCAAGAAA-3' and 5'-CTCCCAACGAGACAATCCCATC-3'); PDR5 (5'-TTTTCTAGTGCCGCCTGGGTTA-3' and 5'-CACCTGGGTTTAGGCAACCATC-3'); PDR12 (5'-TGTCCCGAGCTTGATTTCCATT-3' and 5'-CCTCTTGTGGCGTTATCCCAAG-3'); PDR15 (5'-TGTTGGTGTTGCAGGTGAAGGT-3' and 5'-AGGGTTTGCATCAGGTGGACAT-3'); SNQ2 (5'-AACCGCGTTGACTGATCCAAAT-3' and 5'-AAAAGCACCGGAAGTGGATGAA-3'); and YOR1 (5'-GGAGGCTCCAGAAGATGATCCA-3' and 5'-CTAAATCTTCCGGGCCGTCTCT-3'). For semi-quantitative PCR, reactions were conducted for the indicated number of cycles, and products were visualized on agarose gels using standard techniques. For quantitative PCR, 2X master mixes  were used according to manufacturer's recommendations on a Bio-Rad iCycler instrument. Three independent transformants of each strain were assayed in triplicate (n = 9), and the coefficient of variation of CT for all replicate measurements was < 3%. Relative gene expression was calculated using the Pfaffl method [ACT1 as the reference transcript.Cells induced in YPG were harvested by quick centrifugation and immediately frozen in liquid nitrogen. Cells were disrupted with glass beads in a bead beater, and total RNA was extracted using the RNEasy Mini kit (QIAgen) following the manufacturer's recommendations. RNA was quantified by spectrophotometry, and its integrity verified with a 2100 Bioanalyzer (Agilent). The RNA (450 ng) was treated with the Turbo-DNA free kit (Ambion), and 350 ng of the treated RNA was reverse-transcribed with SuperScript III (Invitrogen) using an oligo(dT) primer following manufacturer's recommendations. The cDNA synthesis reactions were treated with RNAse H, diluted five fold, and 2 μl of the diluted cDNA served as template for PCR. The PCR primers used were the following: l method  with ACT. Labeled cDNA targets were hybridized to cDNA microarray slides double-spotted with the 6,240 predicted ORFs of S. cerevisiae  and washed using standard methods on a hybridization station (TECAN). Images were acquired with a GenePix Professional 4200A scanner (Axon Instruments), and feature fluorescence intensities were extracted with the GenePix Pro 6.1 software. Four or five replicates slides for each time point were hybridized. Artifactual or aberrant spots were excluded, as well as spots with low signal in both channels and spots for which more than 50% of the values were missing. Lowess normalization was performed using BRB-ArrayTools version 3.5.0 developed by Dr. Richard Simon and Amy Peng Lam . Significant differentially expressed genes were extracted using the SAM software [Five independent transformants of each strain were induced in YPG and harvested by centrifugation after 24, 48 and 72 h of induction and cell pellets were immediately frozen in liquid nitrogen. Total RNA was prepared by hot acid phenol extraction  and furtsoftware , with fasoftware .-1 initial glucose concentration, vitamins and trace metals solution in an initial volume of 720 ml. When glucose was depleted after approximately 21 h, a solution of 186 g L-1 of glucose, vitamins and minerals started to be added in the bioreactor with feed-rate addition controlled by a D.O stat algorithm [-1 of galactose, vitamins and trace metals which was added into the bioreactor for 72 h at a feed rate also controlled the D.O. stat.The fermentation was carried using a Sartorius Biostat B+ bioreactor. The temperature was kept at 30°C and pH was kept at 5.5 with addition of 4 M NaOH. The OD was monitored using an Optek ASD19-N single channel NIR absorption probe and the off-gas analysis was monitored by a Thermo Onix VG Prima δB mass-spectrometer. The yeast was firstly grown in batch fermentation with 27 g Llgorithm . After 4DKR, MO, EMP, CP, JDN, and JDK designed experiments. DKR, MO, and EMP conducted plasmid construction, analytical chemistry, and molecular biology. DE and CP conducted yeast genetics. HB conducted the fermentation experiment. All authors contributed to the manuscript writing.A list of genes significantly differentially expressed in EPY330 or EPY338. The additional data file includes a processed data set for differentially expressed genes identified by microarray analysis. The data set has 2,156 genes that were significantly differentially expressed in at least one experimental time points and has 488 and 256 genes that were up-regulated and down-regulated, respectively, in the producer strain by more than 2-fold at least one time points.Click here for file


BCNU (carmustine), VM26 (teniposide) and ARA-A5'P (vidarabin-monophosphate) were compared in their activity against 30 cell lines of primary (N = 21) and metastatic (N = 9) human brain tumours, which were characterized in tissue culture by cytochemical, immunological and cytogenetic criteria. In vivo achievable concentration-time products c X t were correlated with in vitro pharmacokinetic data in order to evaluate in vitro drug sensitivity at relevant exposure doses. A microcytotoxicity assay was employed to screen for drug toxicity in individual tumour cell lines. Following drug exposure and 5 to 8 population doubling times of untreated controls, RNA-synthesis - as a parameter of cell metabolism and proliferation - was determined by incorporation of -uridine into cellular RNA (liquid scintillation counting protocol). The cytotoxic effect of each drug on individual cell lines was expressed in terms of a sensitivity index (SI); by these means effects of different drugs on individual tumour cell lines could be compared. Mean sensitivity indices of ARA-A5'P, BCNU and VM26 for primary brain tumour cell lines were 0.59, 0.82 and 0.54. ARA-A5'P and VM26 had almost similar activities against brain tumour cell lines, whereas BCNU was significantly (P less than 0.001) less active. High grade gliomas were less sensitive to all three agents than low grade and infratentorial gliomas. ARA-A5'P was also able to effectively reduce colony formation in brain tumour cell lines. A cross-resistance of ARA-A5'P to either BCNU or VM26 could not be observed. Clearly, ARA-A5'P is an effective drug in treatment of brain tumour cells in vitro.


