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Sample records for menotropin highly-purified menotropin

  1. A randomized assessor-blind trial comparing highly purified hMG and recombinant FSH in a GnRH antagonist cycle with compulsory single-blastocyst transfer

    DEFF Research Database (Denmark)

    Devroey, Paul; Pellicer, Antonio; Nyboe Andersen, Anders

    2012-01-01

    OBJECTIVE: To compare the efficacy and safety of highly purified menotropin (hphMG) and recombinant FSH (rFSH) for controlled ovarian stimulation in a GnRH antagonist cycle with compulsory single-blastocyst transfer. DESIGN: Randomized, open-label, assessor-blind, parallel groups, multicenter......, noninferiority trial. SETTING: Twenty-five infertility centers in seven countries. PATIENT(S): Seven hundred forty-nine women. INTERVENTION(S): Controlled ovarian stimulation with hphMG or rFSH in a GnRH antagonist cycle with compulsory single-blastocyst transfer on day 5 in one fresh or subsequent frozen......MG versus 27% with rFSH for the per-protocol (PP) population and 29% versus 27% for the intention-to-treat (ITT) population. Noninferiority of hphMG compared to rFSH was established. Considering frozen cycles initiated within 1 year, the cumulative live birth rate for a single stimulation cycle was 40...

  2. Influence of a highly purified senna extract on colonic epithelium

    NARCIS (Netherlands)

    van Gorkom, B A; Karrenbeld, A; van Der Sluis, T; Koudstaal, J; de Vries, E G; Kleibeuker, J H

    2000-01-01

    BACKGROUND: Chronic use of sennoside laxatives often causes pseudomelanosis coli. A recent study suggested that pseudomelanosis coli is associated with an increased colorectal cancer risk. A single high dose of highly purified senna extract increased proliferation rate and reduced crypt length in

  3. Absence of TGF-β Receptor Activation by Highly Purified hCG Preparations.

    Science.gov (United States)

    Koistinen, Hannu; Hautala, Laura; Koli, Katri; Stenman, Ulf-Håkan

    2015-12-01

    Recently, several LH/human chorionic gonadotropin (hCG) receptor-independent activities for hCG have been described, including activation of the TGF-β receptor (TGFβR) by hyperglycosylated hCG and stimulation of trophoblast invasion. Because the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, which act at very low concentrations, may be significant. Several commercial hCG preparations have been found to contain significant amounts of epidermal growth factor (EGF), which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-β1. These hCG preparations were able to activate ERK1/2 in JEG-3 choriocarcinoma cells or TGFβR in mink lung epithelial cells transfected with a reporter gene for TGFβR activation. No such activation was found with highly purified hCG or its free β-subunit (hCGβ), irrespective of whether they were hyperglycosylated or not. Taken together, our results suggest that the growth factor contaminations in the hCG preparations can cause activation of TGFβR and, at least in JEG-3 cells, MAPK signaling. This highlights the importance to carefully control for potential contaminations and that highly purified hCG preparations have to be used for biological studies.

  4. Highly purified HMG versus recombinant FSH for ovarian stimulation in IVF cycles

    DEFF Research Database (Denmark)

    Platteau, P.; Nyboe, Andersen A.; Loft, A.

    2008-01-01

    The objective of this study was to compare the live birth rates resulting from ovarian stimulation with highly purified human menopausal gonadotrophin (HP-HMG), which combines FSH and human chorionic gonadotrophin-driven LH activities, or recombinant FSH (rFSH) alone in women undergoing IVF cycles....... An integrated analysis was performed of the raw data from two randomized controlled trials that were highly comparable in terms of eligibility criteria and post-randomization treatment regimens with either HP-HMG or rFSH for ovarian stimulation in IVF, following a long down-regulation protocol. All randomized...... subjects who received at least one dose of gonadotrophin in an IVF cycle (HP-HMG, n = 491; rFSH, n = 495) were included in the analysis. Subjects who underwent intracytoplasmic sperm injection cycles were excluded. The superiority of one gonadotrophin preparation over the other was tested using...

  5. Isolation of functionally active and highly purified neuronal mitochondria from human cortex.

    Science.gov (United States)

    Khattar, Nicolas K; Yablonska, Svitlana; Baranov, Sergei V; Baranova, Oxana V; Kretz, Eric S; Larkin, Timothy M; Carlisle, Diane L; Richardson, R Mark; Friedlander, Robert M

    2016-04-01

    Functional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently available. We developed and validated a procedure to isolate purified neuronal mitochondria from brain tissue. The method combines Percoll gradient centrifugation to obtain synaptosomal fraction with nitrogen cavitation mediated synaptosome disruption and extraction of mitochondria using anti mitochondrial outer membrane protein antibodies conjugated to magnetic beads. The final products of isolation are non-synaptosomal mitochondria, which are a mixture of mitochondria isolated from different brain cells (i.e. neurons, astrocytes, oligodendrocytes, microglia) and synaptic mitochondria, which are of neuronal origin. This method is well suited for preparing functional mitochondria from human cortex tissue that is surgically extracted. The procedure produces mitochondria with minimal cytoplasmic contaminations that are functionally active based on measurements of mitochondrial respiration as well as mitochondrial protein import. The procedure requires approximately four hours for the isolation of human neuronal mitochondria and can also be used to isolate mitochondria from mouse/rat/monkey brains. This method will allow researchers to study highly enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Osteoinductive potential of highly purified porous β-TCP in mice.

    Science.gov (United States)

    Tsukanaka, Masako; Fujibayashi, Shunsuke; Otsuki, Bungo; Takemoto, Mitsuru; Matsuda, Shuichi

    2015-03-01

    Material-induced osteoinduction of calcium phosphate ceramics has been reported in specific animals. We previously reported that recruitment of tartrate-resistant acid phosphatase (TRAP)-positive cells might be one of the main factors responsible for the difference in the occurrence of material-induced osteoinduction between dogs and rats. In this study, we evaluated the osteoinductive potential of highly purified porous beta-tricalcium phosphate materials (HPP-β-TCP) with two different porosities, 75 and 60 % (Olympus Terumo Biomaterials, Tokyo, Japan), implanted into subcutaneous pockets of FVB and C57BL/6 mice. Twelve weeks after implantation, histological examination and gene expression analysis using reverse transcription-polymerase chain reaction were performed. We observed osteoinduction in half of the HPP-β-TCP materials with 60 % porosity implanted into FVB mice. This group of mice also exhibited the most TRAP-positive cells. Significantly more vessels were found in FVB mice than in C57BL/6 mice, but the greatest number of vessels was counted in implants from materials with 75 % porosity implanted into FVB mice, which did not show osteoinduction. These results indicate that recruitment of TRAP-positive cells is one factor responsible for osteoinduction caused by HPP-β-TCP materials in both FVB mice and dogs. Vessel formation seems to be necessary but appears to be less influential for osteoinduction than TRAP-positive cell recruitment.

  7. Prescription omega-3 fatty acid products containing highly purified eicosapentaenoic acid (EPA).

    Science.gov (United States)

    Brinton, Eliot A; Mason, R Preston

    2017-01-31

    The omega-3 fatty acid eicosapentaenoic acid (EPA) has multiple actions potentially conferring cardiovascular benefit, including lowering serum triglyceride (TG) and non-high-density lipoprotein cholesterol (non-HDL-C) levels and potentially reducing key steps in atherogenesis. Dietary supplements are a common source of omega-3 fatty acids in the US, but virtually all contain docosahexaenoic acid (DHA) in addition to EPA, and lipid effects differ between DHA and EPA. Contrary to popular belief, no over-the-counter omega-3 products are available in the US, only prescription products and dietary supplements. Among the US prescription omega-3 products, only one contains EPA exclusively (Vascepa); another closely related prescription omega-3 product also contains highly purified EPA, but is approved only in Japan and is provided in different capsule sizes. These high-purity EPA products do not raise low-density lipoprotein cholesterol (LDL-C) levels, even in patients with TG levels >500 mg/dL, in contrast to the increase in LDL-C levels with prescription omega-3 products that also contain DHA. The Japanese prescription EPA product was shown to significantly reduce major coronary events in hypercholesterolemic patients when added to statin therapy in the Japan EPA Lipid Intervention Study (JELIS). The effects of Vascepa on cardiovascular outcomes are being investigated in statin-treated patients with high TG levels in the Reduction of Cardiovascular Events With EPA-Intervention Trial (REDUCE-IT).

  8. Altered gene expression in highly purified enterocytes from patients with active coeliac disease

    Directory of Open Access Journals (Sweden)

    Jackson John

    2008-08-01

    Full Text Available Abstract Background Coeliac disease is a multifactorial inflammatory disorder of the intestine caused by ingestion of gluten in genetically susceptible individuals. Genes within the HLA-DQ locus are considered to contribute some 40% of the genetic influence on this disease. However, information on other disease causing genes is sparse. Since enterocytes are considered to play a central role in coeliac pathology, the aim of this study was to examine gene expression in a highly purified isolate of these cells taken from patients with active disease. Epithelial cells were isolated from duodenal biopsies taken from five coeliac patients with active disease and five non-coeliac control subjects. Contaminating T cells were removed by magnetic sorting. The gene expression profile of the cells was examined using microarray analysis. Validation of significantly altered genes was performed by real-time RT-PCR and immunohistochemistry. Results Enterocyte suspensions of high purity (98–99% were isolated from intestinal biopsies. Of the 3,800 genes investigated, 102 genes were found to have significantly altered expression between coeliac disease patients and controls (p Conclusion This study provides a profile of the molecular changes that occur in the intestinal epithelium of coeliac patients with active disease. Novel candidate genes were revealed which highlight the contribution of the epithelial cell to the pathogenesis of coeliac disease.

  9. Addition of highly purified HMG after corifollitropin alfa in antagonist-treated poor ovarian responders: a pilot study.

    Science.gov (United States)

    Polyzos, N P; De Vos, M; Corona, R; Vloeberghs, V; Ortega-Hrepich, C; Stoop, D; Tournaye, H

    2013-05-01

    Will sequential administration of highly purified (hp)-HMG after corifollitropin alfa in a GnRH antagonist protocol benefit women with poor ovarian response according to the Bologna criteria? Corifollitropin alfa followed by hp-HMG in a GnRH antagonist protocol results in very promising pregnancy rates, albeit only in young (effect in young poor ovarian responders fulfilling the Bologna criteria. The data provide the rationale for performing a randomized controlled trial to determine if there is sound evidence for a clinical introduction of this protocol. No conflicts of interest to declare. No specific funding was received for this study.

  10. Clinical, Radiographic, and Histologic Evaluation of Maxillary Sinus Lift Procedure Using a Highly Purified Xenogenic Graft (Laddec®

    Directory of Open Access Journals (Sweden)

    Renzo Guarnieri

    2016-03-01

    Full Text Available Objectives: The aim of this study was to evaluate the clinical, radiographic and histologic results when a highly purified xenogenic bone (Laddec® was used as grafting material in maxillary sinuses. Material and Methods: In fifteen patients requiring unilateral maxillary sinus augmentation, the grafting procedure was performed with Laddec®. Forty-two implants were installed after a 6 month healing period. The height of the augmented sinus was measured radiographically immediately after augmentation and postoperatively up to 36 months. At the time of implant placement, a bone core was harvested in each patient for histological examination. Results: The cumulative implant survival rate was 97.6%. The original height was 3.65 (SD 0.7 mm and the augmented sinus height was 13.8 (SD 1.4 mm after the surgery. The reduced height of grafted xenogenic material (RDL at the implant insertion was 0.83 (SD 0.38 mm, and at the final postoperative visit was 0.91 (SD 0.25 mm, showing no significant correlation with the follow-up periods by Spearman’s test (P = 0.118. In addition, no significant difference in the RDL was observed according to the site of implantation (P = 0.682. The mean implant marginal bone loss was 0.38 (SD 0.24 mm. Histological analysis showed the bone cores were composed of 64.72 (SD 3.44% newly formed bone, 17.41 (SD 2.02% connective tissue, 16.93 (SD 2.83% residual graft particles, and 0.94 (SD 0.11% inflammatory cells. Conclusions: According to our data, the highly purified xenogenic bone (Laddec®, used as graft material in the sinus lift procedure, may create adequate bone volume, and appropriate osseointegration of dental implants.

  11. Mixed protocols: Multiple ratios of FSH and LH bioactivity using highly purified, human-derived FSH (BRAVELLE and highly purified hMG (MENOPUR are unaltered by mixing together in the same syringe

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    Raike Elizabeth

    2005-11-01

    Full Text Available Abstract Background The use of mixed or blended protocols, that utilize both FSH and hMG, for controlled ovarian hyperstimulation is increasing in use. To reduce the number of injections a patient must administer, many physicians instruct their patients to mix their FSH and hMG together to be given as a single injection. Therefore, the goal of this study was to definitively determine if the FSH and LH bioactivities of highly purified, human-derived FSH (Bravelle(R and highly purified hMG (Menopur(R were altered by reconstituting in 0.9% saline and mixing in the same syringe. Methods Bravelle(R and Menopur(R were reconstituted in 0.9% saline and mixed in a Becton Dickinson plastic syringe. The FSH and LH bioactivities of the products were determined after injecting female and male rats, respectively, with Bravelle(R, Menopur(R, or a mixture of Bravelle(R and Menopur(R. Ratios of FSH:LH activity tested were 150:75 IU (1 vial Bravelle(R: 1 vial Menopur(R, 300:75 IU (3 vials Bravelle(R: 1 vial Menopur(R or 300:225 IU (1 vial Bravelle(R: 3 vials of Menopur(R. Results There were no statistically significant changes in either FSH or LH bioactivity that occurred after mixing Bravelle(R with Menopur(R in the same syringe. The theoretical vs. actual FSH bioactivity for Bravelle(R and Menopur(R were 75 vs. 76.58 IU/mL and 75 vs. 76.0 IU/mL, respectively. For the 3 ratios of FSH:LH activity tested, 150:75 IU (1 vial Bravelle(R: 1 vial Menopur(R, 300:75 IU (3 vials Bravelle(R: 1 vial Menopur(R or 300:225 IU (1 vial Bravelle(R: 3 vials of Menopur(R tested, the theoretical vs. actual FSH bioactivities were 150 vs. 156.86 IU/mL, 300 vs. 308.69 IU/mL and 300 vs. 306.58 IU/mL, respectively. The theoretical vs. actual LH bioactivity for Menopur(R in the above mentioned ratios tested were 75 vs. 77.50 IU/mL. For the 3 ratios of FSH:LH activity tested, 150:75 IU (1 vial Bravelle(R: 1 vial Menopur(R, 300:75 IU (3 vials Bravelle(R: 1 vial Menopur(R or 300:225 IU (1

  12. Low dose HP-hMG in an antagonist protocol for IVF in ovulatory and anovulatory patients with high AMH.

    Science.gov (United States)

    Sopa, Negjyp; Larsen, Elisabeth Clare; Nyboe Andersen, Anders

    2018-01-18

    Women with high-AMH levels have an increased risk of ovarian hyperstimulation syndrome (OHSS). Studies have suggested that highly purified menotropin (HP-hMG) Menopur® reduces the risk. We, therefore, studied use of low-dose (112.5 IU/day) HP-hMG in ovulatory and anovulatory patients with high AMH (>32 pmol/L). The primary endpoint was the distribution of patients with appropriate, excessive, and inadequate response (5-14, ≥15, and ≤4 oocytes). Another endpoint was frequency of OHSS. Totally 115 women were included and 78 (67.8%) had an appropriate, 8 (7.0%) an excessive, and 29 (25.2%) an inadequate response. The number of oocytes was independent on AMH levels and ovulatory status but declined significantly with increasing bodyweight (R 2  = 0.07, p HP-hMG in patients with high levels of AMH provides 5-14 oocytes in more than two-thirds of the patients and is safe with low risk of OHSS. The number of aspirated oocytes was independent of AMH levels and ovulatory status, but inversely related to body weight.

  13. High resolution diffraction imaging of mercuric iodide: Demonstration of the necessity for alternate crystal processing techniques for highly purified material

    International Nuclear Information System (INIS)

    Steiner, B.; Berg, L. van den; Laor, U.

    1995-01-01

    The overall crystalline lattice uniformity in recently available, highly purified mercuric iodide single crystals has been shown to be impacted by crystal handling techniques that were previously satisfactory. High resolution diffraction imaging of the surface regularity of crystals of various levels of purity and growth orientation shows: (1) that the newer materials have a generally lower level of precipitates, (2) that the incidence of these precipitates is now closely correlated with growth direction, and (3) that the deformation resistance and resulting sensitivity to crystal handling procedures are also closely correlated with these factors in this soft material. As a result, gentler cutting and polishing procedures have been developed and are shown to be effective in preserving overall lattice regularity in the new material. The polishing required to remove residual surface scratches affect the lattice orientation of the softer, precipitate-free regions, while not affecting those regions with detectable levels of precipitates. These results correlate closely with the electrical properties of devices made from these crystals. Mercuric iodide single crystals have proved to be particularly useful for x and γ ray detectors because their room temperature operation allow for simple, efficient, and compact instrumentation

  14. A highly purified vegetal fraction able to modulate HMGB1 and to attenuate septic shock in mice.

    Science.gov (United States)

    Apetrei, Natalia Simona; Călugăru, Ana; Kerek, F; Panteli, Minerva; Rasit, I; Cremer, Lidia; Szegli, G; Lupu, Andreea-Roxana

    2011-01-01

    High-mobility group box protein 1 (HMGB1) is an intracellular protein that may be released actively from monocytes and macrophages or passively from necrotic or damaged cells. Its inhibition in animal experiments, even in the late phase of septic shock, significantly enhanced the survival rate of rodents. The aim of our study was to investigate the effect of a vegetal fraction isolated and highly purified from Helleborus purpurascens regarding the modulation of HMGB1 release either from tumor cells or human blood mononuclear cells. Our results showed that the vegetal fraction was able to down-regulate the release of HMGB1 from activated human blood mononuclear cells (PBMCs) and tumor cells. By combining the purified fraction with Cyclophosphamide the release of HMGB1 from tumor cells was strongly decreased. This synergism was not noticed when the ve getal product was associated with Doxorubicin. We also studied the effect of the purified fraction in mice with septic shock induced by cecal ligation and puncture (CLP) method. The tested vegetal product increased significantly the survival rate of animals compared to the mice not treated with it. Our data suggest that the purified vegetal fraction may modulate inflammation by down-regulating the HMGB1, which can also explain its efficacy in septic shock in mice.

  15. Highly purified eicosapentaenoic acid ameliorates cardiac injury and adipose tissue inflammation in a rat model of metabolic syndrome

    Science.gov (United States)

    Ito, S.; Sano, Y.; Nagasawa, K.; Matsuura, N.; Yamada, Y.; Uchinaka, A.; Murohara, T.

    2016-01-01

    Summary Introduction n‐3 Polyunsaturated fatty acids such as eicosapentaenoic acid (EPA), which are abundant in fish oil, have been shown to delay the onset of cardiovascular events. We previously established DahlS.Z‐Lepr fa/Lepr fa (DS/obese) rats, which are derived from a cross between Dahl salt‐sensitive and Zucker rats, as a model of metabolic syndrome. This study has now explored the influence of highly purified EPA on cardiac and adipose tissue pathophysiology in this animal model. Materials and methods DS/obese rats were administered EPA (300 or 1,000 mg kg−1 d−1, per os) or vehicle from age 9 to 13 weeks. Homozygous lean (DahlS.Z‐Lepr +/Lepr +, or DS/lean) littermates were studied as controls. Results Whereas EPA had no effect on body weight, food intake or systolic blood pressure in DS/obese rats, it attenuated cardiac fibrosis, diastolic dysfunction, oxidative stress and inflammation in these animals. In addition, EPA did not affect insulin resistance but reduced adipocyte hypertrophy and inflammation in visceral fat of DS/obese rats. Moreover, EPA increased circulating levels of adiponectin as well as attenuated both the down‐regulation of AMP‐activated protein kinase phosphorylation and the up‐regulation of phosphorylation of the p65 subunit of nuclear factor‐kB in the heart of DS/obese rats. Conclusions Treatment of DS/obese rats with EPA did not affect hypertension but reduced cardiac fibrosis and diastolic dysfunction, with the latter effects being accompanied by AMP‐activated protein kinase activation and inactivation of nuclear factor‐kB signalling in the heart, possibly as a result of an increase in adiponectin secretion. EPA may be suitable for the treatment of cardiac injury associated with metabolic syndrome. PMID:27708849

  16. Immune tolerance induction with highly purified plasma-derived factor VIII containing von Willebrand factor in hemophilia A patients with high-responding inhibitors.

    Science.gov (United States)

    Orsini, Frédérique; Rotschild, Chantal; Beurrier, Philippe; Faradji, Albert; Goudemand, Jenny; Polack, Benoît

    2005-09-01

    We retrospectively evaluated the efficacy of immune tolerance induction (ITI) in a homogenous cohort of eight patients with constitutive severe hemophilia A with high-responding factor VIII (FVIII) inhibitors using Facteur VIII-LFB/Factane, a highly purified FVIII concentrate containing von Willebrand factor (VWF).

  17. Incidence of factor VIII inhibitor development in severe hemophilia A patients treated only with one brand of highly purified plasma-derived concentrate.

    Science.gov (United States)

    Guérois, C; Laurian, Y; Rothschild, C; Parquet-Gernez, A; Duclos, A M; Négrier, C; Vicariot, M; Fimbel, B; Fressinaud, E; Fiks-Sigaud, M

    1995-02-01

    The incidence of factor VIII inhibitor was studied in a cohort of 56 previously untreated patients with severe hemophilia A (factor VIII below 1 U/dl). They received only one brand of highly purified factor VIII concentrate (HPSD-VIII) prepared by conventional chromatography with a solvent-detergent step for viral inactivation. Follow-up since the first infusion of HPSD-VIII was from 1 to 76 months (mean = 29) and cumulative exposure days (CED) from 1 to over 100 (median = 26). Five patients (9%) developed an inhibitor after 6 to 19 CED, only one being a high responder (2%), showing a low incidence of inhibitor compared with previous studies using high purity plasma-derived or recombinant products.

  18. The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue.

    Science.gov (United States)

    da Silva Meirelles, Lindolfo; de Deus Wagatsuma, Virgínia Mara; Malta, Tathiane Maistro; Bonini Palma, Patrícia Viana; Araújo, Amélia Goes; Panepucci, Rodrigo Alexandre; Silva, Wilson Araújo; Kashima, Simone; Covas, Dimas Tadeu

    2016-12-10

    Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.

    Science.gov (United States)

    Zonari, Erika; Desantis, Giacomo; Petrillo, Carolina; Boccalatte, Francesco E; Lidonnici, Maria Rosa; Kajaste-Rudnitski, Anna; Aiuti, Alessandro; Ferrari, Giuliana; Naldini, Luigi; Gentner, Bernhard

    2017-04-11

    Ex vivo gene therapy based on CD34 + hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34 + cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E 2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34 + CD38 - cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Randomized controlled trial comparing highly purified (HP-hCG) and recombinant hCG (r-hCG) for triggering ovulation in ART.

    Science.gov (United States)

    Bellavia, Marina; de Geyter, Christian; Streuli, Isabelle; Ibecheole, Victoria; Birkhäuser, Martin H; Cometti, Barbara P S; de Ziegler, Dominique

    2013-02-01

    A randomized controlled trial (RCT) comparing highly purified human Choriogonadotrophin (HP-hCG) and recombinant hCG (r-hCG) both administered subcutaneously for triggering ovulation in controlled ovarian stimulation (COS) for Assisted Reproductive Technology (ART). Multi-centre (n = 4), prospective, controlled, randomized, non-inferiority, parallel group, investigator blind design, including 147 patients. The trial was registered with www.clinicaltrials.gov, using the identifier: NCT00335569. The primary endpoint is the number of oocytes retrieved, while the secondary endpoints include embryo implantation, pregnancy and delivery rates as well as safety parameters. The number of retrieved oocytes was not inferior when HP-hCG was used as compared to r-hCG: the mean number was 13.3 (6.8) in HP-hCG and 12.5 (5.8) in the r-hCG group (p = 0.49) with a 95% CI (-1.34, 2.77). Regarding the secondary outcomes, there were also no differences in fertilization rate at 57.3% (467/815) vs. 61.3% (482/787) (p = 0.11), the number of embryos available for transfer and cryopreservation (2PN stage) and implantation, pregnancy and delivery rates. Furthermore, there were no differences in the number and type of adverse events reported. HP-hCG was therefore not inferior to r-hCG. HP-hCG and r-hCG are equally efficient and safe for triggering ovulation in ART and, both being administered subcutaneously, equally practical and well tolerated by patients.

  1. In vitro fertilisation with recombinant follicle stimulating hormone requires less IU usage compared with highly purified human menopausal gonadotrophin: results from a European retrospective observational chart review

    Directory of Open Access Journals (Sweden)

    Blackmore Stuart

    2010-11-01

    Full Text Available Abstract Background Previous studies have reported conflicting results for the comparative doses of recombinant follicle stimulating hormone (rFSH and highly purified human menopausal gonadotrophin (hMG-HP required per cycle of in vitro fertilisation (IVF; the aim of this study was to determine the average total usage of rFSH versus hMG-HP in a 'real-world' setting using routine clinical practice. Methods This retrospective chart review of databases from four European countries investigated gonadotrophin usage, oocyte and embryo yield, and pregnancy outcomes in IVF cycles (± intra-cytoplasmic sperm injection using rFSH or hMG-HP alone. Included patients met the National Institute for Health and Clinical Excellence (NICE guideline criteria for IVF and received either rFSH or hMG-HP. Statistical tests were conducted at 5% significance using Chi-square or t-tests. Results Of 30,630 IVF cycles included in this review, 74% used rFSH and 26% used hMG-HP. A significantly lower drug usage per cycle for rFSH than hMG-HP (2072.53 +/- 76.73 IU vs. 2540.14 +/- 883.08 IU, 22.6% higher for hMG-HP; p Conclusions Based on these results, IVF treatment cycles with rFSH yield statistically more oocytes (and more mature oocytes, using significantly less IU per cycle, versus hMG-HP. The incidence of all OHSS and hospitalisations due to OHSS was significantly higher in the rFSH cycles compared to the hMG-HP cycles. However, the absolute incidence of hospitalisations due to OHSS was similar to that reported previously. These results suggest that the perceived required dosage with rFSH is currently over-estimated, and the higher unit cost of rFSH may be offset by a lower required dosage compared with hMG-HP.

  2. Efficacy of Platelet-Rich-Plasma (PRP and Highly Purified Bovine Xenograft (Laddec® Combination in Bone Regeneration after Cyst Enucleation: Radiological and Histological Evaluation

    Directory of Open Access Journals (Sweden)

    Sabrina Pappalardo

    2013-10-01

    Full Text Available Objectives: The purpose of the present study was to evaluate the efficacy of adding platelet-rich plasma (PRP to a new highly purified bovine allograft (Laddec® in the bone regeneration of cystic bony defects augmented following cystectomy.Material and Methods: Study sample included 20 patients undergoing cystectomy in which the bone defect was filled with PRP and Laddec®. All patients were examined with periapical radiographs before operation and at follow-up. After 3 months, at re-entry surgery for implant placement, bone core was taken for histological and histomorphometric analysis.Results: The postoperative successive radiographs showed a good regeneration of bone in the height of bony defects with application of PRP to bone graft. By the first postoperative month, about 48% of the defect was filled, which gradually increased in each month and showed about 90% of defect-fill by 6 months. Histological and histomorphometric analysis, showed a significant presence of bone tissue and vessels, with newly formed bone in contact with anorganic bone particles. The mean volume of vital bone was 68 ± 1.6% and the mean percentage of vital bone was 48 ± 2.4%. The mean percentage of inorganic particles in tissues was 20 ± 1.2% of the total volume. All the samples analyzed did not evidence the presence of inflammatory cells.Conclusions: The results of this study showed how the use of Laddec® in association with platelet-rich plasma allows bone regeneration and has a potential for routine clinical use for regeneration of cystic bony defects.

  3. The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

    Energy Technology Data Exchange (ETDEWEB)

    Silva Meirelles, Lindolfo da, E-mail: lindolfomeirelles@gmail.com [Center for Cell-Based Therapy (CEPID/FAPESP), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); Laboratory for Stem Cells and Tissue Engineering, PPGBioSaúde, Lutheran University of Brazil, Av. Farroupilha 8001, 92425-900 Canoas, RS (Brazil); Deus Wagatsuma, Virgínia Mara de; Malta, Tathiane Maistro; Bonini Palma, Patrícia Viana [Center for Cell-Based Therapy (CEPID/FAPESP), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); Araújo, Amélia Goes; Panepucci, Rodrigo Alexandre [Laboratory of Large-Scale Functional Biology (LLSFBio), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); and others

    2016-12-10

    Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. - Highlights: • Non-cultured adipose tissue-derived human pericytes (ncATPCs) exhibit a distinctive gene expression signature. • ncATPCs express key adipose tissue stem cell genes previously described in vivo in mice. • ncATPCs express message for anti-proliferative and antiangiogenic molecules. • Most ncATPC-specific transcripts are absent in culture-expanded pericytes or ATMSCs • Gene expression changes ncATPCs undergo as they acquire a cultured ATMSC phenotype are pointed out.

  4. Immunization of rabbits with highly purified, soluble, trimeric human immunodeficiency virus type 1 envelope glycoprotein induces a vigorous B cell response and broadly cross-reactive neutralization.

    Directory of Open Access Journals (Sweden)

    Gerald V Quinnan

    Full Text Available Previously we described induction of cross-reactive HIV-1 neutralizing antibody responses in rabbits using a soluble HIV-1 gp140 envelope glycoprotein (Env in an adjuvant containing monophosphoryl lipid A (MPL and QS21 (AS02A. Here, we compared different forms of the same HIV-1 strain R2 Env for antigenic and biophysical characteristics, and in rabbits characterized the extent of B cell induction for specific antibody expression and secretion and neutralizing responses. The forms of this Env that were produced in and purified from stably transformed 293T cells included a primarily dimeric gp140, a trimeric gp140 appended to a GCN4 trimerization domain (gp140-GCN4, gp140-GCN4 with a 15 amino acid flexible linker between the gp120 and gp41 ectodomain (gp140-GCN4-L, also trimeric, and a gp140 with the flexible linker purified from cell culture supernatants as either dimer (gp140-L(D or monomer (gp140-L(M. Multimeric states of the Env proteins were assessed by native gel electrophoresis and analytical ultracentrifugation. The different forms of gp140 bound broadly cross-reactive neutralizing (BCN human monoclonal antibodies (mAbs similarly in ELISA and immunoprecipitation assays. All Envs bound CD4i mAbs in the presence and absence of sCD4, as reported for the R2 Env. Weak neutralization of some strains of HIV-1 was seen after two additional doses in AS02A. Rabbits that were given a seventh dose of gp140-GCN4-L developed BCN responses that were weak to moderate, similar to our previous report. The specificity of these responses did not appear similar to that of any of the known BCN human mAbs. Induction of spleen B cell and plasma cells producing immunoglobulins that bound trimeric gp140-GCN4-L was vigorous, based on ELISpot and flow cytometry analyses. The results demonstrate that highly purified gp140-GCN4-L trimer in adjuvant elicits BCN responses in rabbits accompanied by vigorous B cell induction.

  5. Recombinant versus highly-purified, urinary follicle-stimulating hormone (r-FSH vs. HP-uFSH in ovulation induction: a prospective, randomized study with cost-minimization analysis

    Directory of Open Access Journals (Sweden)

    Grassi Giuseppina

    2006-07-01

    Full Text Available Abstract Background Both recombinant FSH (r-FSH and highly-purified, urinary FSH (HP-uFSH are frequently used in ovulation induction associated with timed sexual intercourse. Their effectiveness is reported to be similar, and therefore the costs of treatment represent a major issue to be considered. Although several studies about costs in IVF have been published, data obtained in low-technology infertility treatments are still scarce. Methods Two hundred and sixty infertile women (184 with unexplained infertility, 76 with CC-resistant polycystic ovary syndrome at their first treatment cycle were randomized and included in the study. Ovulation induction was accomplished by daily administration of rFSH or HP-uFSH according to a low-dose, step-up regimen aimed to obtain a monofollicular ovulation. A bi- or tri-follicular ovulation was anyway accepted, whereas hCG was withdrawn and the cycle cancelled when more than three follicles greater than or equal to 18 mm diameter were seen at ultrasound. The primary outcome measure was the cost of therapy per delivered baby, estimated according to a cost-minimization analysis. Secondary outcomes were the following: monofollicular ovulation rate, total FSH dose, cycle cancellation rate, length of the follicular phase, number of developing follicles (>12 mm diameter, endometrial thickness at hCG, incidence of twinning and ovarian hyperstimulation syndrome, delivery rate. Results The overall FSH dose needed to achieve ovulation was significantly lower with r-FSH, whereas all the other studied variables did not significantly differ with either treatments. However, a trend toward a higher delivery rate with r-FSH was observed in the whole group and also when results were considered subgrouping patients according to the indication to treatment. Conclusion Considering the significantly lower number of vials/patient and the slight (although non-significant increase in the delivery rate with r-FSH, the cost

  6. Preformulation study of highly purified inactivated polio vaccine, serotype 3.

    Science.gov (United States)

    Qi, Wei; Zeng, Yuhong; Orgel, Scott; Francon, Alain; Kim, Jae Hyun; Randolph, Theodore W; Carpenter, John F; Middaugh, C Russell

    2014-01-01

    To improve the effectiveness of the polio vaccination campaign, improvements in the thermal stability of the vaccine are being investigated. Here, inactivated polio vaccine, serotype 3 (IPV3) was characterized via a number of biophysical techniques. The size was characterized by transmission electronic microscopy and light scattering. The capsid protein conformation was evaluated by intrinsic fluorescence and circular dichroism (CD), and the D-antigen content by enzyme-linked immunosorbent assay (ELISA). The pH thermal stability of IPV3 (pH 3.0-8.0; 10°C-87.5°C) was evaluated by fluorescence, CD, and static light scattering. The transition temperatures reflect the responses, respectively, of tertiary structure, secondary structure, and size to applied thermal stress. The data were summarized as empirical phase diagrams, and the most stable conditions were found to be pH 7.0 with temperature lower than 40°C. CD detected a higher transition temperature for capsid protein than that for RNA. The effects of certain excipients on IPV3 thermal stability and antigen content were evaluated. The results of their effects, based on intrinsic fluorescence and ELISA, were in good agreement, suggesting the feasibility of applying intrinsic fluorescence as a high-throughput tool for formulation development. The study improves the understanding of IPV3 thermal stability, and provides a starting point for future formulation development of IPV3 and other serotypes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

  7. Preparation of productive and highly purified mogrosides from ...

    African Journals Online (AJOL)

    The mogrosides of Siraitia grosvenorii are natural sweetener and potential chemopreventive agents. In order to obtain high-yield and good-quality mogrosides, the flash extraction method was employed to extract mogrosides from S. grosvenorii. The extraction parameters were optimized by Taguchi's experimental design, ...

  8. A next-generation, serum-free, highly purified Vero cell rabies vaccine is safe and as immunogenic as the reference vaccine Verorab® when administered according to a post-exposure regimen in healthy children and adults in China.

    Science.gov (United States)

    Li, Rongcheng; Huang, Lirong; Li, Jia; Mo, Zhaojun; He, Bin; Wang, Yunpeng; Wu, Xiaohong; Minutello, Maria; Guinet-Morlot, Françoise; Pichon, Sylvie

    2013-12-05

    As an evolution of its currently licensed rabies vaccine Verorab(®), Sanofi Pasteur has developed a next-generation, serum-free, highly purified Vero rabies vaccine (PVRV-NG). Through this Phase III clinical trial, we aimed to demonstrate the non-inferiority of PVRV-NG over Verorab when administered according to a post-exposure regimen and to assess its clinical safety. A total of 816 healthy subjects aged ≥10 years were randomized according to a 2:1 ratio to receive PVRV-NG or Verorab. Half of the subjects were aged 10-17 years, the other half were aged ≥18 years. All subjects were to receive 5 injections on days 0, 3, 7, 14 and 28. Three blood samples were taken for rabies virus neutralizing antibodies (RVNA) assessment, at baseline, on day 14 and day 42. Solicited adverse reactions (between injections 1, 2 and 3, and within 7 days post-injections 4 and 5) and adverse events (up to 28 days after the last injection) were collected for clinical safety assessment; serious adverse events were reported up to 6-months after the last injection. The proportion of subjects with an RVNA titer ≥0.5 IU/mL after the third injection of PVRV-NG was non-inferior to the proportion of those who received Verorab. PVRV-NG was shown to be as immunogenic as Verorab in each age range in the per-protocol and full analysis sets. PVRV-NG induced a strong immune response in both age ranges, with high RVNA levels and increased geometric mean titers compared to baseline after each measured time point. PVRV-NG had a satisfactory safety profile after each injection, similar to Verorab with regards to the nature, frequency, duration and severity of adverse events. Two serious adverse events were reported, none was related to vaccination. This trial demonstrated the immunogenic non-inferiority of PVRV-NG over Verorab and showed that both vaccines have similar safety profiles. This trial is registered at ClinicalTrials.gov (NCT01339312). This manuscript is the first full report of the

  9. Highly purified eicosapentaenoic acid as free fatty acids strongly suppresses polyps in Apc(Min/+) mice.

    Science.gov (United States)

    Fini, Lucia; Piazzi, Giulia; Ceccarelli, Claudio; Daoud, Yahya; Belluzzi, Andrea; Munarini, Alessandra; Graziani, Giulia; Fogliano, Vincenzo; Selgrad, Michael; Garcia, Melissa; Gasbarrini, Antonio; Genta, Robert M; Boland, C Richard; Ricciardiello, Luigi

    2010-12-01

    Although cyclooxygenase (COX)-2 inhibitors could represent the most effective chemopreventive tool against colorectal cancer (CRC), their use in clinical practice is hampered by cardiovascular side effects. Consumption of ω-3-polyunsaturated fatty acids (ω-3-PUFAs) is associated with a reduced risk of CRC. Therefore, in this study, we assessed the efficacy of a novel 99% pure preparation of ω-3-PUFA eicosapentaenoic acid as free fatty acids (EPA-FFA) on polyps in Apc(Min/+) mice. Apc(Min/+) and corresponding wild-type mice were fed control diet (Ctrl) or diets containing either EPA-FFA 2.5% or 5%, for 12 weeks while monitoring food intake and body weight. We found that both EPA-FFA diets protected from the cachexia observed among Apc(Min/+) animals fed Ctrl diet (P acid was replaced by EPA (P < 0.0001), leading to a significant reduction in COX-2 expression and β-catenin nuclear translocation. Moreover, in the EPA-FFA arms, we found a significant decrease in proliferation throughout the intestine together with an increase in apoptosis. Our data make 99% pure EPA-FFA an excellent candidate for CRC chemoprevention. ©2010 AACR.

  10. Highly purified collagen coating enhances tissue adherence and integration properties of monofilament polypropylene meshes.

    Science.gov (United States)

    Siniscalchi, Rodrigo Teixeira; Melo, Marli; Palma, Paulo César Rodrigues; Dal Fabbro, Inácio Maria; Vidal, Benedicto de Campos; Riccetto, Cassio Luiz Zanettini

    2013-10-01

    Complications related to tissue integration of polypropylene implants used in the treatment of pelvic organ prolapse are relatively prevalent. Collagen, a biocompatible, less immunogenic material with modulating properties on the inflammatory process, may improve polypropylene integration. The objective was to study biomechanical and histological effects of monofilament polypropylene mesh coated with purified collagen gel. Forty rats were implanted with two fragments of polypropylene mesh in their abdominal walls (one on each side of the linea alba). One of the fragments had a collagen gel coating (group I) while the other one did not (group II). The animals were euthanized at 7, 14, 90, and 180 days after implantation and their abdominal walls were excised for analysis. The biomechanical study showed that mesh adherence to neighboring tissue increased significantly in group II (p Polypropylene mesh coated with purified collagen gel increases adherence to tissue, promotes a less intense and lasting inflammatory response and triggers a greater organization and packing arrangement of collagen fibers in the late phase of implantation.

  11. Structure and magnetic response of a residual metal catalyst in highly purified single walled carbon nanotubes

    Czech Academy of Sciences Publication Activity Database

    Pacáková Bittová, Barbara; Kalbáč, Martin; Kubíčková, Simona; Mantlíková, Alice; Mangold, S.; Vejpravová, Jana

    2013-01-01

    Roč. 15, č. 16 (2013), s. 5992-6000 ISSN 1463-9076 R&D Projects: GA ČR GAP204/10/1677 Institutional support: RVO:68378271 ; RVO:61388955 Keywords : carbon nanotubes * purification * nanoparticles * magnetic property measurements Subject RIV: BM - Solid Matter Physics ; Magnetism; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 4.198, year: 2013

  12. In vitro and in vivo characterization of highly purified Human Mesothelioma derived cells

    International Nuclear Information System (INIS)

    Melotti, Alice; Daga, Antonio; Marubbi, Daniela; Zunino, Annalisa; Mutti, Luciano; Corte, Giorgio

    2010-01-01

    Malignant pleural mesothelioma is a rare disease known to be resistant to conventional therapies. A better understanding of mesothelioma biology may provide the rationale for new therapeutic strategies. In this regard, tumor cell lines development has been an important tool to study the biological properties of many tumors. However all the cell lines established so far were grown in medium containing at least 10% serum, and it has been shown that primary cell lines cultured under these conditions lose their ability to differentiate, acquire gene expression profiles that differ from that of tissue specific stem cells or the primary tumor they derive from, and in some cases are neither clonogenic nor tumorigenic. Our work was aimed to establish from fresh human pleural mesothelioma samples cell cultures maintaining tumorigenic properties. The primary cell cultures, obtained from four human pleural mesotheliomas, were expanded in vitro in a low serum proliferation-permissive medium and the expression of different markers as well as the tumorigenicity in immunodeficient mice was evaluated. The established mesothelioma cell cultures are able to engraft, after pseudo orthotopic intraperitoneal transplantation, in immunodeficient mouse and maintain this ability to after serial transplantation. Our cell cultures were strongly positive for CD46, CD47, CD56 and CD63 and were also strongly positive for some markers never described before in mesothelioma cell lines, including CD55, CD90 and CD99. By real time PCR we found that our cell lines expressed high mRNA levels of typical mesothelioma markers as mesothelin (MSLN) and calretinin (CALB2), and of BMI-1, a stemness marker, and DKK1, a potent Wingless [WNT] inhibitor. These cell cultures may provide a valuable in vitro and in vivo model to investigate mesothelioma biology. The identification of new mesothelioma markers may be useful for diagnosis and/or prognosis of this neoplasia as well as for isolation of mesothelioma tumor initiating cells

  13. Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

    Science.gov (United States)

    Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs res...

  14. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Science.gov (United States)

    Pinnapireddy, Shashank Reddy; Baghdan, Elias; Jedelská, Jarmila

    2017-01-01

    Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI). PMID:28239294

  15. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Konrad H. Engelhardt

    2017-01-01

    Full Text Available Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc at certain nitrogen-to-phosphorus (N/P ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3. Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS, atomic force microscopy (AFM, and scanning electron microscopy (Cryo-SEM, respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI.

  16. Highly purified water production technology. The influence of water purity on steam quality

    International Nuclear Information System (INIS)

    Ganter, J.

    1975-01-01

    The fundamental question related to high-pressure steam generation, intended for powering steam turbines, concerns steam production conditions based on constant quality standards. The characteristics of water (salinity, silica concentration) are indicated for a given steam quality as a function of the pressure. Two processes for the purification of feedwater for high pressure boilers are described: a treatment using precoated cellulose or resin filters and a treatment using mixed-bed ion exchangers. When ultrapure water is required, the demineralized water is filtred using microfiltration and ultrafiltration processes [fr

  17. Efficacy of Highly Purified Urinary FSH versus Recombinant FSH in Chinese Women over 37 Years Undergoing Assisted Reproductive Techniques

    Directory of Open Access Journals (Sweden)

    Xuemei Liu

    2015-02-01

    Full Text Available Background: Urine derived follicle-stimulating hormone (uFSH contains a higher proportion of acidic isoforms, whereas recombinant FSH (rFSH contains a higher proportion of less-acidic isoforms. Less-acidic isoforms have a faster clearance, and thus a shorter half-life than the acidic FSH isoforms. The slow clearance of the acidic isoforms has a longer half-life and higher biological activity. This study was designed to determine whether uFSH or rFSH is more effective in older Chinese women undergoing assisted reproductive techniques (ART. Materials and Methods: This is a prospective, randomized, controlled cohort study. A total of 508 Chinese women over 37 years were randomized into two following study groups for their in vitro fertilization (IVF or intracytoplasmic sperm injection (ICSI cycles: i. group A (n=254 were treated with rFSH, and ii. group B (n=254 were treated with uFSH. Both groups were suppressed with a gonadotropin-releasing hormone (GnRH analogue using a long down-regulation protocol. The main outcomes for comparison were days of stimulation, estradiol (E2 on the day of human chorionic gonadotropin (hCG administration, number of oocytes collected, amount of FSH used, quantity of FSH/oocyte, endometrial thickness at hCG day, M П oocyte rate, 2PN zygote rate, grade І embryo rate, number of embryos cryopreserved, pregnancy rate, implantation rate, abortion rate and the rate of no transferable embryos. Results: Twenty two cycles including 16 cycles with poor ovarian response and six cycles with ovarian hyperstimulation syndrome were cancelled. There were 243 cycles left in each group. The patients treated with uFSH had a significantly higher 2PN zygote rate (87.4 vs. 76.6%, p0.05. Conclusion: This study showed that uFSH produced a significantly higher proportion of grade І embryos than rFSH in older Chinese women and there was a significantly lower chance of no transferable embryos in uFSH cycles. The clinical efficacy of the two gonadotropins was equivalent.

  18. Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.

    Science.gov (United States)

    Koehl, U; Esser, R; Zimmermann, S; Tonn, T; Kotchetkov, R; Bartling, T; Sörensen, J; Grüttner, H-P; Bader, P; Seifried, E; Martin, H; Lang, P; Passweg, J R; Klingebiel, T; Schwabe, D

    2005-01-01

    Allogeneic natural killer (NK) cells are known to show medium to high cytotoxic activity against HLA-nonidentical leukemia or tumor cells. For a possible benefit of post transplant treatment with NK cells after haploidentical stem cell transplantation (haplo-SCT) we developed a clinical scale procedure for NK cell processing observing Good Manufacturing Practice (GMP). Allogeneic donor NK cells were selected from 15 unstimulated leukaphereses using two rounds of immunomagnetic T cell depletion, followed by an NK cell enrichment step. CD56 (+)CD3 (-) NK cells were stimulated and expanded in vitro according to GMP. Quality control of NK cell purity, residual T cells and cytotoxic activity was done by multi-coloured flow cytometric analyses. Purification led to an absolute number of 234-1 237 x 10 (6) CD56 (+)CD3 (-) NK cells from leukapheresis harvests with a median purity of 95 % and a 4 to 6(1/2) log depletion of T cells. After two weeks stimulation with IL-2 a five-fold expansion of NK cells with a T cell contamination below 0.1 % was reached. Median cell viability was 95 % after purification and 99 % after expansion. The IL-2 stimulated NK cells showed a highly increased lytic activity against the MHC-I deficient K562 cells compared to freshly isolated NK cells and a medium cytotoxicity against patients' leukemic cells. Clinical scale enrichment and activation of allogeneic donor NK cells is feasible. High dose NK cell application may be a new treatment option for pediatric patients with leukemia or solid tumors in case of minimal residual disease or unbalanced chimerism post haplo-SCT as we could show for the first three patients .

  19. Use of ion exchange during preparation of raw materials for production of molybdenum and tungsten of high purify

    International Nuclear Information System (INIS)

    Blokhin, A.A.; Rumyantsev, V.K.; Taushkanov, V.P.; Maksimkov, S.M.; Majorov, D.Yu.; Pak, V.I.