The risk of zoonotic infection by porcine endogenous retroviruses (PERV) has been highlighted in the context of pig-to-human xenotransplantation. The use of receptors for cell entry often determines the host range of retroviruses. A human-tropic PERV subgroup, PERV-A, can enter human cells through either of two homologous multitransmembrane proteins, huPAR-1 and huPAR-2. Here, we characterised human PARs and their homologues in the PERV-A resistant rodent species, mouse and rat .Upon exogenous expression in PERV-A resistant cells, human and rat PARs, but not muPAR, conferred PERV-A sensitivity. Exogenously expressed ratPAR binds PERV-A Env and allows PERV-A infection with equivalent efficiency to that of huPAR-1. Endogenous ratPAR expression in rat cell lines appeared to be too low for PERV-A infection. In contrast, the presence of Pro at position 109 in muPAR was identified to be the determinant for PERV-A resistance. Pro109. was shown to be located in the second extracellular loop (ECL2) and affected PERV-A Env binding to PAR molecules.The basis of resistance to PERV-A infection in two rodent species is different. Identification of a single a.a. mutation in muPAR, which is responsible for mouse cell resistance to PERV-A highlighted the importance of ECL-2 for the viral receptor function. Our updFurther evidence to support the predicted topology was obtained utilising a glycosylation study. Using NetNGlyc 1.0 software , one N-gTo further investigate the mechanism responsible for abrogation of PERV-A infection by Pro109 in muPAR, we analysed the binding properties of the receptors. Parental and receptor-transduced QT6 cells, expressing similar levels of HA-tagged receptors  is defective in PERV-A receptor function, whereas the rat cell encodes a fully functional PAR protein. RatPAR can rescue PERV-A infection in non-permissive cell lines, including the resistant rat cell lines from which it has been cloned. The PERV-A infection of rat cells upon overexpression of ratPAR is reminiscent of results from a previous study which show that overexpression of amphotropic MLV and GALV receptors from Chinese hamster cells and FeLV-C receptor from MDTF cells, supports viral infection in the cell lines of their origin . This tyThe defect in muPAR as a PERV-A receptor is due to the presence of Pro at position 109. Our topology study indicated that a.a.. 109 is most likely to be located in the second extracellular loop (ECL2) and potentially accessible for the direct binding by PERV-A Env. Our binding assay consistently detected soluble PERV-A Env binding to cells expressing 'functional' huPARs and ratPAR, but not muPAR. Furthermore, a Leu-to-Pro mutation at a.a. 109 in huPAR-2 abolished Env binding as well as PERV-A infection, further highlighting the important role of this a.a.. These results identified the ECL2 as the likely target for PERV-A Env binding, leading to PERV-A entry. This, together with recent studies on the determinants in PERV Env for binding and entry ,38 contrThe amino acid sequence positions 108–110 of muPAR ECL2, KPY instead of QLH, is intriguingly unique in murine species. Since rats share QLH at the corresponding positions with diverse non-murine species including primates, horse and dog, it is likely that murine species acquired 3 mutations after separating from rats. Although we cannot exclude the possibility that these changes are a stochastic evolutionary outcome, it is more likely that certain selective pressure, at least partly, caused these changes. It is tempting to speculate that severe epidemics of PERV-A like viruses which target the ECL2-QLH structure may have selected 'PERV-A-resistant' murine species with KPY. Our result showing that Leu-to-Pro 109 change alone blocks PERV-A infection raises the question why changes are also required at positions 108 and 110. It is possible that all three a.a. changes were required to escape viral attacks in the past. Alternatively the acquisition of Lys108 and Tyr110 by murine species might be required to maintain yet unknown physiological function of PAR while escaping deadly viruses. To further gain insight into this hypothesis, as well as to study involvement of PAR in the possible PERV-A pathology, identification of physiological roles of PAR is warranted.Different bases for PERV-A resistance between mice and rats are shown. Expression of endogenous ratPAR in rat cells appear to be under a threshold level to support PERV-A infection. In mice, a single a.a. mutation in muPAR in the ECL2 is responsible for the resistance to PERV-A infection. ECL2 in muPAR has a unique sequence with three a.a. changes compared with a wide range of species. Possible selective pressure may have caused this ECL2 diversion in mice.Mus dunni tail fibroblast), rat NRK [ECACC: 86032002] HSN cells [Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle Medium  supplemented with 15% fetal bovine serum . Quail QT6 cell [ECACC: 93120831], murine MDTF (SN cells  and XC  by two-step PCR using primers G3;G9 and G10;G8, then G3;G8, where primers G9 and G10 contain the nucleotide change. Primer G8 includes a HindIII restriction site which allows the cloning of the mutant receptor into pcDNA3/huPAR-2HA. Chimeric receptors H2M a and f were obtained by mix-and-match cloning between huPAR-2 and muPAR using the restriction sites EcoRI and NheI. The other huPAR-2-derived chimeric receptors were produced in a similar way using mutagenesis primers G11;G12 (H2M e) and G13;G14 (H2M d) in association with the primers G3;G8. Similarly, muPAR-derived chimeric receptors were produced using primers G15;G16 (H2M b) and G17;G18 (H2M c) in combination with primers G6;G7.An NheI restriction site was introduced into huPAR-2 at the site corresponding to that in muPAR . The M1 primer introduced the Kozak sequence in front of the ATG of the receptor. The PCR product was cloned into pcDNA3 using EcoRI and NotI restriction sites present in the primers. HA-tagged C-terminal ratPAR was obtained by PCR using KOD HiFi polymerase and the primers M1;M3 which contain the HindIII restriction site, and introduced into pcDNA/huPAR-2HA. This product was then subcloned into pCFCR.Total RNA from NRK cells was extracted using the RNeasy kit (Qiagen) and incubated with 5 U of RNase-free DNase (Promega) for 30 min at 37°C. First strand cDNA was produced by incubation of 2 μg of DNase-treated RNA with 200 U of Moloney MLV Reverse Transcriptase (Promega), 1 μg of random primers (Promega), 20 U of RNasin Ribonuclease Inhibitor (Promega), 1 mM dNTPs (Qiagen) in a final volume of 20 μl for 10 min at 25°C, 1 hr at 42°C and an inactivation step of 10 min at 70°C. The ratPAR coding sequence was then amplified using HotStart polymerase (Qiagen) and primers M1;M2 with PCR conditions: 95°C 30 sec, 52°C 30 sec, 72°C 90 sec. Primers M1 and M2 were designed to anneal to the rat homologue of huPAR-1 , dog PAR [RefSeq: XP_532355], horse PAR [RefSeq: XP_001505049], pig PAR [Swissprot: Q863Y7], cow PAR [RefSeq: NP_001069369], muPAR [RefSeq: NP_083919] and ratPAR [RefSeq: NP_001103140].The amino acid sequences used in this study are: huPAR-1 [RefSeq: NP_078807] and huPAR-2 [RefSeq: NP_060456], chimpanzee PAR-1 [RefSeq: XP_001156784] and PAR-2 [RefSeq: XP_001164395], Rhesus macaque PAR-1 [RefSeq: XP_001091189] and PAR-2 [RefSeq: XP_001099620], baboon PAR-2 [Swissprot: The author(s) declare that they have no competing interests.YT conceived the study. GM and YT designed the experiments and wrote the manuscript. GM carried out the experiments. MM contributed to cloning and initial characterization of ratPAR. All the authors read and approved the final manuscript.PERV-A receptors cell surface expression. Expression of C-terminal HA-tagged PAR constructs in QT6 cells was demonstrated by flow cytometry analysis following surface immunostaining with an anti-HA antibody.Click here for fileRatPAR function as PERV-A receptor depends on its expression on cell surface. PERV-A infection was measured in quail QT6 cell clones expressing different levels of ratPAR.Click here for file


The dicarboxylic acids and their metal complexes werecharacterized by their physical, analytical and spectral data. The [M(L)] complexes showed a square planargeometry whereas an octahedral geometry was observed for [M(L)Cl2] complexes. The title dicarboxylicacids and their metal complexes have also been screened for their antibacterial activity.Some novel symmetric 1,1′-dimethylferrocene derived amino acids have been prepared by the reaction of1,1′-ferrocenedimethyldichloride with amino acids . TheirCu(II), Co(II) and Ni(II) complexes, of the type [M(L)] where [M = Cu(II) and L = L


The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis. Mutations in the G-CSFR in patients with severe congenital neutropenia (SCN) transforming to acute myelogenous leukemia (AML) have been shown to induce hypersensitivity and enhanced growth responses to G-CSF. Recent studies have demonstrated the importance of the ubiquitin/proteasome system in the initiation of negative signaling by the G-CSFR. To further investigate the role of ubiquitination in regulating G-CSFR signaling, we generated a mutant form of the G-CSFR (K762R/G-CSFR) which abrogates the attachment of ubiquitin to the lysine residue at position 762 of the G-CSFR that is deleted in the Δ716 G-CSFR form isolated from patients with SCN/AML. In response to G-CSF, mono-/polyubiquitination of the G-CSFR was impaired in cells expressing the mutant K762R/G-CSFR compared to cells transfected with the WT G-CSFR. Cells stably transfected with the K762R/G-CSFR displayed a higher proliferation rate, increased sensitivity to G-CSF, and enhanced survival following cytokine depletion, similar to previously published data with the Δ716 G-CSFR mutant. Activation of the signaling molecules Stat5 and Akt were also increased in K762R/G-CSFR transfected cells in response to G-CSF, and their activation remained prolonged after G-CSF withdrawal. These results indicate that ubiquitination is required for regulation of G-CSFR-mediated proliferation and cell survival. Mutations that disrupt G-CSFR ubiquitination at lysine 762 induce aberrant receptor signaling and hyperproliferative responses to G-CSF, which may contribute to leukemic transformation. The granulocyte colony-stimulating factor receptor (G-CSFR) plays a critical role in myeloid cell survival, proliferation, and neutrophilic maturation Ligand binding has been shown to trigger intracellular signaling as well as endocytosis, internalization, and subsequent intracellular degradation of growth factor receptors. For the Type I cytokine receptors to which the G-CSFR belongs, endocytosis and degradation have been best characterized for the growth hormone receptor (GH-R), interleukin-2 receptor (IL-2R), and the erythropoietin receptor (EpoR) We previously examined the role of the ubiquitin/proteasome system in G-CSFR internalization and intracellular sorting To further investigate the role of ubiquitination in G-CSFR-mediated cell proliferation, we generated a mutant form of the G-CSFR (K762R/G-CSFR) which abrogates the attachment of ubiquitin molecules to the lysine residue at position 762 of the receptor that is deleted by mutations in the G-CSFR in patients with SCN/AML. In response to G-CSF, mono-/polyubiquitination was impaired in cells transiently expressing the mutant receptor form. Cells stably transfected with the K762R/G-CSFR exhibited increased growth and enhanced sensitivity to G-CSF, and extended cell survival following G-CSF depletion. Activation of both Stat5 and Akt were also prolonged in K762R/G-CSFR transfected cells in response to G-CSF, and their activation remained prolonged even after G-CSF withdrawal. These data indicate that ubiquitination of the C-terminal lysine in the G-CSFR that is detected in patients with SCN/AML is required for normal G-CSFR-mediated cell proliferation and survival.We previously reported that the WT G-CSFR undergoes both mono- and polyubiquitination in response to G-CSF, which could be detected using the commercially available FK2 antibody We previously reported that BaF3 cells transfected with the Δ716 G-CSFR that is detected in a majority of patients with SCN transforming to AML exhibit hyperproliferative responses to G-CSF 5 cells/ml, and grown in 2 ng/ml of G-CSF for a period of 18 days. As shown in We stably transfected BaF3 cells with either the WT- or the K762R/G-CSFR construct and isolated by limited dilution three independent clones each expressing similar levels of the WT or the K762R/G-CSFR, removed them from IL-3-containing media, washed them, then treated them with varying concentrations of G-CSF for a period of 72 h. As shown in We next examined the survival capacity of the K762R transfectants compared to WT transfectants following removal from G-CSF-containing media. For these experiments, we used pools of three independent clones of WT transfectants or K762R transfectants and analyzed their survival following incubation in media containing 10 ng/ml of G-CSF and subsequent transfer to cytokine-free media. As shown in To confirm that our observations in the BaF3 lymphoid cell line were physiologically relevant, we next examined the growth and survival of 32D myeloid cells transfected with the K762R mutant. As shown in To determine the mechanisms underlying the aberrant response of cells expressing the K762R/G-CSFR mutant to G-CSF, we next examined the activation of Stat5 and Stat3 in these cells, pathways known to be activated by G-CSF. For these experiments, cells were washed out of IL-3-containing media, treated with G-CSF for 10 min, washed, then transferred to cytokine-free media for up to 2 h. Whole cell lysates from WT- and K762R/G-CSFR-transfectants were then analyzed for evidence of phosphorylation of Stat5 and Stat3 by immunoblot analysis with antibodies to phosphor-Stat5 and phosphor-Stat3. As shown in We next investigated activation of the Akt signaling pathway in response to G-CSF, which we have previously shown to be important in G-CSF-mediated cell survival and proliferation. Cells were stimulated with G-CSF for 10 min, lysed, and immunoprecipitated with Akt antibody, then blotted with an antibody recognizing the phospho-serine Akt species. As shown in Receptor desensitization rapidly follows growth factor receptor activation and serves as a mechanism for controlling the amplitude and duration of intracellular signaling. Disruption of the delicate balance between growth factor receptor activation and inactivation has been shown to be associated with severe human pathologies For many receptors, ligand-induced ubiquitination has been shown to be a crucial initial event in attenuation of receptor signaling In the current study, we replaced the lysine residue at position 762 of the G-CSFR with an arginine residue, and examined the effects of this mutation on G-CSF-mediated cellular responses and signaling. CHO cells expressing the K762R/G-CSFR exhibited markedly reduced mono- and polyubiquitination of the G-CSFR in response to G-CSF compared to cells expressing the WT receptor, indicating that lysine 762 is critical for ligand-induced G-CSFR ubiquitination. BaF3 cells stably transfected with the mutant K762R/G-CSFR were found to by hypersensitive to G-CSF and could survive and proliferate in G-CSF concentrations as low as 0.08 ng/ml, a concentration that was insufficient to support the growth and survival of cells expressing the WT receptor. Furthermore, at G-CSF concentrations high enough to support the survival of cells expressing the WT G-CSFR, cells expressing the K762R mutant G-CSFR hyperproliferated in response to G-CSF, which was reproducible in three independent experiments using three different K762R clones. Additionally, following withdrawal of the BaF3 transfectants from G-CSF-containing media, cells expressing the K762R/G-CSFR mutant exhibited prolonged survival over WT G-CSFR-expressing cells. These results suggest that ubiquitination of the G-CSFR at lysine 762 is required for the normal G-CSFR-mediated cell growth and survival.To confirm the physiologic relevance of our findings in BaF3 cells, we next examined the G-CSF-responsiveness of myeloid cells expressing the K762R mutant. For these experiments, we used the 32Dcl3 cell line. 32Dcl3 cells stably expressing the K762R/G-CSFR mutant also