    1988-01-01

    The data on the application of selective ionites for the steep purification of ammonium molybdate and tungstate solutions, are given. It is shown that to purify molybdenum and ammonium tungstate solutions from the impuerities of alkali earth and two- and threevalent transition metals, iminodiacetate ampholites of the ANKB-35 type are the most effective sorbents. To purify from phosphorus, silicon and arsenic impurities composition ionites on the base of hydrated oxides of multivalent metals introduced in the granules of porous cationites should be used. To extract phosphorus, silicon, arsenic impurities from ammonium molybdate and tungstate solutions and tungsten from ammonium molybdate solutions the method of their coprecipitation with iron (3) hydroxide can also be used. The best results on the purification of ammonium molybdate from tungstate provides for the application of structural organomineral ionites as well as weak-basicity anionites of the AN-31 type and its different modifications containing hydroxyl groups along with aminogroups. To purity ammonium tungstate solutions from molybdate a method is developed which transforms molybdenum in the form of thiocomplexes and the following selective sorption by strong-basicity anionites. The data on the quality of molybdenum monocrystals which are taken from the raw material purified using ionites, are given

  20. Studying placental transfer of highly purified non-dioxin-like PCBs in two models of the placental barrier

    DEFF Research Database (Denmark)

    Correia Carreira, S; Cartwright, L; Mathiesen, L

    2011-01-01

    for monolayer formation. Secondly, we performed placental perfusions to validate the in vitro model. To that end, the transport of radiolabelled PCB52 and 180 was investigated in both models. We were not able to grow the ATCC cell line to confluency, but determined monolayer formation using BeWo b30....... A confluent monolayer is present by day 4 post-seeding, transepithelial electrical resistance being 44.65 ± 11.06 O cm(2) and sodium fluorescein transport being 4.1% ± 0.18. Both measures can be used as indicators for monolayer formation. Results from kinetic studies in vitro and ex vivo were in excellent...... model of the placental barrier using BeWo b30. We hypothesise that the observed differences in transport and association patterns of NDL-PCBs may indicate that toxic effects of PCB52 play a more important role regarding placental function, whereas PCB180 may be of greater importance for fetal toxicity....

  1. Heterogeneous hCG and hMG commercial preparations result in different intracellular signalling but induce a similar long-term progesterone response in vitro.

    Science.gov (United States)

    Riccetti, Laura; Klett, Danièle; Ayoub, Mohammed Akli; Boulo, Thomas; Pignatti, Elisa; Tagliavini, Simonetta; Varani, Manuela; Trenti, Tommaso; Nicoli, Alessia; Capodanno, Francesco; La Sala, Giovanni Battista; Reiter, Eric; Simoni, Manuela; Casarini, Livio

    2017-10-01

    Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis? hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro. hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition. Commercial hCG preparations for ART or research purposes were compared in vitro. The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and β-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann-Whitney's U-test as appropriate. Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20-75 KDa range), isoelectric points (4.0-9.0 pI range) and lectin binding (P hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant h

  2. A review of the nonclinical safety of Transcutol®, a highly purified form of diethylene glycol monoethyl ether (DEGEE) used as a pharmaceutical excipient.

    Science.gov (United States)

    Sullivan, Dexter W; Gad, Shayne C; Julien, Marjorie

    2014-10-01

    Transcutol® (Diethylene glycol monoethyl ether, DEGEE), CAS # 111-90-0, is commonly used as a vehicle in the formulation or manufacturing process of pharmaceuticals, cosmetics, and food additives. This paper presents unpublished nonclinical safety data using a form of DEGEE which includes a significantly decreased level of impurities, specifically ethylene glycol and diethylene glycol. It also reviews the history of use, regulatory status, and previously published toxicity data for DEGEE. The review supports that DEGEE is well tolerated across animal species and gender with toxicity occurring only at levels well above those intended for human use. At high levels of exposure, the kidney is identified as the critical target organ of DEGEE toxicity. DEGEE is negative for genotoxicity in in vitro and in vivo studies. Subchronic and chronic toxicity studies produced no reports of preneoplastic changes in organs, but the animal data is insufficient to allow a definitive opinion as to carcinogenicity. In silico data suggested that DEGEE is not carcinogenic or genotoxic. Developmental toxicity was seen in rats but only at levels 200 times greater than the estimated oral Permissible Daily Exposure Level of 10 mg/kg/day. The nonclinical data along with the long history of DEGEE use as a vehicle and solvent by multiple routes provide evidence of its safety. Furthermore, the novel data discussed herein provides evidence that toxicity previously associated with high levels of DEGEE in nonclinical studies conducted prior to 1990 could possibly be attributed to the presence of significant amounts of ethylene glycol or other impurities. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. A randomized, double-blind, placebo-controlled trial of a highly purified equine F(ab)2 antibody black widow spider antivenom.

    Science.gov (United States)

    Dart, Richard C; Bogdan, Gregory; Heard, Kennon; Bucher Bartelson, Becki; Garcia-Ubbelohde, Walter; Bush, Sean; Arnold, Tom; Clark, Richard C; Hendey, Gregory W; Holstege, Christopher; Spradley, Elizabeth A

    2013-04-01

    Black widow spider antivenom has never been tested in a randomized clinical trial, to our knowledge. We explore various efficacy measures for a novel F(ab)2 antivenom in patients with moderate to severe pain caused by black widow spider envenomation. A randomized, placebo-controlled, double-blind, clinical trial was conducted in 12 academic emergency departments. We included patients at least 10 years old with moderate to severe latrodectism. Subjects received either a single intravenous infusion of antivenom or placebo. Pain was assessed with the visual analog scale. The primary efficacy outcome was the difference in pre- and posttreatment visual analog scale score. Prospectively defined secondary outcomes included treatment failures and time to clinically important decrease in pain. Twenty-four subjects were enrolled between October 2005 and October 2006; 13 were randomized to antivenom and 11 to placebo. The median change in visual analog scale at 150 minutes posttreatment was -50.0 mm (Interquartile Range [IQR] -67, -41 mm) in the antivenom treatment group and -46.0 mm (IQR -51, 0 mm) in the placebo treatment group (P=.14). There were 7 treatment failures (64%; 95% confidence interval 35% to 92%) in the placebo group and 3 (23%; 95% confidence interval 0.2% to 46%) in the antivenom group (P=.06). The median time to a clinically important decrease in pain after treatment was shorter in the antivenom group compared with the placebo group (30 minutes [IQR 30, 60 minutes] versus 90 minutes [IQR 30, 90 minutes]; P=.03). No serious adverse events or deaths were reported. Although the overall reduction in pain was similar for antivenom- and placebo-treated subjects, antivenom reduced pain more rapidly than placebo. No significant adverse events occurred in either group. Copyright © 2012. Published by Mosby, Inc.

  4. Treatment of snake envenomations by a new polyvalent antivenom composed of highly purified F(ab')2 : results of a clinical trial in northern Cameroon

    OpenAIRE

    Chippaux, Jean-Philippe; Lang, J.; Amadi-Eddine, S.; Fagot, P.; Le Mener, V.

    1999-01-01

    A clinical trail was conducted in 2 health centers in northern Cameroon to assess the safety and efficacy of a new polyvalent antivenom composed of higly purified and pasteurized F(ab')2 (FAV-Africa). Forty-six patients with objective signs of envenomation, including 67% with hemorrhage, were included in the study. Each patient received at least 20 ml of FAV-Africa by direct, slow intravenous injection ; 172 10-ml ampules were administered. All patients were clinically cured after treatment. ...

  5. Differences in gene expression of granulosa cells from women undergoing controlled ovarian hyperstimulation with either recombinant follicle-stimulating hormone or highly purified human menopausal gonadotropin

    DEFF Research Database (Denmark)

    Grøndahl, Marie Louise; Borup, Rehannah; Lee, Young Bae

    2009-01-01

    groups of granulosa cells. Expression level of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor gene and genes involved in biosynthesis of cholesterol and steroids were expressed at lower levels in the hMG-treated cells; inositol 1,4,5-triphosphate-3-kinase-A and S100-calcium....... Granulosa cells were collected from follicular fluid after oocyte retrieval, and mRNA were isolated for gene expression analysis. MAIN OUTCOME MEASURE(S): General gene expression profile. RESULT(S): Ninety-six probe sets (85 genes) showed statistically significant differences in expression level in the two......-binding-protein-P (anti-apoptosis protein) were expressed at higher levels in hMG than in recombinant FSH. CONCLUSION(S): The different hormone compositions of the two drugs used for COH had a statistically significant impact on the gene expression profile of preovulatory granulosa cells. Some of these genes may...

  6. Environmental effects on irradiation creep behavior of highly purified V-4Cr-4Ti alloys (NIFS-Heats) irradiated by neutrons

    OpenAIRE

    FUKUMOTO, K; MARUI, M; MATSUI, H; NAGASAKA, T; MUROGA, T; LI, M; HOELZER, D.T.; ZINKLE, S.J.

    2009-01-01

    In order to investigate the effect of the environment on the irradiation creep properties ofhighly purified V-4Cr-4Ti alloys, neutron irradiation experiments with sodium-enclosed irradiationcapsules in Joyo and lithium-enclosed irradiation capsules in HFIR-17J were carried out usingpressurized creep tubes (PCTs).It was found that the creep strain rate exhibited a linear relationship with the effective stressup to 150 MPa at 458°C and 598°C in the Joyo irradiation experiments. For HFIR-17J irr...

  7. Simplified process for preparation of schizophyllan solutions for biomaterial applications

    Science.gov (United States)

    Schizophyllan is a biopolymer commercially produced for pharmaceutical and cosmetics uses. However, schizophyllan also has potential biomaterial applications. Schizophyllan is conventionally produced from glucose and recovered by diafiltration and ultrafiltration to produce a highly purified product...

  8. Chloramphenicol-sensitive mitochondrial translation in Trypanosoma brucei

    NARCIS (Netherlands)

    Nabholz, C. E.; Speijer, D.; Schneider, A.

    1999-01-01

    We developed an in organello system to label newly synthesized mitochondrially encoded proteins of Trypanosoma brucei. Highly purified mitochondria, prepared under isotonic conditions, were incubated with radioactive methionine and cysteine in a suitable translation buffer. Analysis of mitochondrial

  9. Affinity-purified human interleukin I is cytotoxic to isolated islets of Langerhans

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

    1986-01-01

    Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets. These e......Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets....... These effects were dose-dependent and reproducible when using three different Interleukin-1 preparations. Highly purified human Interleukin-2, Lymphotoxin, Leucocyte Migration Inhibitory Factor and Macrophage Migration Inhibitory Factor were ineffective. These findings suggest that Interleukin-1 may play...

  10. Localization and Functionality of the Inflammasome in Neutrophils

    DEFF Research Database (Denmark)

    Bakele, Martina; Joos, Melanie; Burdi, Sofia

    2014-01-01

    Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality...... of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein...... and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases...

  11. Kidney function and size in normal subjects before and during growth hormone administration for one week

    DEFF Research Database (Denmark)

    Gammelgaard, Jens; Orskov, H; Andersen, A R

    1981-01-01

    Kidney function and size were studied in seven normal male subjects before and after administration of highly purified human growth hormone for 1 week. Glomerular filtration rate, renal plasma flow (steady-state infusion technique with urinary collections using 125I-iothalamate and 131I-hippuran)......Kidney function and size were studied in seven normal male subjects before and after administration of highly purified human growth hormone for 1 week. Glomerular filtration rate, renal plasma flow (steady-state infusion technique with urinary collections using 125I-iothalamate and 131I...

  12. Use of recombinant nucleoproteins in enzyme-linked immunosorbent assays for detection of virus-specific immunoglobulin A (IgA) and IgG antibodies in influenza virus A- or B-infected patients

    NARCIS (Netherlands)

    J. Groen (Jan); D. van Alphen; E.C.J. Claas (Eric); R. de Groot (Ronald); G.F. Rimmelzwaan (Guus); J.T.M. Voeten; A.D.M.E. Osterhaus (Albert)

    1998-01-01

    textabstractThe nucleoprotein genes of influenza virus A/Netherlands/018/94 (H3N2) and influenza virus B/Harbin/7/94 were cloned into the bacterial expression vector pMalC to yield highly purified recombinant influenza virus A and B nucleoproteins. With these recombinant influenza

  13. Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

    NARCIS (Netherlands)

    Wolvetang, E. J.; Wanders, R. J.; Schutgens, R. B.; Berden, J. A.; Tager, J. M.

    1990-01-01

    Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 +/- 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum

  14. Overproduction and Improved Strategies for Isolation and ...

    African Journals Online (AJOL)

    Yajing Zhou

    2012-04-24

    Apr 24, 2012 ... Mammalian deoxyribonucleic acid (DNA) polymerase delta (pol δ) is well characterized as a tightly associated heterotetrameric complex. It is thought to play a central role in chromosomal DNA replication and various DNA repair processes. However, the availability of highly purified active pol δ becomes ...

  15. Expression, purification and characterization of the interferon ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent ...

  16. Eicosapentaenoic acid ethyl ester (AMR101) therapy in patients with very high triglyceride levels (from the Multi-center, plAcebo-controlled, Randomized, double-blINd, 12-week study with an open-label Extension [MARINE] trial)

    NARCIS (Netherlands)

    Bays, Harold E.; Ballantyne, Christie M.; Kastelein, John J.; Isaacsohn, Jonathan L.; Braeckman, Rene A.; Soni, Paresh N.

    2011-01-01

    AMR101 is an omega-3 fatty acid agent containing ≥96% eicosapentaenoic acid ethyl ester and no docosahexaenoic acid. Previous smaller studies suggested that highly purified eicosapentaenoic acid lowered triglyceride (TG) levels without increasing low-density lipoprotein (LDL) cholesterol levels.

  17. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses...

  18. Method for removal of phosgene from boron trichloride. [DOE patent application; mercury arc lamp

    Science.gov (United States)

    Freund, S.M.

    1981-09-03

    Selective ultraviolet photolysis using an unfiltered mercury arc lamp has been used to substantially reduce the phosgene impurity in a mixture of boron trichloride and phosgene. Infrared spectrophotometric analysis of the sample before and after irradiation shows that it is possible to highly purify commercially available boron trichloride with this method.

  19. Free-flow electrophoresis of plasma membrane vesicles enriched by two-phase partitioning enhances the quality of the proteome from Arabidopsis seedlings

    DEFF Research Database (Denmark)

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet Tempé

    2016-01-01

    using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane...

  20. Method for removal of phosgene from boron trichloride

    Science.gov (United States)

    Freund, S.M.

    1983-09-20

    Selective ultraviolet photolysis using an unfiltered mercury arc lamp has been used to substantially reduce the phosgene impurity in a mixture of boron trichloride and phosgene. Infrared spectrophotometric analysis of the sample before and after irradiation shows that it is possible to highly purify commercially available boron trichloride with this method. 5 figs.

  1. Expression of recombinant Streptokinase from local Egyptian ...

    African Journals Online (AJOL)

    We reported for the first time the expression of a recombinant SK from a local Streptococcus strain. When produced on industrial scale this r-SK may substantially contribute to reducing the costs of thrombolytic therapy in developing countries. In this study, a highly purified r-SK from Streptococcus sp. isolated from Egyptian ...

  2. Retention of lignin in seagrasses: angiosperms that returned to the sea

    NARCIS (Netherlands)

    Klap, V.A.; Hemminga, M.A.; Boon, J.J.

    2000-01-01

    Using Curie-point Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Direct Temperature-resolved Mass Spectrometry (DT-MS), lignin was detected in highly purified preparations (Milled Wood Lignin = MWL) of various tissues of the seagrasses Zostera marina and Posidonia oceanica. The results

  3. Statin Drugs Markedly Inhibit Testosterone Production by Rat Leydig Cells In Vitro: Implications for Men

    Science.gov (United States)

    Statin drugs lower blood cholesterol by inhibiting hepatic 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase. During drug development it was shown that statins inhibit production of cholesterol in the testis. We evaluated testosterone production in vitro, using highly purified rat ...

  4. Gene Expression Profiling of Apoptosis Regulators in Patients with Sepsis

    NARCIS (Netherlands)

    Hoogerwerf, Jacobien J.; van Zoelen, Marieke A.; Wiersinga, W. Joost; van 't Veer, Cornelis; de Vos, Alex F.; de Boer, Anita; Schultz, Marcus J.; Hooibrink, Berend; de Jonge, Evert; van der Poll, Tom

    2010-01-01

    Introduction: Sepsis is associated with a dysregulation of apoptosis in immune cells, which has been implicated in both immunosuppression and multiple organ failure. We describe the expression profiles of genes encoding key regulators of apoptosis in highly purified monocytes, granulocytes and CD4+

  5. Notes on the path and wake of a gas bubble rising in pure water

    NARCIS (Netherlands)

    de Vries, A.W.G.; Biesheuvel, A.; van Wijngaarden, L.; van Wijngaarden, L.

    2002-01-01

    This paper is concerned with the structure of the wake behind gas bubbles rising at high Reynolds numbers in highly purified water. It describes a schlieren optics technique to visualise the wake. The technique does not contaminate the water, and so does not affect the zero-stress condition at the

  6. Extended Abstracts. The Electrochemical Society Fall Meeting (174th) Held in Chicago, Illinois on 9-14 October 1988. Volume 88-2

    Science.gov (United States)

    1988-10-14

    exhibit a complex structure which has been sepa- 3 .Unitat Inorganica , Universitat de BARCELONA SPAIN rated by site selective excitation into two indivi- 4...de Fisicoquimica highly purified Au in a newly machined 6.0 mm I.D. Facultad de Ciencias Quimicas carbon crucible. Polyorystalline Au and Pt electrodes

  7. INACTIVATION OF HEPATITIS A VIRUS AND MS2 BY OZONE AND OZONE-HYDROGEN PEROXIDE IN BUFFERED WATER

    Science.gov (United States)

    Disinfection of drinking water by chlorine is a primary means of preventing the transmission of waterborne disease, and its efficacy is well-established. The comparative inactivation of highly purified hepatitis A virus (HAV) and MS2 by 1 mg water/L, 2.0 and 0.4 mg ozone/L plus 0...

  8. Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

    NARCIS (Netherlands)

    K. Sugasawa (Kaoru); J.M.Y. Ng (Jessica); C. Masutani (Chikahide); T. Maekawa; A. Uchida; P.J. van der Spek (Peter); A.P.M. Eker (André); S. Rademakers (Suzanne); C.E. Visser (Cécile); A. Aboussekhra; R.D. Wood (Richard); F. Hanaoka (Fumio); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1997-01-01

    textabstractXPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a

  9. Induction of osteoarthritis by intra-articular injection of collagenase in mice. Strain and sex related differences

    NARCIS (Netherlands)

    van Osch, G. J.; van der Kraan, P. M.; Vitters, E. L.; Blankevoort, L.; van den Berg, W. B.

    1993-01-01

    To study the effects of strain and sex on the development of injury-induced osteoarthritis (OA) in murine knee joints, two doses of highly purified bacterial collagenase (10 units and 30 units) were injected into male and female mice of two closely related strains, C57BL6 and C57BL10. Frontal

  10. Proteinase 3 carries small unusual carbohydrates and associates with αlpha-defensins

    DEFF Research Database (Denmark)

    Zoega, Morten; Ravnsborg, Tina; Højrup, Peter

    2012-01-01

    with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with α-defensins and oligomers hereof. Irreversible inhibition of PR3 by α1-antitrypsin did not affect its association...

  11. Impact of cereal fibre on glucose-regulating factors

    DEFF Research Database (Denmark)

    Weickert, M O; Mohlig, M; Koebnick, C

    2005-01-01

    24 h after the fibre-enriched diets compared with control (p>0.15). Colonic fermentation was increased only on study days C-OF (p=0.017) and C-RS (p=0.016). CONCLUSIONS/INTERPRETATION: The consumption of highly purified insoluble dietary fibres accelerated the acute GIP and insulin response...

  12. DNA Modification Methylase Activity of Escherichia coli Restriction Endonucleases K and P

    Science.gov (United States)

    Haberman, Allan; Heywood, Janet; Meselson, Matthew

    1972-01-01

    The highly purified restriction endonucleases of E. coli K and coliphage P1 transfer methyl groups from S-adenosylmethionine to adenine residues of unmodified DNA. Incubation of unmodified DNA with endonucleases K or P and S-adenosylmethionine renders the DNA resistant to restriction. The enzymes, therefore, have both restriction endonuclease and modification methylase activities. PMID:4564204

  13. Cell fractionation of parasitic protozoa: a review

    Directory of Open Access Journals (Sweden)

    Souza Wanderley de

    2003-01-01

    Full Text Available Cell fractionation, a methodological strategy for obtaining purified organelle preparations, has been applied successfully to parasitic protozoa by a number of investigators. Here we present and discuss the work of several groups that have obtained highly purified subcellular fractions from trypanosomatids, Apicomplexa and trichomonads, and whose work have added substantially to our knowledge of the cell biology of these parasites.

  14. Adult pneumacoccal vaccination guideline | Feldman | South African ...

    African Journals Online (AJOL)

    Vaccine description. A highly purified vaccine containing 25 ug of each of 23 capsular polysaccharides representing≥85% of the serotypes causing pneumonia and invasive pneumococcal disease in the community. Polysaccharide antigens induce type-specific antibodies that enhance opsonisation, phagocytosis and ...

  15. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membran...

  16. Effect of impurities on mechanical properties of vanadium alloys under liquid-lithium environment during neutron irradiation at HFIR

    Science.gov (United States)

    Fukumoto, K.; Kuroyanagi, Y.; Kuroiwa, H.; Narui, M.; Matsui, H.

    2011-10-01

    Vanadium alloys, including the highly purified V-4Cr-4Ti alloy, were irradiated in liquid lithium up to a damage level of 3.7 dpa in the HFIR at 425 °C and 598 °C. Neutron irradiation caused an increase of the ductile-brittle transition temperature (DBTT) and irradiation hardening was observed. Adding titanium to the V-Cr alloys was effective for increasing irradiation hardening at 425 °C. For highly purified (Zr-treated) V-4Cr-4Ti alloys the irradiation hardening was significantly reduced at both 425 °C and 598 °C. However, microstructural observations after the irradiation experiments showed that there was no significant difference in microstructure between the original and the highly purified specimens. It is suggested that the reduction of irradiation hardening in the highly purified V-4Cr-4Ti alloys was caused by the configuration and distribution of interstitial impurities in the neutron-irradiated specimen matrix. Controlling the impurities in V-4Cr-4Ti alloys has a very important effect for improving their mechanical properties that take place under neutron irradiation at around 400 °C.

  17. Transcriptional Regulation in Haematopoiesis:

    DEFF Research Database (Denmark)

    Lauridsen, Felicia K B

    Haematopoietic stem cells (HSCs) are responsible for the formation of all of the distinct mature cell types found in the blood. HSCs can – as the only cells of the haematopoietic system – regenerate all of the blood cells when transplanted into a irradiated host, because they are endowed...... of distinct lineage affiliated genes in the otherwise highly purified HSCs. Taken together, these studies demonstrate the use of our model as a tool for isolating superior HSCs, and show that low-level expression of mature lineage markers is inherent in the highly purified stem cell compartment. In the second...... in transplantation studies. Consistent with this, transcriptome profiling revealed very low expression of cell cycle genes in these reporter-dim HSCs. Sequencing of >1200 single HSCs confirmed that the main source of transcriptional heterogeneity was the cell cycle. It also revealed a low-level expression...

  18. Kidney function and size in type 1 (insulin-dependent) diabetic patients before and during growth hormone administration for one week

    DEFF Research Database (Denmark)

    Christiansen, JS; Gammelgaard, J; Frandsen, M

    1982-01-01

    Kidney function and size were studied in seven well-controlled male Type 1 (insulin-dependent) diabetic patients before and after administration of highly purified human growth hormone for one week. Glomerular filtration rate, renal plasma flow (steady state infusion technique with urinary...... collections using 125I-iothalamate and 131I-hippuran), kidney size (ultrasonic scanning) and urinary excretion rates of albumin and beta-2-microglobulin were measured. Highly purified growth hormone was injected subcutaneously, 2 IU in the morning and 4 IU in the evening. The growth hormone dosage applied...... induced an elevation in plasma growth hormone concentration from the normal level seen in these very well controlled diabetics to levels within the range previously demonstrated in normally controlled Type 1 diabetic patients. During the week of growth hormone administration, glycaemic control...

  19. Kidney function and size in normal subjects before and during growth hormone administration for one week

    DEFF Research Database (Denmark)

    Gammelgaard, Jens; Orskov, H; Andersen, A R

    1981-01-01

    Kidney function and size were studied in seven normal male subjects before and after administration of highly purified human growth hormone for 1 week. Glomerular filtration rate, renal plasma flow (steady-state infusion technique with urinary collections using 125I-iothalamate and 131I......-hippuran) kidney size (ultrasonic scanning) and urinary excretion rates of albumin and beta 2-microglobulin (radioimmunoassays) were measured. Highly purified growth hormone was injected subcutaneously, 2 IU in the morning and 4 IU in the evening. Glomerular filtration rate increased from (mean +/- SEM) 114 +/- 5...... to 125 +/- 4 ml/min x 1.73 m2 (P less than 0.01) and renal plasma flow increased from 554 +/- 30 to 601 +/- 36 ml/min x 1.73 m2 (P less than 0.01). Kidney size and urinary excretion rates of albumin and beta 2-microglobulin did not change significantly. Our results show that raising plasma growth hormone...