hyperproliferated in response to G-CSF and exhibited enhanced survival following cytokine depletion. These findings are consistent with our previously reported observations with cells expressing the Δ716-G-CSFR Previous studies have demonstrated that both Stat3 and Stat5 are rapidly activated and phosphorylated in response to G-CSF A recent study by Irandoust et al in which all five lysine residues in the G-CSFR were mutated to arginine was reported to result in hyperproliferative responses to G-CSF Activation of the Akt signaling pathway has been reported to protect a variety of cell types from apoptosis Collectively, our data suggest a role for ubiquitination in G-CSF-mediated cell survival and proliferation. Lysine 762 in the cytoplasmic tail of the G-CSFR appears to be critical in regulating the amplitude and duration of Stat5 and Akt activation. Future studies to identify the molecules that interact with the ubiquitinated G-CSFR and the E3 ligase that is recruited to the phosphorylated G-CSFR should provide additional insights into the mechanisms that downregulate G-CSFR signaling, which are disrupted in patients with SCN/AML.G-CSF-responsive murine 32Dcl3 cells were kindly provided by Dr. Giovanni Rovera  and were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 10% WEHI 3B conditioned medium as a source of IL-3. BaF3 cells were maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 10% FBS, and 10% WEHI-3B conditioned media as a source of interleukin 3 (IL-3). Chinese hamster ovary (CHO) cells were grown in αMEM  supplemented with 4.5 g/L glucose and 10% FBS. Penicillin and streptomycin (100 U/mL each) were added to all culture media. Recombinant human G-CSF was a generous gift from Amgen . Media and cell culture reagents were purchased from GIBCO/Invitrogen .5′-CCCCAGCCCCAGGTCCTATGAGAACCTC-3′, and the reverse primer was 5′-GAGGTTCTCATAGGACCTGGGGCTGGGG-3′. The K762R/G-CSFR was also epitope-tagged with V5. The fidelity of the entire open reading frame of each expression vector was confirmed by automated DNA sequencing. The hemagluttinin (HA)-tagged ubiquitin cDNA was generously provided by Dr. Dirk Bohmann .The wild type G-CSFR (WT G-CSFR) cDNA was cloned into the pcDNA3.1D-TOPO mammalian expression vector (Invitrogen) in frame with a V5 epitope tag, as described previously BaF3 cells were stably transfected with the various G-CSFR forms using conditions that have previously been described 3 cells were removed and analyzed using the CellTiter-Glo Cell Viability Luminescence Assay protocol  utilizing the Berthold Technologies Centro XS3 LB 960 plate luminometer and MicroWin 2000 software. Cells were also counted at each time point and cell viability was determined by Trypan Blue staining. For proliferation experiments, cells were grown in different concentrations of G-CSF for 72 h to determine their dose response to G-CSF. Aliquots of cells were subsequently grown in 2 ng/ml of G-CSF for either 72 h (32Dcl3 cells) or 18 days (BaF3 cells) and cell numbers counted at each time point.Stably transfected BaF3 and 32Dcl3 cells (WT and K762R mixed-pools) were removed from IL-3-containing media, washed, and then grown in the presence of 10 ng/ml of G-CSF at 37°C overnight. Cells were then washed and resuspended in cytokine-free media and incubated at 37°C for 0 h, 4 h, 8 h, 12 h, and 24 h. At each time point, 5×10Antibody to V5 was purchased from Invitrogen . Anti-poly/monoubiquitin antibody raised against the FK2 clone was purchased from Affiniti Research . Antibodies to phospho-Stat3 (pStat3), phospho-Stat5 (pStat5), and phospho-serine-Akt (pSerAkt) were purchased from Cell Signal . Antibodies to Stat3, Stat5, Akt, and actin were from Santa Cruz .7/mL) were lysed in ice-cold lysis buffer , 0.1 mM sodium deoxycholate, 500 mM NaCl, 5 mM EDTA, 10 mM N-ethylmaleimide, 25 mM HEPES, pH 7.8) containing a cocktail of protease inhibitors , incubated on ice for 20 min, and centrifuged at 4°C at 10,000 g. The supernatants were collected from each sample and protein concentrations determined using the BCA reagent . A total of 400 mg of protein from each sample in equivalent final volumes was mixed 1∶1 with IP buffer , and incubated with 1–2 µg antibodies and 30 µL of protein G-agarose (Invitrogen) overnight at 4°C. Immunoprecipitates were collected by centrifugation at 2,000 g at 4°C, washed 3× with a 1∶1 mix of lysis and IP buffers, and resuspended in LDS sample buffer (Invitrogen). The samples were heated to 70°C for 10 min, resolved on 4–12% Bis-Tris acrylamide gels using a MOPS running buffer (Invitrogen), and transferred to nitrocellulose. For analysis of whole cell lysates, 20–80 µg of protein from each sample was resolved by SDS-PAGE and subjected to immunoblot analysis. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) reagent . For densitometric analysis, ImageJ software made available through the National Institutes of Health was used.Cells  for 4 h at 4°C, and incubated with 100 ng/mL G-CSF at 37°C for varying times. For signaling studies, cells were washed and incubated in serum-free media (RPMI/0.1%BSA) devoid of all cytokines and incubated at 37°C for 4 hours. Cells were then treated with G-CSF (100 ng/ml) at 37°C for 10 min, washed, incubated in serum and cytokine-free media for varying times, and lysed. Aliquots of cells were stimulated with activated orthovanadate (5 µl/100 µl cell suspension) at room temperature for 20 min as a positive control. Cell lysis, immunoprecipitations and immunoblotting were performed as described above.


We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT).Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments.Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT.The diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP. The human immunodeficiency virus type 1 (HIV-1) epidemic has rapidly evolved to include 6 major circulating subtypes  and numerous recombinant forms, showing 25–35% overall genetic variation, including 10–15% in reverse transcriptase (RT) . SubtypeGenetic divergence in the RT enzyme may also be linked to differential acquisition of resistance to nucleoside or nucleotide RT inhibitors (N(t)RTIs) that are core constituents of antiretroviral (ARV) regimens for treatment of HIV-1 infection. These drugs include the eight N(t)RTIs approved for clinical treatment of HIV-1 infection: zidovudine (ZDV), stavudine (d4T), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), abacavir (ABC), emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) .The RT mutation K65R can be selected by each of tenofovir (TFV), ddI, ddC, ABC and d4T and yields decreased susceptibility to all clinically used NRTIs except ZDV -9. Our lGA while it is AAG → AGG in subtype C. The present study was designed to determine if variations in enzymatic function might be responsible for the higher propensity of K65R to occur in subtype C. In this work, we have characterized the enzymatic properties of recombinant B and wild-type RTs as well as RTs harboring the K65R and K65R/M184V mutations.Although subtype C viruses harbour a unique KKK nucleotide motif, amino acid polymorphisms and codon bias at position 65 cannot explain the differential acquisition of K65R in subtype C variants. In subtype B the mutation required in codon 65 is AAA → ARecombinant heterodimer (p66/p51) RTs from both subtype C and B were purified to > 95% homogeneity; all RT subunits p66 and p51 were processed to similar molar ratio Fig. . To detePrevious cell culture assays showed that viruses of subtypes A/E, B, C harboring K65R exhibited similar 6.5 to 10-fold resistance to TFV .The reduced efficiency of initiation of (-)ssDNA synthesis and tRNA primer usage, associated with subtype B RTs harboring K65R and K65R+M184V is a mechanism responsible for the diminished replicative fitness of viruses containing these substitutions Fig  [18]. InAnalyses of single-cycle processivity were performed with HIV PBS RNA and 5'-end labeled dPR primer under variable dNTP concentrations with heparin as a trap. The products of this primer extension assay were separated on a 6% PAGE-7M urea sequencing gels and subjected to phosphorimager analysis FIG. . At highP ≤ 0.01). Thus, the relative inhibitory capacity (Ki/Km) for TFV-DP was increased by 7.2-fold compared to WT. For the double mutant K65R/M184V, the Km value for dATP was significantly increased compared to that of WT (P ≤ 0.01) and the Ki value for TFV-DP was also increased compared to WT (P ≤ 0.01). Ki/Km was elevated by 4.1-fold compared to WT. These results are in agreement with published data obtained with subtype B RTs  dTTP (70–80 Ci/mmol) was from Perkin Elmer Life Sciences. [α-32P]dNTPs and [γ-32P]ATP were obtained from MP Biomedicals.Tenofovir diphosphate (TFV-DP) was kindly provided by Gilead Sciences . Zidovudine triphosphate (ZDV-TP) was purchased from Trilink Biotechnologies . Poly(rA)/oligo(dT)12–18 ultrapure dNTPs, NTPs and ddATP were purchased from GE Healthcare. dTTP and variable amounts of wild-type or mutated RT. Reactions were performed at 37 w and aliquots of 15 ul were removed at 3 min, 9 min, 15 min and quenched with 0.2 ml of 10% cold trichloractic acid (TCA) and 20 mM sodium pyrophosphate. After 30 min on ice, the precipitated products were filtered onto 96-well plates using glass fiber filters (Millipore) and sequentially washed with 10% TCA and 95% ethanol. The radioactivity of incorporated products was analyzed by liquid scintillation spectrometry. The incorporated [3H] dTTP was plotted as cpm versus time and initial velocities were determined from the slopes of the linear regression analyses using GraphPad Prism 4.0 software. Specific activities were calculated as described previously dATP (> 70–80 Ci/mmol), 5 U of RNase inhibitor and variable concentrations of dATP in the absence or presence of TFV-DP. Reactions were incubated at 37°C. Aliquots were removed at 3 min, 7 min, 15 min and quenched with 10% trichloroacetic acid (TCA) and 20 mM sodium pyrophosphate. After 30 minutes on ice, the precipitated products were filtered onto 96-well glass fibre filter plates (Millipore), washed twice with 10% TCA and once with 95% ethanol. Incorporated radioactivity was measured by liquid scintillation counting. Kinetic constants were determined using Graphpad Prism 4.0 software as described [The Km for dATP and the Keviously . In brieescribed .32P]-dCTP.The efficiency of (-)ssDNA synthesis was determined by cell-free assay as described ,31,37. BAliquots were removed at various time points and quenched with 95% formamide-40 mM EDTA. Samples were resolved in 6% polyacrylamide-7M urea gel and analyzed by using the Molecular Dynamics Typhoon Phosphorimager system . To study the effect of mutated RTs on the initiation of synthesis of (-)ssDNA, the above reactions were initiated with 1 μM dNTPs, except ddATP was employed as a termination nucleotide instead of dATP to give rise to a six-nucleotide initiation product. Products were separated as described above and analyzed by ImageQuant software.Lys – HIV PBS RNA complexes were incubated with either WT or mutant RT enzymes to initiate the RT reaction in the presence of 10 μM dNTPs. Time-course experiments were performed, and products were separated and analyzed by ImageQuant software as described above.Using the same gel-based system as described ,37, we e2P]ATP. The dPR primer (500 nM) containing labeled dPR as tracer was annealed to PBS RNA transcript. RT (50 nM) was then preincubated with the T/P for 5 min at 37°C before initiation of the reaction by the addition of dNTPs using a heparin trap (1 mg/ml). Three concentrations of dNTPs were assayed: 200 μM, 5 μM and 2 μM. After 30 min of incubation at 37°C, aliquots of the reaction mixtures were removed and quenched with 95% formamide-40 mM EDTA. The samples were heated at 100°C for 5 min, then analyzed by 6% polyacrylamide-7M urea gel. Resolved products were analyzed by phosphorimager.The 18-nt DNA primer dPR complementary to the viral PBS was 5'end labeled using [γ-To generate TFV- or ZDV-terminated primers, primers 17D and 19D were first radiolabeled at the 5' end and subsequently extended with TFV-DP and ZDV-TP respectively using cWT RT and annealed to template oligonucleotide 57D as described . ExcisioThe authors declare that they have no competing interests.HX performed experiments and drafted the manuscript. JLM-C aided in drafting the manuscript. MLN performed sequencing reactions. DC performed experiments and aided in drafting the manuscript. FAF performed sequencing experiments. BGB aided in drafting the manuscript. MAW supervised the project, aided in drafting the manuscript, and provided resources for the research.


The 250 kDa P2P-R protein  was cloned over a decade ago and was found to bind both the p53 and Rb1 tumor suppressor proteins. In addition, P2P-R has been associated with multiple biological functions, such as mitosis, mRNA processing, translation and ubiquitination. In the current studies, the online GeneNetwork system was employed to further probe P2P-R biological functions. Molecular studies were then performed to confirm the GeneNetwork evaluations.GeneNetwork and associated gene ontology links were used to investigate the coexpression of P2P-R with distinct functional sets of genes in an adipocyte genetic reference panel of HXB/BXH recombinant strains of rats and an eye genetic reference panel of BXD recombinant inbred strains of mice. The results establish that biological networks of 75 and 135 transcription-associated gene products that include P2P-R are co-expressed in a genetically-defined manner in rat adipocytes and in the mouse eye, respectively. Of this large set of transcription-associated genes, >10% are associated with hormone-mediated transcription. Since it has been previously reported that P2P-R can bind the SRC-1 transcription co-regulatory factor , the possible effects of P2P-R on estrogen-induced transcription were evaluated. Estrogen-induced transcription was repressed 50-70% by the transient transfection of P2P-R plasmid constructs into four different cell types. In addition, knockdown of P2P-R expression using an antisense oligonucleotide increased estrogen-mediated transcription. Co-immunoprecipitation assays confirmed that P2P-R interacts with SRC-1 and also demonstrated that P2P-R interacts with estrogen receptor α.The findings presented in this study provide strong support for the value of systems genetics, especially GeneNetwork, in discovering new functions of genes that can be confirmed by molecular analysis. More specifically, these data provide evidence that the expression of P2P-R co-varies in a genetically-defined manner with large transcription networks and that P2P-R can function as a co-repressor of estrogen-dependent transcription. It is based on the analysis of transcripts whose expression co-vary within genetic populations, such as the BXD strains of mice and the HXB strains of rats or other genetic reference panels. GeneNetwork The importance of defining biological networks and predicting molecular interactions has been emphasized by several reports ,2. Such In 2005, we published the first report documenting the ability of the systems genetics tool GeneNetwork to predict interactions between molecules that could be then confirmed by molecular analysis . The P2PFrom the time when P2P-R was cloned it has been implicated in multiple cellular processes such as mitosis ,9, mRNA The outcome of these systems genetics studies implies that P2P-R is an important member of large genetically-defined transcription networks in adipose and eye tissues. That prediction was then confirmed by data showing that P2P-R can function as a co-repressor of estrogen-induced transcription. Immunoprecipitation experiments strengthen such functional studies by confirming that P2P-R interacts with SRC-1 and by demonstrating that P2P-R also interacts with estrogen receptor α (ERα). These data suggest that P2P-R may be a component of large macromolecular complexes that impact steroid-dependent transcription.Since most studies concerning the cloning and characterization of P2P-R were performed using the 3T3T proadipocyte system , we initWhen the top 1000 gene transcripts that are co-expressed with P2P-R and have correlation coefficients ≥ 0.67 were exported for gene ontology analysis to WebGestalt, 67 gene