  20. Tribosupplementation with Lubricin in Prevention of Post-Traumatic Arthritis

    Science.gov (United States)

    2014-10-01

    utilizing anion exchange, hydroxyapatite and a hydrophobic exchange media resins achieves a high level of purity. Explants of bovine articular cartilage...progress report and results in a highly purified product (Fig 1). It consists of all flow through steps utilizing anion exchange, hydroxyapatite and...cost extension The Effect of Age on Functional ACL Healing Optimizing the healing of adolescent ACL tissue so to match that seen in immature

  1. Amylase inhibits Neisseria gonorrhoeae by degrading starch in the growth medium.

    OpenAIRE

    Gregory, M R; Gregory, W W; Bruns, D E; Zakowski, J J

    1983-01-01

    Highly purified salivary alpha-amylase inhibited the growth of fresh isolates of Neisseria gonorrhoeae on GC agar base medium supplemented with 2% IsoVitaleX (BBL Microbiology Systems). Hydrolysis of starch in the medium by amylase resulted in a negative starch-iodine test. However, purified amylase did not inhibit gonococcal growth on agar plates that contained hemoglobin (chocolate agar). This effect was not caused by inhibition of amylase, since amylase activity was the same in the presenc...

  2. 2014 Salish Kootenai College Equipment Grant

    Science.gov (United States)

    2016-09-14

    and Scott, A.L. (2006). Innate immune responses to lung- stage helminth infection induce alternatively activated alveolar macrophages. Infect Immun ...effects of that protein on human lung cells. The purpose of these experiments are to investigate the immune response when cells are stimulated with...human lung cells. The purpose of these experiments are to investigate the immune response when cells are stimulated with highly purified chilectin

  3. Measurements of neutron cross section of the {sup 243}Am(n,{gamma}){sup 244}Am reaction

    Energy Technology Data Exchange (ETDEWEB)

    Hatsukawa, Yuichi; Shinohara, Nobuo; Hata, Kentaro [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-03-01

    The effective thermal neutron cross section of {sup 243}Am(n,{gamma}){sup 244}Am reaction was measured by the activation method. Highly-purified {sup 243}Am target was irradiated in an aluminum capsule by using a research reactor JRR-3M. The tentative effective thermal neutron cross sections are 3.92 b, and 84.44 b for the production of {sup 244g}Am and {sup 244m}Am, respectively. (author)

  4. Allergenius, an expert system for the interpretation of allergen microarray results

    OpenAIRE

    Melioli, Giovanni; Spenser, Clive; Reggiardo, Giorgio; Passalacqua, Giovanni; Compalati, Enrico; Rogkakou, Anthi; Riccio, Anna Maria; Di Leo, Elisabetta; Nettis, Eustachio; Canonica, Giorgio Walter

    2014-01-01

    Background An in vitro procedure based on a microarray containing many different allergen components has recently been introduced for use in allergy diagnosis. Recombinant and highly purified allergens belonging to different allergenic sources (inhalants, food, latex and hymenoptera) are present in the array. These components can either be genuine or cross-reactive, resistant or susceptible to heat and low pH, and innocuous or potentially dangerous. A large number of complex and heterogeneous...

  5. Microbial composition of purified waters and implications for regrowth control in municipal water systems

    OpenAIRE

    Proctor, Caitlin R.; Edwards, Marc A.; Pruden, Amy

    2015-01-01

    The limits of water treatment to control microbial regrowth were examined using highly purified waters. Measurable microbial genetic material was detected in the product water in a survey of thirteen laboratory pure water systems. Illumina 16S rRNA gene amplicon sequencing revealed surprisingly diverse microbial assemblages, confirmed to be active in bioassays, with no direct relationship to quality or maintenance of the systems. With storage under both light and dark conditions, a 2-log incr...

  6. Immobilization of Enzymes on mesoporous Supports and their Application in continuous-flow Biotransformations

    OpenAIRE

    Jung, Dirk

    2012-01-01

    Green and sustainable chemistry is an approach to reduce or to eliminate the use and the generation of hazardous substances by designing appropriate products and processes. The demand for specific and highly-purified fine-chemicals and pharmaceuticals can not be fulfilled with conventional catalysts alone. A promising alternative is the use of enzymes, the catalysts of biological processes. Enzymes are used to increase the selectivity of reactions; their outstanding advantage is the different...

  7. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    OpenAIRE

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomy...

  8. Factors underlying the natural resistance of animals against snake venoms

    Directory of Open Access Journals (Sweden)

    H. Moussatché

    1989-01-01

    Full Text Available The existence of mammals and reptilia with a natural resistance to snake venoms is known since a long time. This fact has been subjected to the study by several research workers. Our experiments showed us that in the marsupial Didelphis marsupialis, a mammal highly resistant to the venom of Bothrops jararaca, and other Bothrops venoms, has a genetically origin protein, a alpha-1, acid glycoprotein, now highly purified, with protective action in mice against the jararaca snake venom.

  9. Nano-Objects and Ions in Liquid Crystals: Ion Trapping Effect and Related Phenomena

    OpenAIRE

    Yuriy Garbovskiy; Iryna Glushchenko

    2015-01-01

    The presence of ions in liquid crystals is one of the grand challenges that hinder the application of liquid crystals in various devices, which include advanced 3-D and flexible displays, tunable lenses, etc. Not only do they compromise the overall performance of liquid crystal devices, ions are also responsible for slow response, image sticking, and image flickering, as well as many other negative effects. Even highly purified liquid crystal materials can get contaminated during the manufact...

  10. Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain.

    OpenAIRE

    Kato, K; Makino, Y; Kishimoto, T; Yamauchi, J; Kato, S; Muramatsu, M; Tamura, T

    1994-01-01

    The conformational states of the mouse TATA-binding protein (TBP) in solution were studied. A histidine tag and a factor Xa recognition site-carrying mouse TBP was expressed in E. coli, highly purified, and its fundamental functions as a TBP were demonstrated. We analyzed the molecular states of mouse TBP by gel filtration and glycerol gradient sedimentation, and found that TBP forms heterogeneous multimers in solution. Direct binding of TBP molecules to each other was proven by the far-Weste...

  11. Preparation Of Labeled Prolactin By 125I Using Lactoperoxidase And Iodogen

    International Nuclear Information System (INIS)

    ABDEL-MOUHTY, N.R.

    2009-01-01

    The present study was carried out to prepare 125 I-prolactin (PRL) tracer, using different oxidizing agents such as iodogen and lactoperoxidase, which was purified by column sephadex G-100 and used to measure the prolactin in human serum by radioimmunoassay (RIA) technique. The conditions of radioiodination such as concentration of oxidizing agent, reaction time, concentration of prolactin and effect of ph were studied to get maximum yield. Stability study of prolactin tracers was carried out. The prolactin standards were prepared using highly purified PRL antigen with phosphate buffer (ph 7.4, 0.05 M). Optimization of the assay was carried out.

  12. Analysis of the humoral immune response to Chlamydia outer membrane protein 2

    DEFF Research Database (Denmark)

    Mygind, P; Christiansen, Gunna; Persson, K

    1998-01-01

    The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated...... patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P immune responses were not confined to any particular region of the Omp2 protein, and no species-specific anti-Omp2 immunoglobulins were detected....

  13. Inhibition of vitamin D2-induced arteriosclerosis in rats by depletion of complement with cobra venom factor.

    Science.gov (United States)

    Pang, A S; Minta, J O

    1980-01-01

    Widespread calcerous deposits developed in the aorta, heart and kidneys of rats fed for 4 days with purina chow and high doses of vitamin D2 (200,000 IU/kg body wt/day). Decomplementation of rats with highly purified cobra venom factor (CoF) prior to vitamin D2 feeding, almost completely prevented calcium deposition in the aorta and arteritis. The mortality rate in the CoF-treated vitamin D2-fed rats was much lower than in untreated rats. These findings suggest that the complement system may be recruited in the pathogenesis of vitamin D2-induced arteriosclerosis.

  14. Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiation

    International Nuclear Information System (INIS)

    Cabrera-Juarez, E.; Setlow, J.K.

    1977-01-01

    Irradiation at 334 and 365 nm of a highly purified preparation of thymine-labelled transforming DNA from Haemophilus influenzae produced a photoproduct containing label from thymine but different from the cyclobutane dimer. The photoproduct is soluble in water and in ethanol and Rsub(f) values in a number of solvents are presented. The photoproduct has properties similar in a number of respects to those of the spore photoproduct, 5-thyminyl-5,6-dihydrothymine. The near ultraviolet photoproduct is more likely to affect the oxygen independent inactivation of transforming DNA rather than its mutagenesis, as judged by the quantitative relationship between amount of photoproduct and inactivation and mutagenesis

  15. Translation activity of chimeric ribosomes composed of Escherichia coli and Bacillus subtilis or Geobacillus stearothermophilus subunits

    Directory of Open Access Journals (Sweden)

    Sayaka Tsuji

    2017-07-01

    Full Text Available Ribosome composition, consisting of rRNA and ribosomal proteins, is highly conserved among a broad range of organisms. However, biochemical studies focusing on ribosomal subunit exchangeability between organisms remain limited. In this study, we show that chimeric ribosomes, composed of Escherichia coli and Bacillus subtilis or E. coli and Geobacillus stearothermophilus subunits, are active for β-galactosidase translation in a highly purified E. coli translation system. Activities of the chimeric ribosomes showed only a modest decrease when using E. coli 30 S subunits, indicating functional conservation of the 50 S subunit between these bacterial species.

  16. Synthesis and purification of methyl-13C fluoride

    International Nuclear Information System (INIS)

    Cohen, I.; Shulman, J.; Popowicz, A.; Ishida, T.

    1982-01-01

    A 20 mmole quantity of methyl- 13 C fluoride at a 13 C-enrichment level of 90% was prepared by a two-step synthesis starting with methanol- 13 C. The overall yield was 60% of methanol used. A series of gas-chromatographic treatments using Ascarite, silica gel, and Chromosorb-102 columns yielded a highly purified methyl fluoride in which the major impurities were less than 10 parts per million of carbon dioxide and 10 parts per million of methyl chloride. (author)

  17. The existence of two forms of hypertensin.

    Science.gov (United States)

    SKEGGS, L T; MARSH, W H; KAHN, J R; SHUMWAY, N P

    1954-03-01

    Two types of hypertensin have been demonstrated by means of counter-current distribution. The first type is the product of the action of the enzyme, renin, upon its substrate and has been designated hypertensin I. It can be rapidly converted to a second approximately equally pressor compound, hypertensin II, apparently through the action of an enzyme in the plasma which requires halide or nitrate for activation. A highly purified preparation containing horse hypertensins I and II caused an elevation of blood pressure when injected into human beings.

  18. Preparation and characterization of the Li(17)Pb(83) eutectic alloy and the LiPb intermetallic compound

    International Nuclear Information System (INIS)

    Jauch, U.; Karcher, V.; Schulz, B.

    1986-01-01

    Li(17)Pb(83) and LiPb were prepared from the pure elements in amounts of several hundred grams. The resolidified samples were characterized by melting points (eutectic temperature), chemical analysis and metallography. Using differential thermal analysis the heats of fusion were determined and the behaviour of the intermetallic phase LiPb in vacuum and high purified He was studied. The results from these investigations were applied to characterize Li(17)Pb(83) prepared in high amounts for technical application as a potential liquid breeder material. (orig.)

  19. The Isolation and Characterization of Human Prostate Cancer Stem Cells

    Science.gov (United States)

    2015-05-01

    Baylor College of Medicine from a 19 y/o boy who died after a traumatic accident. This line has been maintained in culture for several years and is...Bhatia B, Tang S, Reilly JG, Chandra D, Zhou J, Claypool K, Coghlan L, Tang DG. Highly purified CD44þ prostate cancer cells from xenograft human tumors...androgen receptor? Histochemis- try 1993;100(5):393–398. 24. Huang J, Yao JL, di Santagnese PA, Yang Q, Bourne PA, Na Y. Immunohistochemical

  20. Protective coatings (paints) for the nuclear industry - approved standard 1974 - (revision and redesignation of N5.9-1967)

    International Nuclear Information System (INIS)

    Anon.

    1976-01-01

    A standard is presented which provides a common basis on which protective coatings (paints) for surfaces in a nuclear complex may be compared and selected by reproducible tests. The environmental conditions of these complexes are such that a paint may be deteriorated by exposure to any, all, or a combination of the following conditions: ionizing radiation; contamination by radioactive nuclides and decontamination processes, chemicals; immersion in highly purified water; and abrasion or wear. This standard specifically applies to the surfaces of such facilities as reactors, fuel reprocessing plants, and laboratories and hot cells

  1. Production of isotopes using high power proton beams

    Science.gov (United States)

    Nolen, Jr., Jerry A.; Gomes, Itacil C.

    2015-12-01

    The invention provides for a method for producing isotopes using a beam of particles from an accelerator, whereby the beam is maintained at between about 70 to 2000 MeV; and contacting a thorium-containing target with the particles. The medically important isotope .sup.225Ac is produced via the nuclear reaction (p,2p6n), whereby an energetic proton causes the ejection of 2 protons and 6 neutrons from a .sup.232Th target nucleus. Another medically important isotope .sup.213Bi is then available as a decay product. The production of highly purified .sup.211At is also provided.

  2. Saccharin-induced sister chromatid exchanges in Chinese hamster and human cells

    Energy Technology Data Exchange (ETDEWEB)

    Wolff, S.; Rodin, B.

    1978-05-05

    Since the induction of sister chromatid exchanges in cultured cells has been shown to be the most sensitive mammalian system to detect the effects of mutagenic carcinogens, Chinese hamster ovary cells and human lymphocytes were exposed to the sodium saccharin found to induce bladder cancer in rats. Both that saccharin and a highly purified extract of it increased the yield of sister chromatid exchanges in both types of cells. The results, which were repeatable and statistically highly significant, indicated that the weak carcinogen, saccharin, is also mutagenic in the sense that it induces cytogenetic changes.

  3. [Highly active fractions of the medicinal leech recombinant destabilase-lysozyme].

    Science.gov (United States)

    Fadeeva, Iu I; Antipova, N V; Baskova, I P; Zavalova, L L

    2014-01-01

    From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms.

  4. Endotoxin, thrombin, and the Limulus amebocyte lysate test.

    Science.gov (United States)

    Yin, E T

    1975-09-01

    The Limulus amebocyte lysate, a proteinaceous composite isolated from the hemolymph cells of the horseshoe crab (Limulus polyphemus) is sensitive to picogram quantities of Gram-negative bacterial lipopolysaccharides. However, a controversy currently exists as to whether the Limulus amebocyte lysate is specifically sensitive to Gram-negative bacterial endotoxins as a result of a recent report that the blood coagulation protease, thrombin, can mimic endotoxins in the Limulus amebocyte lysate test. Experiments including those employing two highly purified fractions isolated from the Limulus lystae have provided us with evidence that thrombin per se is unable to mimic endotoxin.

  5. Calculated and measured efficiency of a man-portable 3He neutron detector

    International Nuclear Information System (INIS)

    O'Dell, A.A.

    1976-01-01

    A small man-portable neutron detector was constructed using 18 3 He proportional counters arranged in three layers within a polyethylene moderator. Each counter is 25 mm in diameter by 340 mm long (sensitive length) and is filled with highly-purified 3 He to a pressure of 400 kPa (4 atm). Efficiency measurements were made using a 252 Cf neutron source. Detailed calculations of the detector efficiency were done using the TART Monte Carlo transport code. Calculations and measurements were compared for several source/detector configurations

  6. Counting Characteristics Of Carbon dioxide-Filled Wall-Less Proportional Counter Suitable For Radiocarbon Dating

    International Nuclear Information System (INIS)

    Osadebe, F.A.N.; Hannan, H.H.M.A.; Uwah, E.J.

    1992-01-01

    A wall-less proportional counter that is suitable for radiocarbon dating has been developed. The counter consists of a central counter and an annular chain of twelve ring counters which are all enclosed in a cylindrical copper tube. Highly purified carbon dioxide is used as the counting gas and a chemical system designed for the purification of the gas is briefly described. The counter characteristics obtained with only tank gas as well as the purified gas are illustrated. They demonstrate the effect of electromagnetic impurities on the counter sensitivity and the significant background reduction achieved with the counter

  7. Interaction of 125I-labeled Ca2+-dependent regulator protein with cyclic nucleotide phosphodiesterase and its inhibitory protein

    International Nuclear Information System (INIS)

    Richman, P.G.; Klee, C.B.

    1978-01-01

    The Ca 2+ -dependent regulator protein of cyclic nucleotide phosphodiesterase was labeled with 125 I to the extent of 1 mol of monoiodotyrosine per mol. The iodinated protein showed a small decrease in affinity for phosphodiesterase but gave the same maximal level of activation of the enzyme as did the unmodified regulator protein. Iodinated regulator protein formed complexes with both highly purified cyclic nucleotide phosphodiesterase and phosphodiesterase inhibitory protein in the presence but not in the absence of Ca 2+ as demonstrated by ultracentrifugation in glycerol gradients. Cross-linking experiments indicate that the Ca 2+ -dependent regulator protein interacts with the large subunit of the inhibitory protein

  8. [Anthology of the first clinical studies with hypothalamic hormones: a story of successful international cooperation].

    Science.gov (United States)

    Schally, Andrew V; Gual, Carlos

    2002-01-01

    Our early pioneering clinical trials in Mexico with natural and synthetic thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH) also known as gonadotropin releasing hormone (Gn-RH), were reviewed. Highly purified TRH of porcine origin was shown to stimulate Thyrotropin (TSH) release in hypothyroid cretins. Subsequent tests with synthetic TRH also demonstrated significant increases in plasma TSH in normal men and women as well as in patients with primary hypothyroidism and other endocrine disorders. Even more extensive clinical studies were carried out with highly purified natural porcine LH-RH. Subjects with normal basal serum levels of gonadotropins, low levels (men and women pretreated with steroids) and high levels (e.g. post menopausal women) all responded to LH-RH with a release of LH and FSH. The results of these early studies with the natural LH-RH were confirmed by the use of synthetic LH-RH. These investigations made in Mexico with TRH and LH-RH preceded all other clinical studies by a wide margin. Subsequently various clinical investigations with LH-RH agonists and antagonists were also carried out. All these studies played a major role in introducing hypothalamic-releasing hormones into clinical medicine.

  9. Monoclonal antibodies to the major Lolium perenne (rye grass) pollen allergen Lol p I (Rye I).

    Science.gov (United States)

    Kahn, C R; Marsh, D G

    1986-12-01

    Thirteen monoclonal antibodies (MAbs) were produced against Lol p I (Rye I), the major Lolium perenne (rye grass) pollen allergen. Spleen cells from A/J and SJL mice immunized with highly purified Lol p I (Lol I) were allowed to fuse with cells from the non-secreting Sp2/0-Ag14 myeloma cell line. Each MAb was analyzed for antigenic specificity by radioimmunoassay (RIA) using 125I-Lol I. The epitope specificities of seven of the MAbs were examined by competitive binding against a labelled standard MAb for the Lol I antigen (Ag). The dissociation constant, Kd, of one MAb (No. 3.2) that was studied most extensively was determined by double Ab RIA to be 3.5 X 10(-6) L/M. This MAb recognized the related 27,000-30,000 Group I glycoproteins found in the pollens of nine other species of grass pollens tested, including weak binding to Bermuda grass Group I (Cyn d I), which by conventional analysis using polyclonal anti-Lol I serum shows no detectable binding. Monoclonal antibody No. 3.2 was coupled covalently to Sepharose 4B and used to prepare highly purified Lol I from a partially purified rye pollen extract. Finally, an RIA was developed which permitted the analysis of the Group I components in rye grass and nine other grass pollen species. The latter assay is likely to prove useful in the standardization of grass pollen extracts according to their Group I contents.

  10. Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation.

    Directory of Open Access Journals (Sweden)

    Antoine Ménoret

    Full Text Available Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. The localization of these factors, their bioactivity and natural countermeasures remain unclear. This concept was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA inhalation. Highly purified bronchoalveolar lavage fluid (BALF fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. The Inflammatory and inhibitory potentials of the identified proteins were measured using T cells assays. A potent pro-inflammatory factor was detected in BALF, and we hypothesized SEA could be recovered with its biological activity. Highly purified BALF fractions with bioactivity were subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. Inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show a fraction of bioactive SEA resided in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail.

  11. Iron-related toxicity of single-walled carbon nanotubes and crocidolite fibres in human mesothelial cells investigated by Synchrotron XRF microscopy.

    Science.gov (United States)

    Cammisuli, Francesca; Giordani, Silvia; Gianoncelli, Alessandra; Rizzardi, Clara; Radillo, Lucia; Zweyer, Marina; Da Ros, Tatiana; Salomé, Murielle; Melato, Mauro; Pascolo, Lorella

    2018-01-15

    Carbon nanotubes (CNTs) are promising products in industry and medicine, but there are several human health concerns since their fibrous structure resembles asbestos. The presence of transition metals, mainly iron, in the fibres seems also implicated in the pathogenetic mechanisms. To unravel the role of iron at mesothelial level, we compared the chemical changes induced in MeT-5A cells by the exposure to asbestos (crocidolite) or CNTs at different content of iron impurities (raw-SWCNTs, purified- and highly purified-SWCNTs). We applied synchrotron-based X-Ray Fluorescence (XRF) microscopy and soft X-ray imaging (absorption and phase contrast images) to monitor chemical and morphological changes of the exposed cells. In parallel, we performed a ferritin assay. X-ray microscopy imaging and XRF well localize the crocidolite fibres interacting with cells, as well as the damage-related morphological changes. Differently, CNTs presence could be only partially evinced by low energy XRF through carbon distribution and sometimes iron co-localisation. Compared to controls, the cells treated with raw-SWCNTs and crocidolite fibres showed a severe alteration of iron distribution and content, with concomitant stimulation of ferritin production. Interestingly, highly purified nanotubes did not altered iron metabolism. The data provide new insights for possible CNTs effects at mesothelial/pleural level in humans.

  12. Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma. [/sup 125/I tracer techniques

    Energy Technology Data Exchange (ETDEWEB)

    Fahrenkrug, J.; Schaffalitzky de Muckadell, O.B.

    1977-06-01

    A sensitive and specific radioimmunoassay for vasoactive intestinal polypeptide (VIP) has been developed, which can detect 3.3 pmol x L/sup -1/ of the peptide in plasma. Antisera to highly purified porcine VIP coupled to albumin were raised in eight rabbits. The final dilution, the avidity, and the specificity of each antiserum were determined. /sup 125/I-VIP served as label, and highly purified porcine VIP was used as standard. Separation of antibody-bound and free VIP was achieved by plasma-coated charcoal. Nonspecific interference with the assay system was excluded by extraction of plasm samples with ethanol. The reliability of the assay was investigated by recovery experiments, by serial dilution of plasma samples with high concentration of endogenous VIP, and by immunosorption. The within-and between assay reproducibility at a concentration of 18.3 pmol x L/sup -1/ was 1.6 and 2.3 pmol x L/sup -1/ (1 S.D.), respectively. Median fasting concentration of VIP in plasma from 74 normal subjects was 7.3 pmol x L/sup -1/ (range: 0 to 20.0 pmol x L/sup -1/).

  13. Heterostructured semiconductor single-walled carbon nanotube films for solution-processed high-performance field-effect transistors

    Science.gov (United States)

    Park, Noh-Hwal; Lee, Seung-Hoon; Jeong, Seung-Hyeon; Khim, Dongyoon; Kim, Yun Ho; Yoo, Sungmi; Noh, Yong-Young; Kim, Jang-Joo

    2018-03-01

    In this paper, we report a simple and effective method to simultaneously achieve a high charge-carrier mobility and low off current in conjugated polymer-wrapped semiconducting single-walled carbon nanotube (s-SWNT) transistors by applying a SWNT bilayer. To achieve the high mobility and low off current, highly purified and less purified s-SWNTs are successively coated to form the semiconducting layer consisting of poly (3-dodecylthiophene-2,5-diyl) (P3DDT)-wrapped high-pressure carbon mono oxide (HiPCO) SWNT (P3DDT-HiPCO) and poly (9, 9-di-n-dodecylfluorene) (PFDD)-wrapped plasma discharge (PD) SWNT (PFDD-PD). The SWNT transistors with bilayer SWNT networked film showed highly improved hole field-effect mobility (6.18 ± 0.85 cm2V-1s-1 average), on/off current ratio (107), and off current (˜1 pA). Thus, the combination of less purified PFDD-PD (98%-99%) charge-injection layer and highly purified s-P3DDT-HiPCO (>99%) charge-transport layer as the bi-layered semiconducting film achieved high mobility and low off current simultaneously.