products were found to be associated with transcription with p = 2.81e-4  , Ep300 [+0.8056], Pparbp [+0.7709], Glcci1 [+0.7690], Esrrg [+0.7574], Srebf2 [-0.7568], Ncoa6 [+0.7423], Ncor1 [+0.7115], Ncoa1 (Src-1) [+0.6987] and Ncoa2 [+0.6787] and Nr2f6 [-0.6653] (the parenthetic numbers indicate positive or negative expression correlation coefficients with P2P-R expression).The findings from studies on fat cell-adipose and eye tissues therefore strongly suggest that P2P-R has the potential to function as a member of a genetically-defined transcription complex. Two important questions arise from these findings. Does a P2P-R-associated transcription network exist in all tissues? Can the role of P2P-R in transcription be confirmed by molecular studies? To address the first question, studies were performed on several other tissues in the GeneNetwork database to search for additional P2P-R transcription networks. There was no evidence of a P2P-R-associated transcription network in liver or whole brain databases, however, in a cerebellum database a smaller P2P-R-associated transcription set could be identified (data not shown). These data suggest that the role of P2P-R in transcription may be tissue specific. The studies in the following sections address the second question and show that P2P-R can indeed function as a transcriptional co-repressor.Nuclear Receptor Signaling Atlas online database http://www.nursa.org that P2P-R interacts with Ncoa1 (SRC-1), a well-known steroid receptor co-regulator . In this regard, of special note is the April 1, 2005 report in the see ref. . This daU83913), while HeLa and 293T cells were additionally co-transfected with ERα. Co-transfection experiments were performed in the presence of either vehicle (DMSO) or 100 picomolar 17-beta estradiol (E2) to determine if the transfection of P2P-R influences basal and/or estrogen-dependent transcription. The results presented in Figure Based on these observations, we sought to investigate whether P2P-R impacts estrogen-dependent transcription in cell lines that either contain estrogen receptor α, such as MCF-7 and Ishikawa cells or lack native estrogen receptors, such as HeLa and 293T cells. To this end MCF-7 and Ishikawa cells were transiently co-transfected with an ERα-reporter construct with an empty vector or with a plasmid construct that encodes the near full length P2P-R  The data reported in this paper establish that the function of gene products can be confidently predicted by determining their association with specific genetically-defined biological networks, using systems genetics approaches and tools, such as GeneNetwork. GeneNetwork has been previously used to predict a molecular interaction between P2P-R and Pum2, the gene transcript which is most consistently co-expressed with P2PR in three tissues of the BXD mouse genetic reference panel. That prediction was confirmed by molecular studies showing that Pum2 binds to a specific sequence in the 5'UTR of P2PR mRNA to modulate P2PR expression .The current studies expand the use of GeneNetwork databases in an attempt to identify biological networks in which P2P-R is a member, leading to the prediction of new function(s). Data derived using these systems genetics approaches suggest that P2P-R is an important member of large genetically-defined transcription networks in adipose and eye tissues, thereby having the potential to impact the expression of many hundreds of genes. That prediction was then confirmed by molecular studies showing that P2P-R can indeed function as a transcriptional co-repressor.The results specifically demonstrate that P2P-R acts as a co-repressor of estrogen-dependent transcription in four different cell systems including MCF-7, Ishakawa, HeLa and 293T cells. The co-repressor activity of P2P-R was further confirmed by evidence that knocking down P2P-R protein expression, using an antisense oligonucleotide, facilitates estrogen-induced transcription. Immunoprecipitation experiments support those functional studies by showing that the P2P-R protein is a component of the estrogen-transcription complex that includes ERα and SRC-1. Preliminary data also suggest that P2P-R interacts with the Mad2 mitotic protein , which is of interest in this context, since it has previously been reported that Mad2 associates with estrogen receptor beta .Scaffold/Matrix Attachment Regions) elements of the nuclear matrix, was shown to interact with the C-terminal domain of RNA polymerase II, and with ERα and to suppress ERα-mediated transcription , anti-SRC-1  and anti-ERα . Immunoprecipitations were performed as previously described [4, #SC-72PP and MEB carried out transcriptional assays and co-immunoprecipitation experiments. RAW participated in GeneNetwork analysis. TG and RES designed the study, supervised data collection and analysis and drafted the manuscript. All authors read and approved the final manuscript.List of fat cell transcription network components. Seventy five (75) transcription-associated gene products including P2P-R are genetically co-expressed in fat cells derived from the HXB/BXH rat recombinant inbred genetic reference panel.Click here for fileList of eye tissue transcription network components. One hundred thirty five (135) transcription-associate gene products including P2P-R are genetically co-expressed in the eye tissue derived from the BXD mouse recombinant inbred genetic reference panel.Click here for file


Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission.Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand.Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is connected to apoptosis, a finding that deserves further investigation. The envelope protein (Env) of the human immunodeficiency virus (HIV), a heavily glycosylated type I transmembrane protein, mediates infectious viral entry into target cells . This prReceptor expression levels can limit HIV entry into host cells ,7, and tWe and others have previously shown that apart from dendritic cells, platelets also express DC-SIGN and that these cell fragments bind to HIV in a mainly DC-SIGN-dependent manner ,29. HoweHere, we report that CLEC-2, unlike DC-SIGN, does not bind to the viral Env protein, but to a cellular factor incorporated into the viral envelope. For viruses produced in the kidney-derived cell line 293T, this factor was found to be podoplanin , a cellular mucin-like glycoprotein expressed by kidney podocytes (which are known to be susceptible to HIV infection ) and lym2. Highly purified platelets were obtained from the "Transfusionsmedizinische und Hämostaseologische Abteilung" of the University Hospital Erlangen. Alternatively, platelets were prepared from whole blood by centrifugation at 1200 rpm at RT. The upper platelet-rich plasma was collected and centrifuged at 4000 rpm for 20 min at RT. Subsequently, the supernatant was removed, and platelets were resuspended in RPMI 1640 medium supplemented with 10% FCS and antibiotics. PBMCs were isolated from whole blood or leukocyte filters by centrifugation through a Ficoll gradient and either cultured in RMPI 1640 medium supplemented with 10% FCS and antibiotics or stimulated with PHA (Sigma) at a concentration of 5 μg/ml and IL-2 (Roche) at a concentration of 10 U/ml.293T, 293 T-REx , GP2 293nef was generated by splice overlap extension (SOE) PCR. Briefly, a NL4-3 env fragment was amplified using oligonucleotides pJM206 (binding upstream of the singular HpaI restriction site in env), and pJM394  and pBRNL4-3 [nef was replaced by EGFP. Oligonucleotide sequences : pJM206 5'-TGGAACTTCTGGGACGCAGG-3'; pJM394 5'-GCTCACCAT CTTATAGCAAAATCC;JM395 AGCTATAGATGGTGAGCAAGGGCG-3';JM396 5'-CGTACGCGTTACTTGTACAGC-3'. The gp120-Fc-IgG1 construct [GGATCCCTTATCGTCGTCATCCTTGTAATCC-3' (sense) and gp120_HindIII 5'-GTACGAAGCTTGTGGAGAAGCTGTGGGTGAC-3' (antisense), followed by insertion of the PCR fragment in the BamHI and HindIII restriction sites of the Fc-IgG1 encoding plasmid pAB61 [1 fusion construct, sense primer 5'-GTACGAAGCTTTGCAGCCCCTGTGACACAAAC-3'and antisense primer 5'-GAGTGGATCCAGGTAGTTTCCACCTTGG-3' were used for PCR amplification, and the product was cloned into pAB61 using the