  14. Influence of protein solution in nucleation and optimized formulation for the growth of ARM lipase crystal

    Science.gov (United States)

    Rahman, Raja Noor Zaliha Raja Abd; Masomian, Malihe; Leow, Adam Thean Chor; Ali, Mohd Shukuri Mohamad

    2015-09-01

    ARM lipase is a thermostable and organic solvent tolerant enzyme which was highly purified prior to crystallization. The His-tagged ARM lipase was purified with immobilized metal affinity chromatography followed by anion-exchange chromatography. The effect of different salt concentrations on stability, solubility and crystal nucleation of the protein was studied. The highly purified and homogeneous ARM lipase with protein concentration of 2 mg/mL was successfully crystallized by a sitting drop, vapor diffusion method with the use of 0.1 M MES monohydrate pH 6.5 and 12% (v/v) polyethylene glycol (PEG) 20000 as precipitant. The crystallization conditions were optimized by changing the pH and concentration of the precipitant. The optimum crystallization condition was 2 mg/mL ARM lipase in 0.1 M Tris-HCl, 0.15 M NaCl, pH 8.0 protein solution, crystallized using 0.1 M Tris-HCl, pH 8.0 and 12% (v/v) PEG 20000 as precipitant.

  15. Studies on the glycosome of Trypanosoma brucei

    International Nuclear Information System (INIS)

    Aman, R.A.

    1985-01-01

    Glycosomes (microbodies) have been purified from bloodstream form Trypanosoma brucei by an improved procedure involving freezing and thawing live organisms in 15% glycerol prior to cell disruption. Highly purified organelles of bloodstream form T. brucei contain 11 major proteins of which 8 tentatively identified glycolytic enzymes make up about 90% of the total glycosomal protein. Treatment of these intact isolated organelles with the bisimidoester dimethylsuberimidate (DMSI) resulted in crosslinking of all glycosomal proteins into a large complex suggestive of juxtapositioning of the glycosomal proteins. The crosslinked complex was capable of catalyzing the multienzyme conversion of glucose to glycerol-3-phosphate but did not possess any special kinetic features different from those of the unaggregated enzymes represented by solubilized glycosomes. The multienzyme reaction had a lab phase associated with it and [ 14 C]-glucose label incorporation into sugar phosphate intermediates was effectively competed by unlabeled intermediates. Glycosomes were also purified from culture form T. brucei by several different procedures. Comparison of highly purified organelles from the two different life stages of the organism showed reduced specific activities and contents of the early glycolytic enzymes in organelles from the culture form with a decrease from 87% to 35% of the contribution of glycolytic enzymes to the total glycosomal protein

  16. Mitochondrial DNA polymerase from embryos of Drosophila melanogaster: purification, subunit structure, and partial characterization

    International Nuclear Information System (INIS)

    Wernette, C.M.; Kaguni, L.S.

    1986-01-01

    The mitochondrial DNA polymerase has been purified to near-homogeneity from early embryos of Drosophila melanogaster. Sodium dodecyl sulfate gel electrophoresis of the highly purified enzyme reveals two polypeptides with molecular masses of 125,000 and 35,000 daltons, in a ratio of 1:1. The enzyme has a sedimentation coefficient of 7.6 S and a stokes radius of 51 A. Taken together, the data suggest that the D. melanogaster DNA polymerase γ is a heterodimer. DNA polymerase activity gel analysis has allowed the assignment of the DNA polymerization function to the large subunit. The DNA polymerase exhibits a remarkable ability to utilize efficiently a variety of template-primers including gapped DNA, poly(rA).oligo(dT) and singly primed phiX174 DNA. Both the crude and the highly purified enzymes are stimulated by KCl, and inhibited by dideoxythymidine triphosphate and by N-ethylmaleimide. Thus, the catalytic properties of the near-homogeneous Drosophila enzyme are consistent with those of DNA polymerase γ as partially purified from several vertebrates

  17. Preparation Of Liquid Phase-Double Antibodies Radioimmunoassay For The In Vitro Determination Of Prolactin Hormone In Human Serum

    International Nuclear Information System (INIS)

    MEHANY, N.L.; EL-KOLALY, M.T.; EBEID, N.H.; MEKY, N.H.

    2009-01-01

    In the present study, the preparation of the basic reagents of prolactin (PRL) radioimmunoassay (RIA) technique using liquid phase double antibody with low cost is considered to be the main objective. Three primary components were prepared and characterized to obtain valid and accurate system. These components were polyclonal antibody (anti-PRL), 125 I-prolactin ( 125 I-PRL) radio-iodinated tracer and PRL standards. The production of polyclonal anti-PRL was undertaken by immunizing eight males of white New-Zealand rabbits (two groups) with highly purified PRL antigen through primary injection and five booster doses subcutaneously and intramuscular. The preparation of radio-iodinated ( 1 '2 5 I-PRL) tracer was carried out using chloramine-T method. The preparation of PRL standards were carried out using highly purified PRL antigen in assay buffer. The obtained PRL-antisera were characterized in terms of titer, immuno response and displacement profile. Formulation, optimization and validation of the local liquid phase RIA system were carried out. The results obtained provide a highly sensitive, specific and accurate RIA system of PRL. In conclusion, this technique could be used in diagnosis of pituitary dysfunction such as hyperprolactinaemia and hyperprolactinaemia, prolactinoma, galactorrhoea, amenorrhea and diagnosis of infertility in males and females.

  18. Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

    International Nuclear Information System (INIS)

    Cameron, R.S.; Cameron, P.L.; Castle, J.D.

    1986-01-01

    A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion

  19. [Oleksandr Solomonovych Tsyperovych--gifted enzymologist, scientist and practician].

    Science.gov (United States)

    Vynohradova, R P; Kolodzeĭs'ka, M V

    2007-01-01

    pepsin, preparation gastric juice, crystalline trypsin, highly purified chemotrypsin, highly purified trypsin, highly purified amylase which were introduced into industry. New methods of silver regeneration from photographic and cinema films with the use of proteinase of S. griseus were developed, as well as the enzyme-antibiotic preparation protezym for hydrolysis of fodder proteins which gave positive results in breeding calves and in feeding adult animals. O. S. Tsyperovich has proposed two simple structures of automatic collector of fractions for the column chromatography, published 178 scientific works including 3 monographs, 10 author's certificates; 12 candidates of science were brought up under his supervision.

  20. Latex beads internalization and quantitative proteomics join forces to decipher the endosomal proteome.

    Science.gov (United States)

    Guimarães de Araújo, Mariana Eça; Huber, Lukas Alfons; Stasyk, Taras

    2011-06-01

    The proteome analysis of endocytic compartments has been constrained by the limited purity of the organelle fractions obtained by current biochemical methods. Duclos and coworkers have developed a novel method to isolate highly purified endosomal organelles based on small latex beads internalization followed by gradient centrifugation and successfully combined it with a redundant peptide counting method to compare the relative abundance of proteins in organelles. The presence of bona fide markers in their respective subcellular organelles and the identification of several new endosomal-associated proteins, attested the applicability of their combinatory approach. Future applications of this strategy may deliver a comprehensive endosomal proteome chart: from the identification of the key players to the determination of time and signaling-dependent proteome changes. As a long-term perspective, such an approach may unveil new clues to the molecular mechanisms underlining human diseases associated with endosomal biogenesis defects.

  1. The Potency of Hyaluronan of Different Molecular Weights in the Stimulation of Blood Phagocytes

    Directory of Open Access Journals (Sweden)

    Barbora Safrankova

    2010-01-01

    Full Text Available The regulatory functions of glycosaminoglycan hyaluronan (HA are suggested to be dependent on its molecular weight (MW. Proinflammatory and stimulatory effects are proposed mainly for the low MW HA. However, the complex response of blood phagocytes to HA of different MW is unclear. Herein, the effects of highly purified HA of precisely defined MW (52, 250, and 970 kDa on human blood phagocytes were tested. All MW HA activated blood phagocytes, including the spontaneous production of ROS, degranulation, and the production of tumor necrosis factor alpha, with low MW HA 52 kDa having the highest potency and high MW HA 970 kDa having the lowest potency. Interestingly, HA inhibited ROS production stimulated by opsonized zymosan particles and, in contrast, potentiated starch-activated ROS production, mostly independent of MW. Data showed a significant effect of HA of different MW on blood phagocytes, including high MW HA.

  2. The potency of hyaluronan of different molecular weights in the stimulation of blood phagocytes.

    Science.gov (United States)

    Safrankova, Barbora; Gajdova, Silvie; Kubala, Lukas

    2010-01-01

    The regulatory functions of glycosaminoglycan hyaluronan (HA) are suggested to be dependent on its molecular weight (MW). Proinflammatory and stimulatory effects are proposed mainly for the low MW HA. However, the complex response of blood phagocytes to HA of different MW is unclear. Herein, the effects of highly purified HA of precisely defined MW (52, 250, and 970  kDa) on human blood phagocytes were tested. All MW HA activated blood phagocytes, including the spontaneous production of ROS, degranulation, and the production of tumor necrosis factor alpha, with low MW HA 52  kDa having the highest potency and high MW HA 970  kDa having the lowest potency. Interestingly, HA inhibited ROS production stimulated by opsonized zymosan particles and, in contrast, potentiated starch-activated ROS production, mostly independent of MW. Data showed a significant effect of HA of different MW on blood phagocytes, including high MW HA.

  3. The Borexino detector: Methodology for a large-scale low background liquid scintillator

    Energy Technology Data Exchange (ETDEWEB)

    Ianni, Andrea, E-mail: ianni@princeton.ed [Physics Department, Princeton University, Jadwin Hall, Princeton, NJ 08544 (United States)

    2010-05-21

    Borexino, a real-time device for low energy neutrino spectroscopy, has completed the construction phase in the middle of 2006, in the underground laboratories at Gran Sasso, Italy (LNGS). The detector has been filled with about 300 tons of highly purified scintillator surrounded by an internal buffer of 1000 tons of a purified organic solvent buffer, 2400 tons of ultra-pure water as an external shield and located underground with the equivalent shielding of 3500 m of water. Since May 16, 2007, Borexino is under data acquisition. The experimental goal is the direct measurement of the flux of {sup 7}Be solar neutrinos of all flavours via neutrino-electron scattering in an ultra-pure scintillation liquid. The paper describes the design of the Borexino detector, the construction phases and the various facilities that play an essential role in its operation. This paper describes the design requirements and methods for reducing background impurities for the Borexino detector.

  4. Recent results on reactions with radioactive beams at RIBRAS (Radioactive Ion Beams in Brazil)

    International Nuclear Information System (INIS)

    Lépine-Szily, A; Lichtenthäler, R; Guimarães, V; Barioni, A; Benjamim, E A; De Faria, P N; Gasques, L R; Leistenschneider; Morais, M C; Morcelle, V; Condori, R Pampa; Pires, K C C; Scarduelli, V; Arazi, A; Descouvemont, P; Mendes, D R Jr; Moro, A M; Rodriguez-Gallardo, M; Shorto, J M B

    2015-01-01

    We present a quick description of RIBRAS (Radioactive Ion beams in Brazil), which is a superconducting double solenoid system, installed at the Pelletron Laboratory of the University of São Paulo and extends the capabilities of the original Pelletron Tandem Accelerator of 8MV terminal voltage (8UD) by producing secondary beams of unstable nuclei. The experimental program of the RIBRAS covers the study of elastic and inelastic scattering with the objective to study the interaction potential and the reaction mechanisms between weakly bound (RIB) and halo ( 6 He and 8 B) projectiles on light, medium and heavy mass targets. With highly purified beams, the study of resonant elastic scattering and resonant transfer reactions, using inverse kinematics and thick targets, have also been included in our recent experimental program. (paper)

  5. Free dendritic growth in viscous melts - Cyclohexanol

    Science.gov (United States)

    Singh, N. B.; Glicksman, M. E.

    1989-01-01

    Experiments were carried out to measure the growth speed, V, and dendritic tip radius, R, of highly purified cyclohexanol. The data show that VR-squared = constant over the entire experimentally observed supercooling range, Delta T is between 0.1 and 1 K. The stability parameter estimated from this result indicates that sigma(asterisk) = 0.027, a value in good agreement with the values of sigma(asterisk) found for the cubic plastic crystals succinonitrile pivalic acid. Cyclohexanol differs from other carefully measured plastic crystals in that the viscosity of its melt at the melting point is about 20 times higher, so gravity-induced convection remains weak even at small supercoolings.

  6. Amylase inhibits Neisseria gonorrhoeae by degrading starch in the growth medium.

    Science.gov (United States)

    Gregory, M R; Gregory, W W; Bruns, D E; Zakowski, J J

    1983-12-01

    Highly purified salivary alpha-amylase inhibited the growth of fresh isolates of Neisseria gonorrhoeae on GC agar base medium supplemented with 2% IsoVitaleX (BBL Microbiology Systems). Hydrolysis of starch in the medium by amylase resulted in a negative starch-iodine test. However, purified amylase did not inhibit gonococcal growth on agar plates that contained hemoglobin (chocolate agar). This effect was not caused by inhibition of amylase, since amylase activity was the same in the presence or absence of blood products. Moreover, survival of N. gonorrhoeae in buffered saline was not affected by amylase. These results suggest that amylase inhibited the growth of N. gonorrhoeae on GC agar base plates by hydrolyzing starch.

  7. Soya Saponins Induce Enteritis in Atlantic Salmon (Salmo salar L.).

    Science.gov (United States)

    Krogdahl, Åshild; Gajardo, Karina; Kortner, Trond M; Penn, Michael; Gu, Min; Berge, Gerd Marit; Bakke, Anne Marie

    2015-04-22

    Soybean meal-induced enteritis (SBMIE) is a well-described condition in the distal intestine of salmonids, and saponins have been implicated as the causal agent. However, the question remains whether saponins alone cause SBMIE. Moreover, the dose-response relationship has not been described. In a 10 week feeding trial with Atlantic salmon, a highly purified (95%) soya saponin preparation was supplemented (0, 2, 4, 6, or 10 g/kg) to two basal diets, one containing fishmeal as the major protein source (FM) and the other 25% lupin meal (LP). Saponins caused dose-dependent increases in the severity of inflammation independent of the basal diet, with concomitant alterations in digestive functions and immunological marker expression. Thus, saponins induced inflammation whether the diet contained other legume components or not. However, responses were often the same or stronger in fish fed the corresponding saponin-supplemented LP diets despite lower saponin exposure, suggesting potentiation by other legume component(s).

  8. Expression, purification, and characterization of almond (Prunus dulcis) allergen Pru du 4.

    Science.gov (United States)

    Zhang, Yuzhu; Du, Wen-Xian; Fregevu, Cécile; Kothary, Mahendra H; Harden, Leslie; McHugh, Tara H

    2014-12-31

    Biochemical characterizations of food allergens are required for understanding the allergenicity of food allergens. Such studies require a relatively large amount of highly purified allergens. The level of Pru du 4 in almond is low, and its expression in a soluble form in Escherichia coli required an expression tag. An MBP tag was used to enhance its expression and solubility. Sumo was used for the first time as a peptidase recognition site. The expression tag was removed with a sumo protease, and the resulting wild-type Pru du 4 was purified chromatographically. The stability of the allergen was investigated with chemical denaturation. The Gibbs free energy of Pru du 4 folding-unfolding transition was determined to be 5.4 ± 0.7 kcal/mol.

  9. An Improved method for separation of leucocytes from peripheral blood of the little skate (Leucoraja erinacea)

    Science.gov (United States)

    Tomana, Mitsuru; Parton, Angela; Barnes, David W.

    2008-01-01

    Cartilaginous fish, especially sharks, rays and skates (elasmobranchs) hold interest as comparative models in immunology because they are thought to be among the organisms most closely related to the ancestor animal that first developed acquired immunity. The aim of this study was to improve methods used for the purification of viable leucocytes from peripheral blood of elasmobranchs. Here we describe modifications of density gradient centrifugation and medium formulation that improve isolation and analysis of highly-purified leucocytes from peripheral blood of a model elasmobranch, Leucoraja erinacea, the little skate. These techniques contribute to the preparation of elasmobranch immune cells that can be reliably analyzed by a variety of means, including the study of immune function. PMID:18474431

  10. Exosome-associated hepatitis C virus in cell cultures and patient plasma

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ziqing [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Zhang, Xiugen [Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Yu, Qigui [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); He, Johnny J., E-mail: johnny.he@unthsc.edu [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2014-12-12

    Highlights: • HCV occurs in both exosome-free and exosome-associated forms. • Exosome-associated HCV is infectious and resistant to neutralizing antibodies. • More exosome-associated HCV than exosome-free HCV is present in patient plasma. - Abstract: Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell–cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.

  11. Imobilização de enzimas em suportes cromatográficos: uma ferramenta na busca por substâncias bioativas Immobilization of the enzymes on chromatographic supports: a tool to research of inhibitor compounds

    Directory of Open Access Journals (Sweden)

    Carmen Lúcia Cardoso

    2009-01-01

    Full Text Available The development and characterization of bioreactors or IMER (immobilized enzyme reactors as research tools are important in the scope of medicinal chemistry and constitute an alternative for the rational development of drugs. This approach does not require highly purified enzymes or a great amount of protein, but increase the enzymatic stability against heat, organic solvents and pH, without too much loss of catalyst activity. Immobilized enzyme reactors (IMER can be used for the accomplishment of high efficiency screening on-line and, thus inhibitors can be quickly identified. Here, we emphasize the development of IMER by use of different methods of immobilization and chromatographic supports. Their applications, in different areas of research, are also fully discussed.

  12. Small-scale extraction and radioiodination of human hormones for the substitution of imported radioimmunoassay reagents

    International Nuclear Information System (INIS)

    Gimbo, E.K.; Ribela, M.T.C.P.; Borghi, V.C.; Schwarz, I.; Morganti, L.; Araujo, E.A.; Bartolini, P.

    1988-01-01

    The methods for national production of radioimmunoassay reagents to substitute imported kits of: highly purified unlabelled hormones for radioiodination; 125 I-labelled hormones; and specific high titre antisera are presented. The extraction and purification of human growth hormone (hGH) and human luteinizing hormone (hGH) were done from human pituitaries. The 125 I-labelled hormones are obtained by stoichiometric methods. The 125 I-hGH, 125 I-hLH, I-hTSH and 125 I- h calcitonin were prepared and tested in internal and external quality control, in comparison with imported products. The parameters such as: maximum binding to specific antiserum (Bo), nonspecific binding (NSB), mean effective dose (ED 50), sensitivity and accuracy were evaluated. (M.C.K.) [pt

  13. A systematic approach for the chromatographic fractionation and purification of major steroid saponins in commercial extracts of Yucca schidigera Roezl.

    Science.gov (United States)

    Sastre, F; Ferreira, F; Pedreschi, F

    2017-03-01

    Yucca schidigera Roezl. (yucca) is one of the major industrial sources of steroid saponins used as animal and human food additives. This work describes a new, systematic and reproducible three-step method by medium and high-pressure liquid chromatography (under RP, NP and RP conditions), for the isolation and purification of three groups of saponins, which were further purified in six sub-fractions, and finally into twelve individual steroid saponins previously reported in Y. schidigera. In accordance to the increasing applications of yucca extracts, further analytical, biological and physicochemical studies are still required. The presented method is applicable to the preparation of steroids saponins previously reported in commercial extracts of Y. schidigera, both as highly purified mixtures of defined composition, including twelve pure components. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

    Science.gov (United States)

    He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

    2015-10-01

    An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Studies on a radioimmunoassay for human erythopoietin.

    Science.gov (United States)

    Lertora, J J; Dargon, P A; Rege, A B; Fisher, J W

    1975-07-01

    A highly purified erythropoietin (ESF) preparation (12,000 units per milligram of protein) was labeled with Na'125I using the Chloramine-T method. Undamaged immunoreative labeled ESF was separated from the damaged, nonimmunologically receiveESF by Sephadex G-150 fractionation. This undamaged immunreactive ESF was usedin radioimmunoassay for human erythropoietin. Separation of bound from free antigen was acheived using the double-antibody technique. Approximately 55 per cent binding wasobserved at an antiserum dilution of 1:1500. This assay appears to be sensitive enough to detect as little as 0.025 milliunits of the International Reference Preparation erythropoietin. The estimated levels of thid hormone in normal and anemic uremic human subjects suggests that immunoreactive serum erythropoietin levels are elevated above normal in anemia of uremia.

  16. Characteristics of Lipoprotein(a)-Containing Circulating Immune Complexes as Markers of Coronary Heart Disease.

    Science.gov (United States)

    Klesareva, E A; Afanas'eva, O I; Donskikh, V V; Adamova, I Yu; Pokrovskii, S N

    2016-12-01

    We studied the composition of circulating immune complexes precipitated in the presence of various concentrations of polyethylene glycol in patients with coronary heart disease (CHD) and high concentration of lipoprotein(a) - Lp(a). Precipitation of highly purified Lp(a) preparation with polyethylene glycol was evaluated. The contents of Lp(a), autoantibodies to Lp(a), IgG, and IgM in circulating immune complexes isolated from the sera of donors and CHD patients with normal and high levels of Lp(a) were measured. Circulating immune complexes containing Lp(a) were detected in the plasma of CHD patients with high Lp(a) concentrations. The presence of high concentrations of Lp(a), autoantibodies to Lp(a), and circulating immune complexes in CHD patients suggests that immunological factor contributes to high atherothrombogenicity of Lp(a).

  17. Electrophoretic and immunological properties of folate-binding protein isolated from bovine milk

    International Nuclear Information System (INIS)

    Iwai, Kazuo; Tani, Masako; Fushiki, Tohru

    1983-01-01

    Changes of the folate-binding protein (FBP) concentration in bovine milk after parturition were investigated. The FBP was highly purified from mature milk by affinity chromatography. The purified FBP showed a single protein band in polyacrylamide gel electrophoresis and was immunologically homogenous in double immunodiffusion. However, in two-dimensional gel electrophoresis, the FBP was separated into several spots in isoelectric focusing in the first dimension, and each spot also showed two molecular weights in SDS-gel electrophoresis in the second dimension. But these FBP molecules were immunologically identical with each other. The neuraminidase treatment obviously diminished the number of isoelectric points of the FBP. Thus, the variety of FBP molecules was at least partially due to the variability of the sialic acid content in the carbohydrate moieties. Moreover, the milk FBP showed species-specificity among mammals immunologically as well as physicochemically. (author)

  18. Purification of Sarcocystis neurona sporocysts from opossum (Didelphis virginiana) using potassium bromide discontinuous density gradient centrifugation.

    Science.gov (United States)

    Elsheikha, Hany M; Murphy, Alice J; Fitzgerald, Scott D; Mansfield, Linda S; Massey, Jeffrey P; Saeed, Mahdi A

    2003-06-01

    This report describes a new, inexpensive procedure for the rapid and efficient purification of Sarcocystis neurona sporocysts from opossum small intestine. S. neurona sporocysts were purified using a discontinuous potassium bromide density gradient. The procedure provides a source of sporocyst wall and sporozoites required for reliable biochemical characterization and for immunological studies directed at characterizing antigens responsible for immunological responses by the host. The examined isolates were identified as S. neurona using random amplified polymorphic DNA primers and restriction endonuclease digestion assays. This method allows the collection of large numbers of highly purified S. neurona sporocysts without loss of sporocyst viability as indicated by propidium iodide permeability and cell culture infectivity assays. In addition, this technique might also be used for sporocyst purification of other Sarcocystis spp.

  19. Determination of hCG-alpha subunit in threatened pregnancy

    International Nuclear Information System (INIS)

    Talas, M.; Pohanka, J.; Fingerova, H.; Janouskova, M.; Krikal, Z.; Prasilova, J.; Zupkova, H.

    1987-01-01

    Radioimmunoassay of the hCG-alpha subunit was made using an antibody anti hCG-alpha serum, highly purified hCG-alpha for 125 I-labelling and the standard hCG-alpha. Sera of healthy pregnant women sampled throughout the whole pregnancies were used to determine x-bar±S.D. of hCG-alpha for 14-day intervals. Included in the study were groups of women with high risk of premature labor, late toxemia of pregnancy, twins and fetal hypotrophy. It was shown that increased hCG-alpha is found in pregnant women in whom signs of late toxemia of pregnancy are combined with high risk of premature labor, or with twin pregnancies, while in those with fetal hypotrophy hCG-alpha is within normal limits. (author). 3 figs., 7 refs

  20. A revisionist history of adult marrow stem cell biology or 'they forgot about the discard'.

    Science.gov (United States)

    Quesenberry, P; Goldberg, L

    2017-08-01

    The adult marrow hematopoietic stem cell biology has largely been based on studies of highly purified stem cells. This is unfortunate because during the stem cell purification the great bulk of stem cells are discarded. These cells are actively proliferating. The final purified stem cell is dormant and not representative of the whole stem cell compartment. Thus, a large number of studies on the cellular characteristics, regulators and molecular details of stem cells have been carried on out of non-represented cells. Niche studies have largely pursued using these purified stem cells and these are largely un-interpretable. Other considerations include the distinction between baseline and transplant stem cells and the modulation of stem cell phenotype by extracellular vesicles, to cite a non-inclusive list. Work needs to proceed on characterizing the true stem cell population.

  1. EspP, a Type V-secreted serine protease of enterohaemorrhagic Escherichia coli O157:H7, influences intestinal colonization of calves and adherence to bovine primary intestinal epithelial cells.

    Science.gov (United States)

    Dziva, Francis; Mahajan, Arvind; Cameron, Pamela; Currie, Carol; McKendrick, Iain J; Wallis, Timothy S; Smith, David G E; Stevens, Mark P

    2007-06-01

    Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic diarrhoeal pathogens of worldwide importance. Cattle are a key reservoir; however the molecular mechanisms that promote persistent colonization of the bovine intestines by EHEC are ill-defined. The large plasmid of EHEC O157:H7 encodes several putative virulence factors. Here, it is reported that the pO157-encoded Type V-secreted serine protease EspP influences the intestinal colonization of calves. To dissect the basis of attenuation, a bovine primary rectal epithelial cell line was developed. Adherence of E. coli O157:H7 to such cells was significantly impaired by espP mutation but restored upon addition of highly purified exogenous EspP. Data of this study add to the growing body of evidence that cytotoxins facilitate intestinal colonization by EHEC.

  2. Optimization of protein samples for NMR using thermal shift assays

    Energy Technology Data Exchange (ETDEWEB)

    Kozak, Sandra [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany); Lercher, Lukas; Karanth, Megha N. [European Molecular Biology Laboratory (EMBL), SCB Unit (Germany); Meijers, Rob [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany); Carlomagno, Teresa, E-mail: teresa.carlomagno@oci.uni-hannover.de [European Molecular Biology Laboratory (EMBL), SCB Unit (Germany); Boivin, Stephane, E-mail: sboivin77@hotmail.com, E-mail: s.boivin@embl-hamburg.de [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany)

    2016-04-15

    Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor{sup ®} provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

  3. Simultaneous measurement of 239Pu, 240Pu, 241Pu, and 242Pu by high resolution inductively coupled plasma mass spectrometer (HR ICP-MS) in marine sediments

    International Nuclear Information System (INIS)

    Bruneau, F.

    1999-01-01

    Transuranics elements are of particular interest in radioecological studies because of their radiotoxicity and their potential use to decipher source fingerprints and transport processes. The simultaneous measurement of 239 Pu, 240 Pu, 241 Pu, and 242 Pu in environmental samples requires a specific chemical procedure. This work deals with an analytical procedure which yields a very high grade of purification of Pu suitable for ultra low level detection by HR ICP-MS, from marine sediments. After the elimination of major elements (Fe, Al, Mg...) by a first chromatographic separation, a new device of purification by solvent extraction and concentration by a second chromatographic separation is used to obtain a concentrated and high purified solution of plutonium. The chemical procedure have been validated on IAEA certified sediment samples and on sediment samples collected in the roads of Cherbourg which had been previously analysed by other techniques (a spectrometry and thermo-ionisation mass spectrometer). (author)

  4. A fusion protein of the synthetic IgG-binding domain and aequorin: Expression and purification from E. coli cells and its application.

    Science.gov (United States)

    Inouye, Satoshi; Sahara-Miura, Yuiko

    2017-09-01

    Aequorin is a Ca 2+ -binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties. ZZ-AQ could be used as a reporter for detecting IgG and the measurable range of IgG coated on a 96-well plate was 1-1000 ng/mL. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Analysis of LSD in human body fluids and hair samples applying ImmunElute columns.

    Science.gov (United States)

    Röhrich, J; Zörntlein, S; Becker, J

    2000-01-10

    Immunoaffinity extraction units (LSD ImmunElute) are commercially available for the analysis of lysergic acid diethylamide (LSD) in urine. The ImmunElute resin contains immobilized monoclonal antibodies to LSD. We applied the ImmunElute procedure to serum and also to human hair samples. For hair analysis the samples were first extracted with methanol under sonication. The extracts were then purified using the ImmunElute resin. LSD analysis was carried out with HPLC and fluorescence detection. The immunoaffinity extraction provides highly purified extracts for chromatographic analysis. The limit of detection (signal-to-noise ratio = 3) has been determined to be hair samples from drug abusers (n = 11). One of these samples tested positive with an amount of 110 pg LSD in 112 mg extracted hair corresponding to a concentration of 1 pg/mg.

  6. Sensitivity of Vi phages to γ- radiation in the presence of cisplatin

    International Nuclear Information System (INIS)

    Dabrowski, T.; Kwiatkowski, B.

    2005-01-01

    In this study we determined Vi bacteriophage III sensitivity to native cisplatin, γradiation ( 60 Co) or to irradiated cisplatin, and checked the possibility of enhanced Vi bacteriophage III inactivation under combined exposure to cisplatin and γ radiation. We used highly purified phage suspensions in 0.9% NaCl solution or phosphate-buffered saline. Phage suspensions were titrated using a double agar layer method. Our study implies that survival of Vi bacteriophage III shows an exponential inverse correlation with cisplatin concentration in incubation medium and the time of phage incubation in the presence of cisplatin. The use of irradiated cisplatin reduces phage survival in comparison with suspensions containing non-irradiated cisplatin. Irradiation of phage suspension with cisplatin causes a significant increase of phage inactivation in comparison with either treatment alone. Our results suggest that presence of cisplatin in irradiated medium enhances the radiobiological effect on Vi bacteriophages III. (author)

  7. Capillary detectors for high resolution tracking

    CERN Document Server

    Annis, P

    1997-01-01

    We present a new tracking device based on glass capillary bundles or layers filled with highly purified liquid scintillator and read out at one end by means of image intensifiers and CCD devices. A large-volume prototype consisting of 5 × 105 capillaries with a diameter of 20 μm and a length of 180 cm and read out by a megapixel CCD has been tested with muon and neutrino beams at CERN. With this prototype a two track resolution of 33 μm was achieved with passing through muons. Images of neutrino interactions in a capillary bundle have also been acquired and analysed. Read-out chains based on Electron Bombarded CCD (EBCCD) and image pipeline devices are also investigated. Preliminary results obtained with a capillary bundle read out by an EBCCD are presented.

  8. Systematic high-yield production of human secreted proteins in Escherichia coli

    International Nuclear Information System (INIS)

    Dai Xueyu; Chen Qiang; Lian Min; Zhou Yanfeng; Zhou Mo; Lu Shanyun; Chen Yunjia; Luo Jingchu; Gu Xiaocheng; Jiang Ying; Luo Ming; Zheng Xiaofeng

    2005-01-01

    Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies

  9. Neonatal epicardial-derived progenitors aquire myogenic traits in skeletal muscle, but not cardiac muscle

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Jensen, Charlotte H; Skovrind, Ida

    2016-01-01

    heart missing regenerative signals essential for directed differentiation of EPDCs. Herein, we aimed to evaluate the myogenic potential of neonatal EPDCs in adult and neonatal mouse myocardium, as well as in skeletal muscle. The two latter tissues have an intrinsic capability to develop and regenerate......, in contrast to the adult heart. METHODS: Highly purified mouse EPDCs were transplanted into damaged neonatal and adult myocardium as well as regenerating skeletal muscle. Co-cultures with skeletal myoblasts were used to distinguish fusion independent myogenic conversion. RESULTS: No donor EPDC......-derived cardiomyocytes were observed in hearts. In contrast, a remarkable contribution of EPDCs to skeletal muscle myofiber formation was evident in vivo. Furthermore, co-cultures of EPDCs with myoblasts showed that EPDCs became part of multinucleated fibers and appeared to acquire myogenic traits independent...

  10. Isolation and characterization of the normal crossreacting antigen: homology of its NH2-terminal amino acid sequence with that of carcinoembryonic antigen.

    Science.gov (United States)

    Engvall, E; Shively, J E; Wrann, M

    1978-01-01

    The normal antigen, NCA, which crossreacts with the carcinoembryonic antigen, CEA, was purified from normal spleen tissue by an immunochemical purification method using insolubilized antibodies to either CEA or NCA. The highly purified NCA obtained was extensively characterized by immunological tests. The molecular weight of NCA determined by chromatography on Sephadex G-200 was approximately 100,000. The total amount of carbohydrate in NCA was 30%, compared to 60% in CEA. NCA and CEA also differed in sugar composition. The amino acid composition of NCA was nearly identical to that of CEA, except for the apparent presence of methionine in NCA but not in CEA. The sequence of the first 26 NH2-terminal amino acids in NCA was identical to that of CEA except at position 21, where alanine was found in NCA instead of valine in CEA. Images PMID:77016

  11. Proteome analysis of potato juice and tuber vacuoles from cv. Kuras

    DEFF Research Database (Denmark)

    Jørgensen, Malene

    from the vacuoles of mature potato tuber. A comprehensive investigation of the vacuolar proteome and a detailed analysis of the identified proteins and their possible roles in vacuolar function were carried out. A primary requirement of any proteomic analysis of an organelle is purity of the isolated...... organelle. A method was developed for isolation of highly purified intact vacuoles. The proteome analysis of the purified vacuoles involved separation of native soluble proteins by gel filtration with Superdex 200 into nine fractions. Each fraction was analyzed in two ways, by SDS-PAGE followed by protein...... open reading frames and expressed sequence tag databases were identified.   The putative vacuolar proteins were categorized into nine major functional groups (number of proteins in brackets): storage proteins (8), stress or defense related proteins (102), protein signaling and degradation processes (25...

  12. Simultaneous measurement of {sup 239}Pu, {sup 240}Pu, {sup 241}Pu, and {sup 242}Pu by high resolution inductively coupled plasma mass spectrometer (HR ICP-MS) in marine sediments; Mesure des isotopes du plutonium des sediments marins par spectrometrie de masse a plasma couple inductivement haute resolution (HR ICP-MS)

    Energy Technology Data Exchange (ETDEWEB)

    Bruneau, F

    1999-07-01

    Transuranics elements are of particular interest in radioecological studies because of their radiotoxicity and their potential use to decipher source fingerprints and transport processes. The simultaneous measurement of {sup 239}Pu, {sup 240}Pu, {sup 241}Pu, and {sup 242}Pu in environmental samples requires a specific chemical procedure. This work deals with an analytical procedure which yields a very high grade of purification of Pu suitable for ultra low level detection by HR ICP-MS, from marine sediments. After the elimination of major elements (Fe, Al, Mg...) by a first chromatographic separation, a new device of purification by solvent extraction and concentration by a second chromatographic separation is used to obtain a concentrated and high purified solution of plutonium. The chemical procedure have been validated on IAEA certified sediment samples and on sediment samples collected in the roads of Cherbourg which had been previously analysed by other techniques (a spectrometry and thermo-ionisation mass spectrometer). (author)

  13. Tailored enzymatic production of oligosaccharides from sugar beet pectin and evidence of differential effects of a single DP chain length difference on human faecal microbiota composition after in vitro fermentation

    DEFF Research Database (Denmark)

    Holck, Jesper; Hjernø, K.; Lorentzen, A.

    2011-01-01

    Sugar beet pectin was degraded enzymatically and separated by ion exchange chromatography into series of highly purified homogalacturonides and rhamnogalacturonides. MALDI-TOF/TOF mass-spectrometry was used to determine sizes and structural features. The methodology was based on the sequential use...... of monocomponent enzymes that were selected to target specific substructures in the sugar beet pectin. Notably pectin lyase and rhamnogalacturonan I lyase were used, which allowed detection of the resulting cleavage products by UV spectroscopy. Seven different homogalacturonides (HG) with degrees of polymerization...... (DP) from 2 to 8 and six different rhamnogalacturonide (RGI) structures, ranging from DP4 to 6 with defined galactose substitutions were purified. Total recoveries of 200 mg homogalacturonides and 67 mg rhamnogalacturonides per gram sugar beet pectin were obtained. This integrated biorefining method...

  14. Expression and characterization of recombinant tyramine beta-monooxygenase from Drosophila: a monomeric copper-containing hydroxylase.

    Science.gov (United States)

    Gray, Erin E; Small, Shiloh N; McGuirl, Michele A

    2006-05-01

    We report here the development of a robust recombinant expression system for Drosophila melanogaster tyramine beta-monooxygenase (TbetaM), the insect analog of mammalian dopamine beta-monooxygenase. Recombinant TbetaM is rapidly purified from the host cell media in three chromatographic steps. The expression system produces approximately 3-10 mg of highly purified, active protein per liter of culture. Recombinant TbetaM requires copper for activity and has a typical type 2 copper EPR spectrum. While TbetaM efficiently hydroxylates the aliphatic carbon of phenolic amines such as tyramine (the physiological substrate) and dopamine, phenethylamine is a poor substrate. TbetaM is most likely a monomer under physiological conditions, although under conditions of high pH and low ionic strength the dimeric form predominates. The lower oligomeric state of TbetaM may provide an advantage for structural studies over DbetaM, which exists as a mixture of dimer and tetramer.

  15. Oxidation of N-methyl substituted hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives by bovine milk xanthine oxidase.

    Science.gov (United States)

    Bergmann, F; Levene, L

    1976-05-13

    1. The oxidation of six series of purines (hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives) by a highly purified bovine milk xanthine oxidase (EC 1.2.3.2) has been studied, using a variety of N-methyl derivatives. 2. N-Methyl substituents can either enhance or reduce enzymic rates. Enhancement is ascribed to blockade of groups which mediate unfavorable modes of binding of substrate to enzyme. Introduction of N-methyl groups can also inhibit enzymic oxidation, either by occluding essential binding groups or by preventing spontaneous or enzyme-induced tautomerisation processes, which create suitable binding sites in the substrates. 3. In all purines which are rapidly attacked by xanthine oxidase, proper attachment to the active center is mediated by the groupings (3) NH, (9) N or (3) N, (9) NH. 4. Reduced rates usually express lowered substrate affinity, which finds its expression in weak competitive inhibition of xanthine oxidation.

  16. Ovarian imaging by the binding of radioiodinated chorionic gonadotropin: concise communication

    International Nuclear Information System (INIS)

    Lee, C.Y.; Lee, S.L.; Loken, M.K.

    1979-01-01

    Previous studies have demonstrated the presence of specific, high-affinity receptors for human chorionic gonadotropin (HCG) in human and rat ovaries. In the present study, highly purified HCG was labeled with iodine-131 and used for the imaging of luteinized rat ovaries. Excellent images were obtained. The accumulation of radioactivity in the ovaries was completely inhibited by previous administration of excess unlabeled HCG. Time-course studies show that radioiodinated HCG was rapidly accumulated within 1 hr and largely cleared from the ovary at 24 hr. These results show the potential for imaging of endocrine target tissues by hormone-receptor binding, and the possibility of quantitation of hormone receptors in tissues under pathological conditions

  17. Luteinizing hormone and human chorionic gonadotropin: origins of difference.

    Science.gov (United States)

    Choi, Janet; Smitz, Johan

    2014-03-05

    Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are widely recognized for their roles in ovulation and the support of early pregnancy. Aside from the timing of expression, however, the differences between LH and hCG have largely been overlooked in the clinical realm because of their similar molecular structures and shared receptor. With technologic advancements, including the development of highly purified and recombinant gonadotropins, researchers now appreciate that these hormones are not as interchangeable as once believed. Although they bind to a common receptor, emerging evidence suggests that LH and hCG have disparate effects on downstream signaling cascades. Increased understanding of the inherent differences between LH and hCG will foster more effective diagnostic and prognostic assays for use in a variety of clinical contexts and support the individualization of treatment strategies for conditions such as infertility. Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  18. Recent results on reactions with radioactive beams at RIBRAS (Radioactive Ion Beams in Brazil)

    Science.gov (United States)

    Lépine-Szily, A.; Lichtenthäler, R.; Guimarães, V.; Arazi, A.; Barioni, A.; Benjamim, E. A.; de Faria, P. N.; Descouvemont, P.; Gasques, L. R.; E; Leistenschneider; Mendes, D. R., Jr.; Morais, M. C.; Morcelle, V.; Moro, A. M.; Pampa Condori, R.; Pires, K. C. C.; Rodriguez-Gallardo, M.; Scarduelli, V.; Shorto, J. M. B.; Zamora, J. C.

    2015-04-01

    We present a quick description of RIBRAS (Radioactive Ion beams in Brazil), which is a superconducting double solenoid system, installed at the Pelletron Laboratory of the University of São Paulo and extends the capabilities of the original Pelletron Tandem Accelerator of 8MV terminal voltage (8UD) by producing secondary beams of unstable nuclei. The experimental program of the RIBRAS covers the study of elastic and inelastic scattering with the objective to study the interaction potential and the reaction mechanisms between weakly bound (RIB) and halo (6He and 8B) projectiles on light, medium and heavy mass targets. With highly purified beams, the study of resonant elastic scattering and resonant transfer reactions, using inverse kinematics and thick targets, have also been included in our recent experimental program.

  19. Effect of the hinge protein on the heme iron site of cytochrome c1

    International Nuclear Information System (INIS)

    Kim, C.H.; Yencha, A.J.; Bunker, G.; Zhang, G.; Chance, B.; King, T.E.

    1989-01-01

    X-ray absorption spectroscopic (XAS) studies on cytochrome c 1 from beef heart mitochondria were conducted to identify the effect of the hinge protein on the structure of the heme site in cytochrome c 1 . A comparison of XAS data of highly purified one-band and two-band cytochrome c 1 demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c 1 : a conformational change occurs within a radius of approximately 5 angstrom from the heme iron in cytochrome c 1 when the hinge protein is bound to cytochrome c 1 . This result may be correlated with the previous observations that the structure and reactivity of cytochrome c 1 are affected by the hinge protein

  20. Detection of IgE insulin antibody with radioallergosorbent test

    International Nuclear Information System (INIS)

    Nakagawa, S.; Nakayama, H.; Sasaki, T.; Watanabe, T.; Aoki, S.; Saito, N.

    1978-01-01

    An in vitro method for detecting IgE insulin antibody using the principle of the radioallergosorbent test (RAST) is described. In six patients with insulin allergy, the RAST values were higher than in normal persons or insulin-treated diabetics without insulin allergy. No differences were observed between normal persons and insulin-treated diabetics without insulin allergy. Moreover, it was observed that in one patient treated with highly purified insulin, there was a gradual decrease of RAST value parallel to the radioinsulin binding activity and clinical allergic symptoms. The RAST value of insulin is slightly inhibited by non-IgE antibodies and is, therefore, a semiquantitative value. However, the RAST is simple to perform and reproducible; it is therefore very useful in the detection of IgE insulin antibodies. (orig.) [de

  1. The isolation and characterization of the rat liver glucocorticoid hormone receptor complex

    International Nuclear Information System (INIS)

    Hapgood, J.P.

    1986-06-01

    The untransformed rat liver glucocorticoid hormone receptor complex (GHRC) is purified by affinity chromatography and ion-exchange chromatography, with a 46% yield. The methodological problems encountered in the affinity purification of the highly labile GHRC were examined in detail. Some of the properties of the highly purified untransformed and transformed GHRC, such as size on SDS-gel electrophoresis, DNA-cellulose and nuclear binding, and sedimentation coefficients on sucrose gradients, is examined. A detailed investigation of protein kinase activity in purified GHRC preparations was performed. A preliminary investigation on the possible association of RNA with the GHRC was also conducted. The receptor protein was covalently modified with biotin and the properties of biotinyl-GHRC were examined. In order to identify the glucocorticoid receptor during isolation, the receptor was covalently labelled with 3 H dexamethasone-mesylate and the procedure described

  2. Ultrastructural studies of time-course and cellular specificity of interleukin-1 mediated islet cytotoxicity

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Egeberg, J; Nerup, J

    1987-01-01

    Previous electron-microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin-1 for 7 days have indicated that interleukin-1 is cytotoxic to all islet cells. To study the time-course and possible cellular specificity of interleukin-1 cytotoxicity to islets exposed...... to interleukin-1 for short time periods, isolated rat or human islets were incubated with or without 25 U/ml highly purified human interleukin-1 for 24 h. Samples of rat islets were taken after 5 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and samples of human islets after 5 min, 30 min and 24 h...... of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin-1. After 16 h...

  3. Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain.

    Science.gov (United States)

    Kato, K; Makino, Y; Kishimoto, T; Yamauchi, J; Kato, S; Muramatsu, M; Tamura, T

    1994-04-11

    The conformational states of the mouse TATA-binding protein (TBP) in solution were studied. A histidine tag and a factor Xa recognition site-carrying mouse TBP was expressed in E. coli, highly purified, and its fundamental functions as a TBP were demonstrated. We analyzed the molecular states of mouse TBP by gel filtration and glycerol gradient sedimentation, and found that TBP forms heterogeneous multimers in solution. Direct binding of TBP molecules to each other was proven by the far-Western procedure. Analyses using TBPs truncated at the N- and C-termini demonstrated that the functionally important C-terminal domain was responsible for homomultimer formation, and the N-terminal domain enhances multimerization. Furthermore, it was found that the TATA sequence dissociates homomultimers, and only monomeric TBP binds to the TATA-box. We suggest that TBP shares structural motifs in the C-terminal conserved domain for intermolecular interaction and TATA-binding.

  4. Improving our national security readiness posture in high-technology materials

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, H.A. Jr. (Bureau of Mines, U.S. Dept. of Interior, Washington, DC (US))

    1989-05-01

    Having a stockpile is one of the principal options in ameliorating the effect of a supply disruption. The U.S. Government has had most of its present large stockpile of metals and minerals for over 40 years. During the past 10 to 15 years, there has been a steady increase in the use of new, highly purified or highly processed materials that have critical military-related applications, and many of these are substantially imported, making supply vulnerable during a defense emergency. The properties of these materials often make stockpiling difficult. The production and use technology of these materials significantly influences the ease of stockpiling. This paper examines a number of new materials in terms of technology and ease of stockpiling and consider other options for dealing with supply disruptions where necessary.

  5. Efficacy of rabies immunoglobulins in an experimental post-exposure prophylaxis rodent model.

    Science.gov (United States)

    Servat, Alexandre; Lutsch, Charles; Delore, Valentine; Lang, Jean; Veitch, Keith; Cliquet, Florence

    2003-12-12

    In a recently published Syrian hamster animal challenge study [Vaccine 19 (2001) 2273], a highly purified, heat-treated equine rabies immunoglobulin (pERIG HT, Favirab) did not elicit satisfactory protection. The efficacies of this batch, a second stage pERIG HT batch and reference RIG preparations (Imorab, Imogam Rage pasteurised, Berna antiserum) were compared in mice challenged with either Ariana canine field strain or CVS strain. Survival rates against Ariana challenge with the second pERIG HT batch were indistinguishable from those of other licensed preparations (83-90% survival), but the deficient batch did not provide satisfactory protection (53%). These data confirm the inadequate response to a first stage pERIG HT batch, but a current batch provides equivalent protection to that afforded by licensed HRIG and ERIG preparations.

  6. Process for preparing organoclays for aqueous and polar-organic systems

    Science.gov (United States)

    Chaiko, David J.

    2001-01-01

    A process for preparing organoclays as thixotropic agents to control the rheology of water-based paints and other aqueous and polar-organic systems. The process relates to treating low-grade clay ores to achieve highly purified organoclays and/or to incorporate surface modifying agents onto the clay by adsorption and/or to produce highly dispersed organoclays without excessive grinding or high shear dispersion. The process involves the treatment of impure, or run-of-mine, clay using an aqueous biphasic extraction system to produce a highly dispersed clay, free of mineral impurities and with modified surface properties brought about by adsorption of the water-soluble polymers used in generating the aqueous biphasic extraction system. This invention purifies the clay to greater than 95%.