HindIII and BamHI restriction sites. CLEC-2 mutants bearing single amino acid changes were generated by overlap extension PCR. The oligonucleotides 5'-GCCGGATCCACCATGCAGGATGAAGATGGATACATC-3' (sense) and GCCGAATTCTTAAGGTAGTTGGTCCACCTTGG (antisense) were used as outer primers and combined with the following inner primers:5'-GATGGAAAAGGAGCCATGAATTGTGC-3' (sense) and 5'-AGCACAATTCATGGCTCCTTTTCCAT-3' (antisense) for generation of mutant CLEC-2 N192A, 5'-TTGAGTTTTTGGCCGATGGAAAAGG-3' (sense) and 5'-TCCTTTTCCATCGGCCAAAAACTCA-3' (antisense) for mutant CLEC-2 E187A, 5'-GTTTTTGGAAGATGGAGCCGGAAATATGAATTGTG-3' (sense) and 5'-AATTCATATTTCCGGCTCCATCTTCCAAAA-3' (antisense) for mutant CLEC-2 K190A, 5'-GCAACATTGTGGAATATATTGCGGCGCGCACCCATCTGATTC-3' (sense) and 5'-GCGCCGCAATATATTCCACAATG-3' (antisense) for mutant CLEC-2 K150A. For generation of DC-SIGN-Fc-IgG1, primers 5'-GTACGAAGCTTGAACGCCTGTGCCACCCCTG-3' (sense) and 5'-GAGTGGATCCCGCAGGAGGGGGGTTTGGGG-3' (antisense) were used. The resulting PCR fragment was cloned into pAB61, using the HindIII and BamHI restriction sites. A PCR fragment encoding the extracellular domain of podoplanin fused to the Fc portion of human immunoglobulin was generated as described above, employing primers 5'-GCCAAGCTTGCCAGCACAGGCCAGCCAGAAGATG-3' (sense) and 5'-GCGGGATCCTGTTGACAAACCATCTTTCTCAAC-3' (antisense) and inserted into the pAB61 plasmid via the HindIII and BamHI restriction sites . The identity of all PCR amplified sequences was confirmed by sequencing with an ABI3700 genetic analyzer (Applied Biosystems) according to the manufacturer's instructions. The plasmid used for transient expression of podoplanin (podoplanin in pcDNA3) has been previously described [The NL4-3-based reporter virus bearing EGFP in place of pBRNL4-3  as templpBRNL4-3  resultinonstruct  was geneonstruct  with priid pAB61 . For genescribed .Replication-competent HIV-1 NL4-3, NL4-3 luc  and NL4-5 cells were incubated with Fc-fusion proteins and Fc-control protein at 4°C for 45 minutes. Subsequently, the cells were washed with FACS buffer and stained with Cy5-conjugated anti-human IgG secondary antibody for 30 minutes at 4°C. Cell-staining was then analyzed by flow cytometry, employing a Cytomics FC500 flow cytometer , and data were analyzed with FCS Express FACS analysis software .For generating soluble Zaire Ebolavirus glycoprotein (ZEBOV-GP)-Fc- , DC-SIGNAnalyses of podoplanin surface expression were performed by flow cytometry, using the podoplanin specific antibodies NZ-1 or 18H5  in combination with secondary anti rat/mouse antibody coupled to Cy5 . Cells were incubated with 10 μg/ml antibody in PBS supplemented with 5% FCS for 30 minutes at 4°C. Subsequently, PBS supplemented with 5% FCS was added, and the cells were pelleted by centrifugation . Finally, cells were resuspended in fixans  and incubated for 30 minutes at 4°C before staining was analyzed by flow cytometry. For all measurements 20,000 gated events were collected.GATCCGCGAAGATGATGTGGTGACTTTCAAGAGAAGTCACCACATCATCTTCGTTTTTTACGCGTG3' and shRNA137antisense_EcoRI: 5'AATTCACGCGTAAAAAACGAAGATGATGTGGTGACTTCTCTTGAAAGTCACCACATCATCTTCGCG3' followed by insertion of the double stranded fragment into the retroviral vector pSIREN-IRES-EGFP-RetroQ [6 293T cells. At 24 h post transduction, cells were washed and incubated for 3 days. Subsequently, transduced cells were selected in medium containing 10 μg/ml puromycin .For stable knock-down of podoplanin in 293T cells, shRNAs were constructed by using shRNA Hairpin Oligonucleotide Sequence Designer Tool . The podoplanin specific shRNA 137 contained the target shRNA sequence, a hairpin loop region "TTCAAGAGA" and an antisense shRNA sequence followed by a pol III terminator sequence. The shRNA was constructed by annealing shRNA137sense_BamHI: 5'P-RetroQ , using rFor apoptosis induction cells were incubated with 1 μM staurosporine , 25 μg/ml cycloheximide  or 0.1% DMSO as a control in culture medium for 14 h unless otherwise stated. Cells were stained for apoptosis with PE-conjugated annexin V  and for necrosis with 7-aminoactinomycin D . Specifically, cells were incubated with 5 μl annexin V or 7-AAD for 20 min at room temperature and then washed with PBS supplemented with 5% FCS. Subsequently, cells were fixed in 1.5% paraformaldehyde for 30 minutes at 4°C. Staining was analyzed within 30 minutes after completion of fixation by flow cytometry. For all measurements 20,000 gated events were collected.The podoplanin specific antibodies 18H5 and NZ-1  were pre-incubated with concentrated, soluble podoplanin-Fc fusion protein for 30 minutes at 4°C before staining of apoptotic cells for subsequent FACS analysis.Statistical significance was determined by employing a two-tailed student's t-test for paired samples.In order to better understand HIV-1 interactions with CLEC-2, we first asked if CLEC-2, like DC-SIGN , binds tThe cellular mucin podoplanin was recently shown to interact with CLEC-2 . PodoplaO-glycans [N-acetylglucosaminyltransferase I (CHO Lec1) or the trans Golgi localized CMP-sialic acid transporter (CHO Lec2) have lost their abilities to produce complex N-glycans and sialylated glycoconjugates, respectively [N-glycans are exclusively of the high-mannose type if proteins are expressed in Lec1 cells, this finding provides evidence that sialylated O-glycans are involved in mediating the contact to CLEC-2. Based on the knowledge that EDTA influences binding properties of DC-SIGN [We next sought to elucidate the determinants governing efficient interactions between podoplanin and CLEC-2. For instance, it is at present unclear if glycosylation of podoplanin is required for binding to CLEC-2. Watson and colleagues demonstrated that binding of CLEC-2 to the snake venom protein rhodocytin is glycosylation independent, and defined several amino acids in CLEC-2 which contributed to efficient rhodocytin binding ,34. Thus-glycans , and we ectively -41. Solu DC-SIGN , we nextOur results so far indicated that podoplanin is expressed by 293T cells and that podoplanin specifically interacts with CLEC-2. We next assessed if podoplanin is incorporated into HIV-1 released from transfected 293T cells and if the virion incorporation of podoplanin is required for HIV-1 interactions with CLEC-2. To address these questions, particularly the potential relevance of podoplanin for HIV-1 interactions with CLEC-2, we employed shRNA knock-down. We first tested a panel of podoplanin-specific shRNAs and identified one shRNA which efficiently reduced podoplanin expression in transiently transfected 293T cells (data not shown). Subsequently, this shRNA was stably introduced into 293T cells by employing a retroviral vector, which also contained an expression cassette for EGFP. As control, cells were transduced with a retroviral vector encoding a non-sense shRNA. After cultivation in selection antibiotics, all cells were positive for EGFP and thus harboured the vector genome Fig. . PodoplaPodocytes, which are visceral epithelial cells of the kidney, express podoplanin and were found to be infected in HIV-1 patients and to proliferate in HIV-1 associated nephropathy . We analApoptosis of infected and bystander cells is a prominent feature of HIV infection . We therOur expression studies indicated that podoplanin is not expressed on stimulated, viable PBMCs and T-cell lines Fig. , and thaSeveral cellular lectins interact with the highly glycosylated HIV Env protein ,25,65-67++ ions for its structural integrity [DC-SIGN recognizes mannose-rich carbohydrates on the surface of the HIV Env protein and requires Cantegrity -19. Consntegrity  and suggThe discovery of Suzuki-Inoue and colleagues  that podThe endogenous expression of podoplanin on 293T cells and the specific interaction of podoplanin with CLEC-2 raised the questions if podoplanin was incorporated into virions produced in 293T cells, and if incorporation of podoplanin was required for CLEC-2 binding of these virions. Western blot analysis and knock-down of podoplanin expression by shRNA provided affirmative answers to both questions: Podoplanin depletion reduced CLEC-2-, but not DC-SIGN-, dependent HIV-1 transmission by B-THP cells, and