  7. The gram-negative sensing receptor PGRP-LC contributes to grooming induction in Drosophila.

    Science.gov (United States)

    Yanagawa, Aya; Neyen, Claudine; Lemaitre, Bruno; Marion-Poll, Frédéric

    2017-01-01

    Behavioral resistance protects insects from microbial infection. However, signals inducing insect hygiene behavior are still relatively unexplored. Our previous study demonstrated that olfactory signals from microbes enhance insect hygiene behavior, and gustatory signals even induce the behavior. In this paper, we postulated a cross-talk between behavioral resistance and innate immunity. To examine this hypothesis, we employed a previously validated behavioral test to examine the function of taste signals in inducing a grooming reflex in decapitated flies. Microbes, which activate different pattern recognition systems upstream of immune pathways, were applied to see if there was any correlation between microbial perception and grooming reflex. To narrow down candidate elicitors, the grooming induction tests were conducted with highly purified bacterial components. Lastly, the role of DAP-type peptidoglycan in grooming induction was confirmed. Our results demonstrate that cleaning behavior can be triggered through recognition of DAP-type PGN by its receptor PGRP-LC.

  8. Preparation of radioinsulins for radioimmunoassay

    International Nuclear Information System (INIS)

    Pinto, H.; Wajchenberg, B.L.; Souza, I.T.T.; Lerario, A.C.; Pieroni, R.R.

    1977-01-01

    Studies using lactoperoxidase for insulin iodination, in order to obtain better sensitive and immunoreactivity assays, were performed. Equal amounts of highly purified porcine insulin were reacted with 2 μ1 of 125 I (S.A = 250 μCi/μg) by:a) Chloramine-T method of Hunter and Greenwood; b) Lactoperoxidase method of Thorell modified. Purification was performed in both BioGel P-60 or Sephadex G-50 column, after starch gel electrophoresis. Immunoreactivity assays were carried out with the labelled insulin. After 3 days incubation B/F ratio was 50% higher with lactoperoxidase iodination after both purification systems. Greater purity (99%) of the labelled hormone, was achieved from Sephadex G-50 column after starch gel electrophoresis than BioGel P-60 column (90%). The standard curves demonstrate higher sensitivity and precision when labelled insulin was iodinated by lactoperoxidase method giving greater discrimination for basal insulin levels [pt

  9. Polarization curves of solutions of high cadmium concentration

    International Nuclear Information System (INIS)

    Zosimovich, D.P.

    1979-01-01

    At electrochemical cadmium deposition on solid cathode of highly purified cadmium (out of solutions, containing 1.07m, 0.445; 0.312; 0.223; 0.107 mol/l of cadmium, 1 mol/l of sulfuric acid and 0.38 mol/l of zinc) it has been detected, that, cadmium deposition from these solutions agrees with the dependence epsilon - lg/isub(d)-i and for all cadmium concentrations investigated presents a straight line. It has been found that dependence of limiting current on cadmium concentration is a straight line; the mean value of the angle coefficient is 0.026; the mean value of semiwave potential constitutes 0.474+-6 mV as to hydrogen scale; the mean coefficient of cadmium diffusion equals to 2.37x10 -5 cm 2 /s -1

  10. Purification and crystallization of Cor a 9, a major hazelnut allergen

    International Nuclear Information System (INIS)

    Guo, Feng; Kothary, Mahendra H.; Wang, Yang; Yu, Xiaoping; Howard, Andrew J.; Fu, Tong-Jen; Zhang, Yu-Zhu

    2008-01-01

    The major hazelnut allergen Cor a 9 was purified from the natural source and crystallized. Diffraction data were collected to 1.9 Å resolution using a synchrotron-radiation source. Hazelnut (Corylus avellana) is one of the food sources that induce allergic reaction in a subpopulation of people with food allergy. The 11S legumin-like seed-storage protein from hazelnut has been identified as one of the major hazelnut allergens and named Cor a 9. In this study, Cor a 9 was extracted from hazelnut kernels using a high-salt solution and was purified by desalting out and FPLC to a highly purified state. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected and a structure solution has been obtained by molecular-replacement calculations. Further refinement of the structure is currently in progress

  11. Immunological properties of N-acetyl-β-d-glucosaminidase of normal human liver and of GM2-gangliosidosis liver

    Science.gov (United States)

    Carroll, M.; Robinson, D.

    1973-01-01

    Antisera were raised to a partially purified preparation of human liver hexosaminidase and to highly purified preparations of hexosaminidase isoenzymes A and B. All the antisera precipitated the enzyme in an enzymically active form, which could be located on immunodiffusion and immunoelectrophoretic gels by using a histochemical substrate. The antisera to the purified isoenzymes were shown to react with hexosaminidase from human liver, kidney, brain and spleen, but did not cross-react with human liver β-glucosidase, β-galactosidase, α-mannosidase, β-xylosidase, arylsulphatase or acid phosphatase. Hexosaminidases A and B were immunologically identical. The immunological properties of the hexosaminidases from livers of patients with three types of GM2-gangliosidoses were closely similar. No evidence could be found for cross-reacting material in enzyme-deficient states. ImagesPLATE 1 PMID:4198585

  12. Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.

    Directory of Open Access Journals (Sweden)

    Paulina Miernikiewicz

    Full Text Available Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS. In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage and of gpsoc (chaperone TF of E. coli. Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride, protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.

  13. Heterogeneity of L-type calcium channel alpha 1 subunits: stereoselective discrimination of different populations by the novel 1,4-dihydropyridine B 874-67.

    Science.gov (United States)

    Lakitsch, M; Knaus, H G; Topar, G; Romanin, C; Boer, R; Flockerzi, D; Striessnig, J; Schindler, H; Hoeltje, H D; Glossmann, H

    1993-02-01

    The basic (pKa = 8.49) 1,4-dihydropyridine B 874-67 [[3-(C1R,2S)- 2-methylamino-1-phenylpropyl]-5-methyl-1,4-dihydro-2,6-dimethyl-(4R)-4-( 3-nitrophenyl)pyridine-3,5-dicarboxylate hydrochloride] has unique properties; it can discriminate two populations of alpha 1 subunits in 1,4-dihydropyridine-sensitive calcium channels labeled with the neutral 1,4-dihydropyridine (+)-[3H]PN 200-110. The two populations, which occur in proportions of approximately 2:1 in rabbit skeletal muscle membranes and highly purified calcium channel preparations, differ approximately 20-fold in their affinity. The corresponding diastereomer, B 874-66, and other 1,4-dihydropyridines (neutral, basic, or permanently charged) do not share this property. The two populations were observed at 2 degrees, 22 degrees, and 37 degrees in similar proportions. Heterogeneity was also observed for guinea pig heart membrane calcium channels labeled with (+)-[3H]PN 200-110. Heterotropic allosteric regulators, Ca2+, and Mg2+, but not Ba2+ and Ni2+, abolished the discriminatory activity of B 874-67 at 2 degrees and 22 degrees, regardless of whether binding of the neutral 1,4-dihydropyridine was stimulated or inhibited. It is proposed that the two alpha 1 subunit populations differ with respect to their 1,4-dihydropyridine binding domain. The structural basis for the two populations is unclear but may relate to the functional heterogeneity of membrane-bound and highly purified calcium channel preparations previously observed by others.

  14. Some biological properties of human chorionic follicle stimulating hormone

    International Nuclear Information System (INIS)

    Tojo, Shimpei; Ashitaka, Yoshihiko; Maruo, Takeshi; Nishimoto, Hiroyuki

    1975-01-01

    The biological properties of human chorionic FSH (hCFSH) for rat ovaries were investigated. Highly purified hCFSH had similar response to the ovarian augmentation test as bovine FSH and significantly enhanced 3 H-thymidine uptake by granulosa cells and theca cells in the ovary of hypophysectomized rat. In contrast, highly purified hCG little responded to the ovarian augmentation test and had no effect on 3 H-thymidine uptake by the ovary. These results indicate that hCFSH may promote the follicular growth of ovary resulting from granulosa cell proliferation and its enlargement. In addition, freshly harvested porcine granulosa cells were employed in an in vitro system to investigate specific binding of hCFSH to ovarian receptor. Radioiodinated hCFSH ( 125 I-hCFSH) and hCG ( 125 I-hCG) were respectively incubated with cell suspensions. Binding of these hormone preparations was proportional to the cell number and increased with the time of incubation through 120 minutes. The binding ability of 125 I-hCFSH to the cells was greater than that of 125 I-hCG. Increasing concentrations of unlabeled hCFSH in the incubation mixture progressively inhibited the uptake of 125 I-hCFSH by granulosa cells. Unlabeled hCG was not able to compete with 125 I-hCFSH binding. The similar phenomenon to inhibit the binding of 125 I-hCG to the cells was also recognized in the presence of unlabeled hCG. These findings suggest that granulosa cell has at least two different types of receptor sites: one for hCFSH and the other for hCG. (auth.)

  15. Neutron sources for neutrino investigations with the lithium converter

    International Nuclear Information System (INIS)

    Lyashuk, V.I.; Lutostansky, Yu.S.

    2012-01-01

    Creation of the powerful antineutrino source with a hard spectrum is possible on the base of β - -decay of the short lived 8 Li (T 1/2 = 0.84 s) isotope formed in the reaction 7 Li(n,γ) 8 Li. The 8 Li. isotope is a prime perspective antineutrino source taking into account that neutrino cross section depends as σ ∼ E ν 2 at the considered energy. The creation of this type powerful neutrino source (neutrino factory) is possible by (n,γ)-activation of high-purified 7 Li isotope under intensive neutron flux. As a neutron source for this purpose can be used the nuclear reactors (of steady-state flux and pulsed one), neutron sources on the base of accelerators and neutron generating targets, beam-dumps of large accelerators. The capabilities and perspectives of neutron sources are considered for the purpose of creation of the neutrino factory. Different realizations of lithium antineutrino sources (lithium converter on the base of high purified 7 Li isotope) are discussed: static regime (i.e., without transport of 8 Li isotope to the detector); dynamic regime (pumping of activated lithium to a remote detector in a closed cycle); lithium converter on the base of (a) a pulse reactors and (b) constructed as tandem of an antineutrino source and accelerator with a neutron-producing target. Heavy water solution of LiOD is proposed as a substance for the lithium converter. The expressions for neutrino fluxes in the detector position are obtained

  16. Superconductivity of divalent Chevrel phases at very high pressures

    International Nuclear Information System (INIS)

    Yao, Y.S.; Guertin, R.P.; Hinks, D.G.; Jorgensen, J.; Capone II, D.W.

    1988-01-01

    The electrical resistivity and the superconducting transition temperatures were examined for three representative divalent Chevrel phase systems, SnMo 6 S 8 , EuMo 6 S 8 , and BaMo 6 S 8 , as a function of hydrostatic pressure to 2 GPa and in quasihydrostatic pressures to 10 GPa. In all systems, T/sub c/ is depressed to 0 K for sufficiently large pressures. For the Sn- and Eu-based systems, both highly purified samples and samples with controlled oxygen content were used. In an oxygenated SnMo 6 S 8 sample (less than 3% O 2 substituted for the S atoms) the pressure threshold and maximum T/sub c/ are 40% lower than in the pure sample, but for P>3.5 GPa the T/sub c/-P phase diagrams nearly coincide, with T/sub c/ reaching zero at an extrapolated pressure of about 12 GPa. In pure EuMo 6 S 8 , superconductivity appears only above a threshold pressure of about 1 GPa and is depressed to 0 K above 4.5 GPa. In an oxygenated sample the maximum T/sub c/ and the threshold pressure are depressed, and above about 3.5 GPa the T/sub c/-P phase diagrams coincide, as in the Sn-based system, although T/sub c/ is then rapidly depressed to 0 K at about 4.5 GPa. In a highly purified BaMo 6 S 8 sample superconductivity appears above about 2 GPa and is depressed to 0 K at extrapolated pressures above 12 GPa. A full transition to the zero-resistance superconducting state is observed in BaMo 6 S 8 . The data are discussed in terms of a model linking the rhombohedral-to-triclinic structural transition, the superconducting transition temperature, and the role of pressure in suppressing the structural transition

  17. Ovarian cancer stem cells are enriched in side population and aldehyde dehydrogenase bright overlapping population.

    Directory of Open Access Journals (Sweden)

    Kazuyo Yasuda

    Full Text Available Cancer stem-like cells (CSCs/cancer-initiaiting cells (CICs are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDH(Br cells from ovarian cancer cells. Both SP cells and ALDH(Br cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2, than those of main population (MP cells and ALDH(Low cells, respectively. We analyzed an SP and ALDH(Br overlapping population (SP/ALDH(Br, and the SP/ALDH(Br population exhibited higher tumor-initiating ability than that of SP cells or ALDH(Br cells, enabling initiation of tumor with as few as 10(2 cells. Furthermore, SP/ADLH(Br population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDH(Low, MP/ALDH(Br and MP/ALDH(Low cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDH(Br population was detected in several gynecological cancer cells with ratios of 0.1% for HEC-1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDH(Br overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs.

  18. On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution.

    Science.gov (United States)

    Wölfle, K; Michenfelder, M; König, A; Hull, W E; Rétey, J

    1986-05-02

    When (methyl-2H3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 1H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion. When [methyl-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA). By addition of [14C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)- and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using (2H4)succinyl-CoA. When (1-13C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (less than 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place. (1,4-13C2)Succinic acid and (1,4-13C2)succinyl-CoA were synthesised and their 13C-NMR chemical shifts were exactly determined. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonyl-CoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3H to water and slow washing-in from the medium. Whenever migration of

  19. Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli

    Science.gov (United States)

    Farshadpour, Fatemeh; Taherkhani, Reza; Makvandi, Manoochehr; Rajabi Memari, Hamid; Samarbafzadeh, Ali Reza

    2014-01-01

    Background: Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential. Objectives: The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate. Materials and Methods: The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni2+-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results: The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media. Conclusions: In this study, the optimized truncated ORF2 protein

  20. A study of the human immune response to Lolium perenne (rye) pollen and its components, Lol p I and Lol p II (rye I and rye II). I. Prevalence of reactivity to the allergens and correlations among skin test, IgE antibody, and IgG antibody data.

    Science.gov (United States)

    Freidhoff, L R; Ehrlich-Kautzky, E; Grant, J H; Meyers, D A; Marsh, D G

    1986-12-01

    In a stratified random sample of 320 white adults, the prevalence of puncture skin test positivity (ST +) to Lolium perenne (rye grass)-pollen extract (LPE) was 16%. Fifteen percent of all subjects (or 84% of subjects classified LPE IgE antibody positive [Ab +]) was classified IgE Ab + to highly purified Lol p I (Rye I), and 4% of all subjects (or 26% of subjects classified LPE IgE Ab +) was classified IgE Ab + to highly purified Lol p II (Rye II). These data and similar results obtained in an allergy-enriched group of 361 subjects are consistent with previous studies that Lol I is a major allergen and Lol II is a minor allergen of LPE. Whether we studied LPE, Lol I, or Lol II, responder subjects were younger than nonresponder subjects and more male than female subjects were responders. We then investigated the quantitative interrelationships among ST, IgE, and IgG Ab responsiveness to LPE, Lol I, and Lol II in the allergy-enriched group. For each allergen, log-log correlations were strong and significant for ST versus IgE Ab and for IgE Ab versus IgG Ab. All subjects IgE Ab + to Lol I or Lol II were IgG Ab + to that allergen, supporting other evidence for a commonality in the genetic control influencing the production of IgE and IgG Abs to a given allergen. Log-log correlations among ST end points, IgE Ab levels, or IgG Ab levels were strong for LPE versus either Lol I or Lol II but weak between Lol I and Lol II, consistent with the reported lack of cross-reactivity between Lol I and Lol II. Despite these findings, almost all Lol II + subjects were Lol I + by ST (98%), IgE Ab (91%), and IgG Ab (83%), suggesting that the Ia-restricted immune recognition of both these molecules is at least in part under a common genetic control.

  1. Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli.

    Science.gov (United States)

    Farshadpour, Fatemeh; Taherkhani, Reza; Makvandi, Manoochehr; Rajabi Memari, Hamid; Samarbafzadeh, Ali Reza

    2014-07-01

    Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential. The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate. The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media. In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified

  2. Conserved properties of Drosophila and human spermatozoal mRNA repertoires

    Science.gov (United States)

    Fischer, Bettina E.; Wasbrough, Elizabeth; Meadows, Lisa A.; Randlet, Owen; Dorus, Steve; Karr, Timothy L.; Russell, Steven

    2012-01-01

    It is now well established that mature mammalian spermatozoa carry a population of mRNA molecules, at least some of which are transferred to the oocyte at fertilization, however, their function remains largely unclear. To shed light on the evolutionary conservation of this feature of sperm biology, we analysed highly purified populations of mature sperm from the fruitfly, Drosophila melanogaster. As with mammalian sperm, we found a consistently enriched population of mRNA molecules that are unlikely to be derived from contaminating somatic cells or immature sperm. Using tagged transcripts for three of the spermatozoal mRNAs, we demonstrate that they are transferred to the oocyte at fertilization and can be detected before, and at least until, the onset of zygotic gene expression. We find a remarkable conservation in the functional annotations associated with fly and human spermatozoal mRNAs, in particular, a highly significant enrichment for transcripts encoding ribosomal proteins (RPs). The substantial functional coherence of spermatozoal transcripts in humans and the fly opens the possibility of using the power of Drosophila genetics to address the function of this enigmatic class of molecules in sperm and in the oocyte following fertilization. PMID:22378807

  3. Emerging options for the management of scorpion stings

    Directory of Open Access Journals (Sweden)

    Chippaux JP

    2012-07-01

    Full Text Available Jean-Philippe ChippauxUMR 216 (Institute of Research for Development and University Paris Descartes, Sorbonne Paris Cité, Cotonou, Bénin, FranceAbstract: Scorpion stings are common in many tropical countries. Although most scorpion stings cause only localized pain without life-threatening envenoming, about one third of stings cause systemic envenoming which can result in death. Children are particularly sensitive to scorpion envenoming. The severity of scorpion stings is related to the presence of neurotoxins in the venom that cause a sudden release of neurotransmitters from the autonomic nervous system, predominantly sympathetic. There is also a strong inflammatory response that worsens symptoms, including those of a respiratory nature. Several vital functions may be directly affected, including the cardiovascular, respiratory, and neuromuscular systems. Hypertension is constant at the beginning of systemic envenoming and sometimes has a severe cardiac and respiratory impact. Although controversial, immunotherapy is the only etiological treatment. Administered early, it prevents many complications and improves the outcome. New antivenoms are highly purified immunoglobulin fragments, the efficacy and safety of which are excellent. As a consequence, adverse reactions to antivenoms are now very rare and usually mild, which should limit any reluctance regarding their routine use. Symptomatic treatment is still necessary to support immunotherapy, especially in cases of delayed arrival at hospital. A combination of both approaches should be considered, based on local resources and constraints.Keywords: scorpion, envenoming, treatment, antivenom

  4. Data of self-made Taq DNA polymerase prepared for screening purposes

    Directory of Open Access Journals (Sweden)

    E.V. Konovalova

    2017-04-01

    Full Text Available DNA analysis is a key procedure in genetic engineering. Nowadays the analysis is often done by PCR with Taq DNA polymerase. Although the last enzyme price is quite low, demand for numerous analyses results in much money expenditure which are not affordable for many laboratories. In a meanwhile, many screening tasks do not require the highly purified enzyme. Taking into account the enzyme unique properties it makes possible to marginally simplify its production without resorting to costly or lengthy techniques such as column chromatography and/or dialysis. Here the data of routine usage of Taq DNA polymerase prepared according to the protocol developed in our laboratory is presented. The protocol takes only several hours to realize and does not need qualified personnel or expensive equipment. Yet it gives the enzyme preparation suitable for most screening purposes. The isolated Taq DNA polymerase stock can be stored as ammonium sulfate suspension in a refrigerator for prolonged period, not less than 6 months. The working enzyme solution is prepared from the stock suspension on demand, not more than once in a month and can be stored also in a refrigerator.

  5. The characterization of exosome from blood plasma of patients with colorectal cancer

    Science.gov (United States)

    Yunusova, N. V.; Tamkovich, S. N.; Stakheeva, M. N.; Afanas'ev, S. G.; Frolova, A. Y.; Kondakova, I. V.

    2016-08-01

    Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of "International Society for Extracellular Vesicles". The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. The results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.

  6. Polyunsaturation in cell membranes and lipid bilayers and its effects on membrane proteins.

    Science.gov (United States)

    Slater, S J; Kelly, M B; Yeager, M D; Larkin, J; Ho, C; Stubbs, C D

    1996-03-01

    The effect of variation of the degree of cis-unsaturation on cell membrane protein functioning was investigated using a model lipid bilayer system and protein kinase C (PKC). This protein is a key element of signal transduction. Furthermore it is representative of a class of extrinsic membrane proteins that show lipid dependent interactions with cell membranes. To test for dependence of activity on the phospholipid unsaturation, experiments were devised using a vesicle assay system consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) in which the unsaturation was systematically varied. Highly purified PKC alpha and epsilon were obtained using the baculovirus-insect cell expression system. It was shown that increased PC unsaturation elevated the activity of PKC alpha. By contrast, increasing the unsaturation of PS decreased the activity of PKC alpha, and to a lesser extent PKC epsilon. This result immediately rules out any single lipid bilayer physical parameter, such as lipid order, underlying the effect. It is proposed that while PC unsaturation effects are explainable on the basis of a contribution to membrane surface curvature stress, the effects of PS unsaturation may be due to specific protein-lipid interactions. Overall, the results indicate that altered phospholipid unsaturation in cell membranes that occurs in certain disease states such as chronic alcoholism, or by dietary manipulations, are likely to have profound effects on signal transduction pathways involving PKC and similar proteins.

  7. Determination of extinction coefficients of human hemoglobin in various redox states.

    Science.gov (United States)

    Meng, Fantao; Alayash, Abdu I

    2017-03-15

    The role of hemoglobin (Hb) redox forms in tissue and organ toxicities remain ambiguous despite the well-documented contribution of Hb redox reactivity to cellular and subcellular oxidative changes. Moreover, several recent studies, in which Hb toxicity were investigated, have shown conflicting outcomes. Uncertainties over the potential role of these species may in part be due to the protein preparation method of choice, the use of published extinction coefficients and the lack of suitable controls for Hb oxidation and heme loss. Highly purified and well characterized redox forms of human Hb were used in this study and the extinction coefficients of each Hb species (ferrous/oxy, ferric/met and ferryl) were determined. A new set of equations were established to improve accuracy in determining the transient ferryl Hb species. Additionally, heme concentrations in solutions and in human plasma were determined using a novel reversed phase HPLC method in conjugation with our photometric measurements. The use of more accurate redox-specific extinction coefficients and method calculations will be an invaluable tool for both in vitro and in vivo experiments aimed at determining the role of Hb-mediated vascular pathology in hemolytic anemias and when Hb is used as oxygen therapeutics. Published by Elsevier Inc.

  8. Anti-Adhesive Activities of Flavan-3-ols and Proanthocyanidins in the Interaction of Group A-Streptococci and Human Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Aneta Janecki

    2010-10-01

    Full Text Available Bacterial adhesion to epithelial cells is a key step in infections, allowing subsequent colonization, invasion and internalization of pathogens into tissues. Anti-adhesive agents are therefore potential prophylactic tools against bacterial infections. The range of anti-adhesive compounds is largely confined to carbohydrate analogues. Tannins are generously recognized as potent antimicrobials, but little data exist on their anti-adherence potency. Using a model for mucosal pathogenesis with labeled group A-streptococci (GAS and human laryngeal HEp-2 cells, a series of flavan-3-ols (epicatechin, epigallocatechin, epigallocatechin-3-O-gallate and highly purified and chemically characterized proanthocyanidin samples including procyanidins based on epicatechin, catechin or ‘mixed’ constituent flavanyl units, prodelphinidins made up of (epigallocatechin monomeric unts as well as oligomers possessing A-type units in their molecules was evaluated for anti-adhesive effects. Reduced microbial adherence was observed exclusively for prodelphinidins, suggesting that pyrogallol-type elements, i.e., (epigallocatechin units are important structural features. This is the first report on structure-activity relationships regarding the anti-adhesive potency of proanthocyanidins. In addition, the structures of the first chemically defined proanthocyanidins from Pelargonium sidoides are disclosed.