diminished transmission by platelets by about 50% Fig. . The latThe potential relevance of podoplanin incorporation for HIV spread in infected individuals is critically determined by the overlap of the podoplanin expression pattern with the cellular tropism of HIV. Analysis of T-cell lines and PBMCs for podoplanin expression yielded negative results Fig. , at leasPodoplanin was not detected on viable CEM×174 cells (a T/B cell hybrid) and PBMCs, as determined by our gating strategy and by co-staining with the apoptosis and necrosis markers annexin V and 7-AAD, respectively Fig.  suggestsCollectively, our data help to understand how HIV interacts with CLEC-2, an HIV attachment factor on platelets. Several lines of evidence suggest that this interaction could impact HIV spread in infected patients. For one, thrombocytopenia (reduced platelet count) is frequent in HIV/AIDS patients , and it Our results highlight that incorporation of cellular factors can alter HIV attachment to cells and cell to cell transmission. While podoplanin is unlikely to be incorporated into HIV particles produced in infected patients, our results indicate that HIV might incorporate a functional analogue of podoplanin in vivo, and that this process might promote virus binding to CLEC-2 positive cells. The identification of the respective factor and the clarification of the potential connection between podoplanin expression and apoptosis are interesting tasks for future research.The authors declare that they have no competing interests.CC analyzed binding of soluble CLEC-2 and soluble DC-SIGN to HIV Env, determined podoplanin expression by 293T cells, generated and characterized podoplanin knock-down cells, compared transmission of HIV generated in podoplanin-knock-down and control cells, and analyzed CLEC-2-dependent transmission of HIV generated in 293T cells and PBMCs, IS analyzed podoplanin expression on apoptotic cells, determined inhibition of antibody binding to apoptotic cells by soluble podoplanin, analyzed podoplanin expression on uninfected cell lines and HIV infected C8166-SEAP cells, TST analyzed binding of soluble podoplanin to CLEC-2 mutants, determined the impact of glycosylation and divalent ions on podoplanin binding to CLEC-2, analyzed podoplanin expression on cell lines and determined podoplanin incorporation into virions, SB and IG determined podoplanin RNA expression in apoptotic cells and analyzed podoplanin expression by viable cells, JS analyzed podoplanin expression on HIV infected PBMCs, YK provided critical reagents and contributed to the interpretation of experiments, JM, GS and RGS contributed to the design and the interpretation of experiments, SP planned and supervised the research and wrote the manuscript. All authors read and approved the final manuscript.Evidence that apoptotic PBMCs express podoplanin. (A) Apoptotic and necrotic PBMCs express podoplanin. Podoplanin expression on viable and apoptotic PBMCs, as determined by forward and sideward scatter, was analyzed by flow cytometry . Alternatively, the cells were co-stained with podoplanin-specific antibody and the apoptosis marker annexin V or the necrosis marker 7-AAD, and staining analyzed by flow cytometry including both, live and dead cells. Black filled histogram: unstained cells, grey line: cells stained with isotype control antibody, grey filled histogram: cells stained with 18H5. (B) PBMCs were incubated with 1 μM staurosporine for the indicated times and podoplanin expression (black bars) and annexin V binding (white bars) were determined by flow cytometry. The results were confirmed in two independent experiments.Click here for fileThe podoplanin-specific antibody 18H5, but not antibodies with other specificities recognize non-viable cells. (A) CEM×174 cells were analyzed for their distribution in the forward and sideward scatter, and a gate was defined which comprised both viable and non-viable cells. (B) The CEM×174 cells were stained with the indicated monoclonal antibodies and staining of the cells gated as shown in (A) was analyzed. The results of a representative experiment are shown and were confirmed in an independent experiment. IgG1, IgG2a and IgG2b are commercially available isotype control antibodies. The anti-AU1 antibody is specific for the AU1 antigenic tag. ACE2, MER and Axl are cell surface receptors, which are used for cell entry by SARS-coronavirus (ACE2) and Ebola virus . PDPN: podoplanin.Click here for file


Lissodendorix sponge, inhibits GTP binding to tubulin, preventing microtubule assembly. Cell cycle perturbations and apoptosis induced by IHB were investigated on selected human cancer cell lines by using flow cytometric and biochemical techniques. Monoparameter flow cytometric analysis showed that 1 h IHB exposure caused a delayed progression through S-phase, a dramatic block in G2M phase of the cell cycle and the appearance of tetraploid cell population in LoVo, LoVo/DX, MOLT-4 and K562 cells. At 24 h after IHB exposure, the majority of cells blocked in G2M were in prophase as assessed by morphological analysis and by the fact that they expressed high levels of cyclin A/cdc2 and cyclin B1/cdc2. At 48 h, all cells were tetraploid as assessed by biparameter cyclin A/DNA and cyclin B1/DNA content analysis. Apoptotic death was detected in both leukaemic MOLT-4 and K562 cells, which express wild-type and mutated p53 respectively, when the cells were blocked in mitotic prophase. In conclusion, IHB is a novel potent anti-tumour drug that causes delayed S-phase progression, mitotic block, tetraploidy and apoptosis in cancer cell lines. © 1999 Cancer Research CampaignIsohomohalichondrin B (IHB), a novel marine compound with anti-tumoral activity, extracted from the


Bacillus anthracis were used to place a Sverdlovsk patient’s B. anthracis genotype into 1 of 12 subgroups. Reconstruction of the pagA gene also showed a unique SNP that defines a new lineage for B. anthracis.A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for  Bacillus anthracis spores in the northeastern quadrant of Sverdlovsk (today’s Ekaterinburg) in the former Soviet Union resulted in a relatively large-scale incident of inhalation anthrax  marker (vrrA), which suggests that the material contained multiple strains of this species –rich region (307 bp) of the samples from Sverdlovsk found 7 SNPs in this gene ((pagA SNP 3602) placed 7 of 10 Sverdlovsk tissue samples into a large diversity group called western North America (WNA) . The mass spectrometry resequencing technique is described in detail elsewhere , following protocols recommended for formalin-fixed tissue samples. A Genomeplex Whole Genome Amplication Kit  was used to preamplify the extracted DNA sample. Short DNA fragments were amplified from these DNA samples by using the following primer sets: canSNP.A.Br.008 F: 5′-GCCAAGATATTCGTGACATT-3′; canSNP.A.Br.008 R: 5′-TTTGGACCAGGTTTCTGTATTT-3′; canSNP A.Br.009 F: 5′-TGCGGAATATCGTTAAGTAAT-3′; canSNP A.Br.009R: 5′-TGGACGTGAATTAGGAAAAGT-3′ ; canSNP A.Br.008 F: 5′-TCTAAGAAAGATTCGCAACTACGCTATAC-3′; canSNP A.Br.008 R: 5′-TGCATTCGCAACTACGCTATACGTTTTAGATG-3′; canSNP A.Br.009 F: 5′-TGCCGGGGTTTCTACTGTGTATGTTGT-3′; canSNP A.Br.009 R: 5′-TGGGTTAGGTATATTAACTGCGGATGATGC-3′ ; B. anthracis isolates , as measured by both the quantity and the quality of the PCR products, was found in 9 of 13 samples from the tissue specimens  for the WNA subgroup appeared in 7 of 10 samples of the Sverdlovsk tissues  isolates by using a custom made real-time PCR assay: TaqMan Minor Groove Binding probes and primers for SNPs (Applied Biosystems)  This real-time PCR protocol is applicable to low copy–number templates and, somewhat, to mixed sample analyses. We applied it to DNA extracted from the remaining 12 Sverdlovsk tissue samples , except for 1 sample  that contained a mixture of the vrrA 2, 4, and 5 repeats. With this exception, a mixed signal could not be predicted from the previous results  that are found in Europe and Asia. Our findings are consistent with claims that the weaponized strain released in Sverdlovsk (Anthrax 836) was originally isolated from a rodent in the city of Kirov, Soviet Union, in the mid-1950s (