  9. The role of cytochrome b5 structural domains in interaction with cytochromes P450.

    Science.gov (United States)

    Sergeev, G V; Gilep, A A; Usanov, S A

    2014-05-01

    To understand the role of the structural elements of cytochrome b5 in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b5 - microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b5 is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b5 to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b5 are mainly determined by the structure of the heme-binding domain.

  10. Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

    Science.gov (United States)

    Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

    2016-02-01

    Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams). Copyright © 2015 Elsevier Inc. All rights reserved.

  11. In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.

    Science.gov (United States)

    Yonetani, Saori; Moriyama, Mariko; Nishigori, Chikako; Osawa, Masatake; Nishikawa, Shin-ichi

    2008-02-01

    Elucidation of the molecular mechanisms underlying stem cell regulation is of great importance both for basic biology and for clinical applications. Melanocyte stem cells (MSCs) are an excellent model in which to study the molecular basis of stem cell regulation, as the genetic alterations involved in the maintenance of the stem cells are readily identifiable by a premature hair graying phenotype. Research on MSCs has been hampered by the lack of a reliable system to assay their function. Here, by co-culturing highly purified melanoblasts (MBs) with XB2 keratinocytes, we describe an efficient culture method that allows the expansion of immature MBs in vitro. These MBs are also capable of undergoing terminal differentiation into mature melanocytes (MCs) when differentiation is induced. Furthermore, by performing a hair-follicle reconstitution assay in which expanded MBs in a mixture of epidermal and dermal cells were grafted to reconstitute a hair follicle, we demonstrate that the expanded MBs retain their capacity to become incorporated into newly developed hair follicles and repopulate the MC stem cell population there. Thus, by integrating genetic manipulations in cultured MBs in vitro, this method provides a powerful tool with which to study the molecular basis of stem cell regulation.

  12. Formation of oriented membrane multilayers of Na/K-ATPase

    International Nuclear Information System (INIS)

    Pachence, J.M.; Knott, R.; Edelman, I.S.; Schoenborn, B.P.; Wallace, B.A.

    1982-01-01

    The isolated membrane-bound enzyme retains its ouabain-sensitive ATP hydrolysis activity, and produces ATP-dependent Na + and K + fluxes when incorporated into phospholipid vesicles. The ultimate goal of this work is to determine its low resolution structure using both X-ray and neutron diffraction. A number of methods were used to impart lamellar stacking order to highly purified pig Na/K-ATPase membranes. Upon partial dehydration, x-ray diffraction from Na/K-ATPase membrane multilayers at 98% relative humidity yielded discrete reflections of 118 A periodicity, diffracting to 1/14.8 A -1 , additionally, continuous diffraction to 1/10 A -1 was obtained. Subjecting the membrane multilayers to high magnetic fields improved the quality of the lamellar diffraction dramatically. Neutron diffraction studies of the partially dehydrated Na/K-ATPase membrane multilayers detected a mosaic spread of 2 0 when the samples were subjected to a magnetic field of 5 Tesla perpendicular to the membrane surface; the reflections were narrower than the camera line width; hence, the lattice disorder has also decreased significantly, although only four orders were measured

  13. Application of Enzymatic Extracts from a CALB Standard Strain as Biocatalyst within the Context of Conventional Biodiesel Production Optimization

    Directory of Open Access Journals (Sweden)

    Carlos Luna

    2017-11-01

    Full Text Available The application of biocatalysts in the transesterification process of triglycerides (TG allows integrating the glycerol in the form of monoglyceride (MG, sharply increasing the yield and the environmental sustainability of the conventional biodiesel production process. This is known as Ecodiesel. To overcome the inconvenient of the high cost of the currently employed highly purified commercial enzymes, the use of scarcely purified extracts obtained from standard strains of the same species of commercial lipases currently applied in this process is being investigated. Thus, Candida antarctica type B (CALB was chosen to determine the optimal conditions of culture of this yeast. The standard strain was obtained from the Spanish Type Microbial Cultures Collection (CECT and has been used to carry out several studies to elucidate its optimum growth conditions. Through a process of lyophilization with prior dialysis of the liquid cultures, the enzymatic extracts were obtained. The different obtained cultures have been applied as biocatalysts in the 1,3-selective transesterification reaction of sunflower oil with ethanol to obtain Ecodiesel (FAEE + MG. Selectivity and reaction yields were obtained by gas chromatography. Acceptable yields are obtained during the reaction time as well as in successive reactions, demonstrating the feasibility of using these CALB enzymatic extracts as biocatalysts.

  14. G-protein based ELISA as a potency test for rabies vaccines.

    Science.gov (United States)

    Chabaud-Riou, Martine; Moreno, Nadège; Guinchard, Fabien; Nicolai, Marie Claire; Niogret-Siohan, Elisabeth; Sève, Nicolas; Manin, Catherine; Guinet-Morlot, Françoise; Riou, Patrice

    2017-03-01

    The NIH test is currently used to assess the potency of rabies vaccine, a key criterion for vaccine release. This test is based on mice immunization followed by intracerebral viral challenge. As part of global efforts to reduce animal experimentation and in the framework of the development of Sanofi Pasteur next generation, highly-purified vaccine, produced without any material of human or animal origin, we developed an ELISA as an alternative to the NIH test. This ELISA is based on monoclonal antibodies recognizing specifically the native form of the viral G-protein, the major antigen that induces neutralizing antibody response to rabies virus. We show here that our ELISA is able to distinguish between potent and different types of sub-potent vaccine lots. Satisfactory agreement was observed between the ELISA and the NIH test in the determination of the vaccine titer and their capacity to discern conform from non-conform batches. Our ELISA meets the criteria for a stability-indicating assay and has been successfully used to develop the new generation of rabies vaccine candidates. After an EPAA international pre-collaborative study, this ELISA was selected as the assay of choice for the EDQM collaborative study aimed at replacing the rabies vaccine NIH in vivo potency test. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas

    International Nuclear Information System (INIS)

    Nigam, S.K.; Towers, T.

    1990-01-01

    Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with Stains-All. Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER

  16. Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

    Science.gov (United States)

    Morre, D. M.; Morre, D. J.

    2000-01-01

    Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum Salt concentrations and temperature affect partitioning behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for preparation of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodic NADH oxidase, a new enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions for measurement of activity.

  17. FY1995 study to create the high density magnetic recording devices by using an ultra clean sputtering process; 1995 nendo choseijo sputter process ni yoru chokomitsudo jiki kiroku device no sosei

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-03-01

    It is important to control microstructure of thin film magnetic devices such as recording heads and media, in order to induce excellent magnetic properties. Since the impurities in the sputtering atmosphere is easily thought to affect strongly on the initial film growth, we will develop the highly purified sputtering atmosphere to establish a fabrication technology of ultra thin metallic films with desirable microstructure. A specialized multi-sputtering system which has extremely clean atmosphere (impurity level: 1/10000 compared to conventional systems) were realized by (a) decreasing out-gassing rate from vacuum chamber, pumping system, cathode, robot, etc. and (b) using ultra-clean processing gas. The base pressure was 8 x 10{sup -12} Torr (XHV) and the build-up rate was less than 1 x 10{sup -8} Torrl/sec. From the correlation between the microstructure and magnetic properties of a part of spin-valve GMR films, the guiding principle for the microstructural design were clarified to induce the exchange coupling effectively at the ferro/antiferromagnetic interface and to enhance the GMR effect at the magnetic/non-magnetic interface. The mechanism of' Cr segregation on the grain boundaries was clarified, in thin film media deposited under ultra clean sputtering process. The material specification of the magnetic ultra thin film media for high density recording with low media noise were designed from view of the thermal agitation. (NEDO)

  18. Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. Characterization of a soluble suppressor of B cell immunoglobulin production

    International Nuclear Information System (INIS)

    Fleisher, T.A.; Greene, W.C.; Blaese, R.M.; Waldmann, T.A.

    1981-01-01

    Human peripheral blood mononuclear cells (PBMC) activated with the mitogenic lectin concanavalin A (Con A) elaborate a soluble immune suppressor supernatant (SISS) that contains at least 2 distinct suppressor factors. One of these, SISS-B, inhibits polyclonal B cell immunoglobulin production, whereas the other, SISS-T, suppresses T cell proliferation to both mitogens and antigens. The latter mediator is discussed in the companion paper. Characteristics of the human soluble suppressor of B cell immunoglobulin production (SISS-B) include: 1) inhibition by a noncytotoxic mechanism, 2) loss of activity in the presence of the monosaccharide L-rhamnose, 3) appearance within 8 to 16 hr after the addition of Con A, 4) elaboration by cells irradiated with 500 or 2000 rads, 5) production by highly purified T cells, 6) stability at pH 2.5 but instability at 56/sup o/C, and 7) m.w. of 60 to 80,000. These data indicate that after Con A activation, selected T cells not only become potent suppressor cells, but also generate a soluble saccharide-specific factor(s) that inhibits polyclonal immunoglobulin production by human B cells

  19. Recombinant virus like particles as drug delivery system.

    Science.gov (United States)

    Georgens, Christiane; Weyermann, Jörg; Zimmer, Andreas

    2005-02-01

    The drug delivery system described here is based on a virus like particle consisting of the recombinant expressed major capsid protein of Polyomavirus, VP1. Polyoma, a murine virus belonging to the Papovaviridae, forms a non-enveloped icosahedral capsid. These capsids are organized as a double shell composed of three different proteins: VP1,VP2 and VP3. The outer shell of the vision is composed of 360 VP1 molecules arranged as 72 pentamers. These capsids have a diameter of about 50 nm. The VP1 protein acts as a major ligand for certain membrane receptors during virus infection. Furthermore, the N-terminus of the VP1 protein contains a DNA-binding domain and a nuclear localization sequence. The recombinant production of the VP1 protein offers a save way to obtain a highly purified, non pathogenic pharmaceutical excipient. Combining these aspects, VP1 proteins provide a targeting as well as a drug binding site when used as a save drug carrier for gene therapy. Current applications are also including oligonucleotides as well as small molecules as well as vaccines.

  20. Captopril and lisinopril only inhibit matrix metalloproteinase-2 (MMP-2) activity at millimolar concentrations.

    Science.gov (United States)

    Kuntze, Luciana B; Antonio, Raquel C; Izidoro-Toledo, Tatiane C; Meschiari, Cesar A; Tanus-Santos, Jose E; Gerlach, Raquel F

    2014-03-01

    Matrix metalloproteinase-2 (MMP-2) shares structural similarities with the angiotensin-converting enzyme (ACE). ACE inhibitors have been described to inhibit MMP-2, but this inhibitory potential was not shown using a highly purified MMP-2. This study aimed to investigate the inhibitory potential of captopril and lisinopril regarding MMP-2 activity. The first objective was to test the potential of captopril to change the pH of the buffer solution. The second objective was to test the direct inhibitory effect of captopril and lisinopril on plasma MMP-2 and on recombinant human MMP-2 (rhMMP-2). The in vitro activity assays included gelatin zymography and a fluorimetric assay. Captopril solubilization significantly decreased the pH of the 50 mM Tris buffer solution at the following concentrations: 2 mM (p captopril concentrations ≥ 4 and 1 mM, respectively (p captopril led to significant inhibition of the rhMMP-2 activity at concentrations ≥2 mM (p captopril (p captopril and lisinopril concentrations found to inhibit MMP-2 are 3 orders of magnitude higher than those present in vivo after drug administration. We also discuss possible pitfalls for gelatinase inhibitory assays (besides the obvious pH problem already cited). In conclusion, this study's data show that captopril and lisinopril did not inhibit MMP-2 directly at the concentrations reached in vivo.

  1. Diffusion in solids with holographic interferometry

    Science.gov (United States)

    Liu, Dingyu

    1996-12-01

    It is of great importance for the formation of p-n junction in semiconductors by penetrating some impurities through the depth near the surface, so it has long been paid attention to control the concentration distribution of impurities during the diffusion process. In recent years, ionic carburizing, and ion bombardment penetration etc. for the treatment of metal surface have also attracted by material sciences. It requires that the diffusion depth and the diffusion time of the impurities should be under precise control. Different methods, such as the method of radioisotopic detection and the method of chemical analysis have been adopted, however, the reports of different workers are very different, especially in the real time measurement, so, finding new method is never ending. In 1984, H. Fenichel have performed experiments on the solutions of table salt and sugar with the method of holographic interferometry. As for metals which are opaque for the visible light, but they become transparent by making them into a very thin film so that, in principle, the diffusion of atoms within a film is capable of measure by holographic interferometry. Alternatively, the electromagnetic waves within 1 - 70 micrometers wavelengths may be utilized, some materials, such as high purified germanium and silicon are good materials for infrared transmission. Some fluorides of alkaline-earth metals have high transmittance in the range of 1 - 8 micrometers , the concentration of impurities in the semiconductor and metal surface treatment are of 1015 - 1020 atoms per cubic cm, which is capable of detection.

  2. Elimination of contaminant Escherichia coli chromosomal DNA from preparations of P1 artificial chromosome recombinants facilitates directed subcloning.

    Science.gov (United States)

    Davidson, H; Doherty, A; Stevenson, B; Boyd, A C; Porteous, D J

    1999-06-01

    The subcloning of large inserts (>50 kbp) from P1-derived artificial chromosomes (PACs) was found to be hindered by the presence of contaminating Escherichia coli chromosomal fragments which, because of their smaller median size, are recovered preferentially as unwanted subclones. A significant fraction of contaminating DNA was seen to persist after conventional plasmid purification methods. We describe a rigorous protocol for eliminating the bulk of contamination that involves plasmid isolation on commercially available silica-based columns followed by three pulsed field gel electrophoresis steps. Using this, we were able to subclone 55, 85 and 90 kbp PAC inserts but failed to subclone a 195 kbp PAC insert. After surveying a range of DNA purification methods, we devised an optimised protocol that allowed us to subclone the 195 kbp insert. The optimised protocol, which reliably yields DNA with essentially no contaminating material, consists of plasmid isolation on silica-based columns followed by treatment with highly purified DNaseI and retrieval by electroelution of restriction-digested DNA electrophoresed on a single pulsed field gel. By inference it is applicable to the purification of large inserts from other single-copy plasmid vectors such as bacterial artificial chromosomes (BACs).

  3. Acceptable Knowledge Summary Report for Waste Stream: SR-T001-221F-HET/Drums

    Energy Technology Data Exchange (ETDEWEB)

    Lunsford, G.F.

    1999-08-23

    Since beginning operations in 1954, the Department of Energy's Savannah River Site FB-Line conducted atomic energy defense activities consistent with the listing in Section 10101(3) of the Nuclear Waste Policy Act of 1982. The facility mission was to process and convert dilute plutonium solution into highly purified weapons grade plutonium metal. As a result of various activities conducted in support of the mission (e.g., operation, maintenance, repair, clean up, and facility modifications), the facility generated transuranic waste. This document, along with referenced supporting documents, provides a defensible and auditable record of acceptable knowledge for one of the waste streams from the FB-Line. The waste was packaged in 55-gallon drums, then shipped to the transuranic waste storage facility in ''E'' area of the Savannah River Site. This acceptable knowledge report includes information relating to the facility's history, configuration,equipment, process operations, and waste management practices.

  4. In vitro study of histamine and histamine receptor ligands influence on the adhesion of purified human eosinophils to endothelium.

    Science.gov (United States)

    Grosicki, Marek; Wójcik, Tomasz; Chlopicki, Stefan; Kieć-Kononowicz, Katarzyna

    2016-04-15

    It is a well-known fact that histamine is involved in eosinophil-dependent inflammatory responses including cellular chemotaxis and migration. Nevertheless, the relative role of histamine receptors in the mechanisms of eosinophils adhesion to endothelial cells is not known. Therefore the aim of presented study was to examine the effect of selective histamine receptors ligands on eosinophils adhesion to endothelium. For that purpose the highly purified human eosinophils have been isolated from the peripheral blood. The viability and functional integrity of isolated eosinophils have been validated in several tests. Histamine as well as 4-methylhistamine (selective H4 agonist) in concentration-dependent manner significantly increased number of eosinophils that adhere to endothelium. Among the selective histamine receptors antagonist or H1 inverse agonist only JNJ7777120 (histamine H4 antagonist) and thioperamide (dual histamine H3/H4 antagonist) had direct effect on eosinophils adhesion to endothelial cells. Antagonists of H1 (diphenhydramine, mepyramine) H2 (ranitidine and famotidine) and H3 (pitolisant) histamine receptors were ineffective. To the best of our knowledge, this is the first study to demonstrate that histamine receptor H4 plays a dominant role in histamine-induced eosinophils adhesion to endothelium. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. The characterization of exosome from blood plasma of patients with colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yunusova, N. V., E-mail: Bochkarevanv@oncology.tomsk.ru [Tomsk Cancer Research Institute, Kooperativny Street 5, Tomsk, 634009 (Russian Federation); Siberian State Medical University, Moskovsky Trakt 2, Tomsk, 634050 (Russian Federation); Tamkovich, S. N., E-mail: s.tamk@niboch.nsc.ru [Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Avenue 8, Novosibirsk, 630090 (Russian Federation); Novosibirsk State University, Pirogov Street 2, Novosibirsk, 630090 (Russian Federation); Stakheeva, M. N., E-mail: StakheyevaM@oncology.tomsk.ru; Afanas’ev, S. G., E-mail: Afanasievsg@oncology.tomsk.ru; Kondakova, I. V., E-mail: Kondakova@oncology.tomsk.ru [Tomsk Cancer Research Institute, Kooperativny Street 5, Tomsk, 634009 (Russian Federation); Frolova, A. Y., E-mail: Frolovalenya@mail.ru [Siberian State Medical University, Moskovsky Trakt 2, Tomsk, 634050 (Russian Federation)

    2016-08-02

    Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of “International Society for Extracellular Vesicles”. The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. The results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.

  6. Canada's nuclear fuel industry: An overview. Background paper

    International Nuclear Information System (INIS)

    Nixon, A.

    1993-11-01

    Canada was among the first countries to mine and process uranium-bearing ores. Such ores contain trace amounts of radium, which was in great demand for medical treatment and for use by research laboratories in the early part of the century. For the last half century, the same basic processes have been used to extract uranium from its ores and convert it to a form suitable for use in nuclear reactors. The process described here is that currently in use in Canada. Mining can take a variety of forms, from open-pit to deep, hard-rock. Mining is typically the most costly step in the process, particularly for lower-grade ores. The ore is crushed and ground in the mill to the consistency of fine sand from which the uranium is extracted chemically to produce the impure concentrate known as yellowcake. In the next step, the impure uranium concentrate is chemically refined into highly purified, nuclear-grade, uranium trioxide (UO 3 ). Uranium trioxide is then converted, in two separate chemical processes, into uranium dioxide (UO 2 ) which is destined for domestic consumption and uranium hexafluoride (UF 6 ) which is exported. In Canada, fabrication is the final step of the fuel production process. Uranium dioxide powder is compressed and sintered into very dense ceramic pellets which are then sealed in zirconium tubes and assembled into fuel bundles for Candu reactors. This background paper will review the Canadian nuclear fuels industry. 1 fig

  7. [Milk and dairy products for human nutrition: contribution of technology].

    Science.gov (United States)

    Maubois, Jean-Louis

    2008-04-01

    The complex composition of milk has led to the development of innovative technological processes such as membrane separation. The dairy industry is now able to offer consumers safe classical products (liquid milk, raw-milk cheeses) with little or no heat treatment. Indeed, heat treatment undermines the organoleptic qualities and bioactivity of many molecules found in milk. New technologies, and especially membrane microfiltration, have allowed researchers to identify two groups of milk proteins in terms of their human absorption kinetics: slow micellar casein and fast whey proteins. The highly purified products thus obtained are used for infant foods and slimming aids, and as functional ingredients. The same technologies have been applied to colostrum, yielding a sterile "serocolostrum" containing biologically active immunoglobulins, growth factors, and polypeptides. Combined with other separation techniques, membrane technologies should soon allow the separation and purification of minor milk proteins described as having essential roles in bone calcium uptake and vitamin transport, for example. The use of enzymatic membrane reactors has led to the identification of several bioactive peptides, such as--kappa-caseinomacropeptide, which induces CCK (cholecystokinin) secretion and thus regulates food intake and lipid assimilation,--alpha(S1) CN (91-100), a compound with benzodiazepine activity,-- kappaCN (106-116), which has anti-thrombotic activity by inhibiting blood platelet binding to fibrinogen, and--alpha(S) and beta casein phosphopeptides, which are thought to increase iron and calcium absorption.

  8. Frog oocytes to unveil the structure and supramolecular organization of human transport proteins.

    Directory of Open Access Journals (Sweden)

    Marc J Bergeron

    Full Text Available Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM and single particle analysis (SPA of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4 and the aquaporin-1 (AQP1 water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12 family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

  9. The importance of the time interval between insulin injection and breakfast in determining postprandial glycaemic control--a comparison between human and porcine insulin.

    Science.gov (United States)

    Patrick, A W; Collier, A; Matthews, D M; Macintyre, C C; Clarke, B F

    1988-01-01

    This study assessed the effect of the time interval between insulin injection and breakfast in determining subsequent postprandial glycaemic control and also whether this differed between highly purified porcine insulin and human insulin (crb) in six diabetic patients (age range 24-36 years, duration of diabetes greater than 10 years) usually treated with twice daily Actrapid MC and Monotard MC and with stable insulin requirements and diabetic control. On separate mornings each patient was given, after an overnight fast, their usual dose of either Actrapid MC and Monotard MC or Humulin S and Humulin Zn injected 5, 20, or 40 min before a standard breakfast. The postprandial glycaemic profile was not significantly different at any of the three time intervals with Actrapid MC and Monotard MC. However, with the human insulin the profile was significantly better at the 40 min interval than at the 5 min interval (p less than 0.05) and this was also better than any of the profiles with the porcine insulin, there being a significant difference between the two types of insulin (p less than 0.05). These findings suggest that the time interval between insulin injection and breakfast may be more important with human insulin than with porcine insulin.

  10. Effect of nephrotoxicants on renal membrane transport: In vitro studies

    International Nuclear Information System (INIS)

    Ansari, R.A.; Berndt, W.O.

    1990-01-01

    It is possible to study the effects of nephrotoxicants on membrane function free of other cellular influences. By the use of Percoll gradient centrifugation, highly purified preparations of right-side-out basolateral (BL) and brush border (BB) membrane vesicles can be obtained from rat (male, Sprague-Dawley) renal cortex. Membrane function can be monitored by evaluation of sodium driven transport: 14 C-p-aminohippurate (PAH) for BL and 14 C-glucose for BB. Transport was measured by the rapid filtration technique. Each vesicle preparation was preincubated with the nephrotoxicant for five minutes before initiation of transport. Control vesicles showed a prominant overshoot 1 to 2 minutes after start of transport. Mercuric ion (Hg) had no effect on transport by BB at concentrations as high as 10μM. Transport by BL was reduced significantly at Hg concentrations as low as 100 nM. Chromate (Cr) also reduced BL transport at 100 nM and had no effect on BB transport. Citrinin significantly reduced both BB and BL transport, but the sensitivity of the membrane preparations differed. These data are consistent with the hypothesis that some nephrotoxicants may act on either side of the renal tubular cell membrane

  11. Purification of recombinant Aβ(1-42) and pGlu-Aβ(3-42) using preparative SDS-PAGE.

    Science.gov (United States)

    Spahn, Claudia; Wermann, Michael; Eichentopf, Rico; Hause, Gerd; Schlenzig, Dagmar; Schilling, Stephan

    2017-08-01

    Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aβ, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was  >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aβ peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Heterogeneity Determination and Purification of Commercial Hen Egg-White Lysozyme

    Science.gov (United States)

    Thomas, B. R.; Vekilov, P. G.; Rosenberger, F.

    1998-01-01

    Hen egg-white lysozyme (HEWL) is widely used as a model protein, although its purity has not been adequately characterized by modern biochemical techniques. We have identified and quantified the protein heterogeneities in three commercial HEWL preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis with enhanced silver staining, reversed-phase fast protein liquid chromatography (FPLC) and immunoblotting with comparison to authentic protein standards. Depending on the source, the contaminating proteins totalled 1-6%(w/w) and consisted of ovotransferrin, ovalbumin, HEWL dimers, and polypeptides with approximate M(sub r) of 39 and 18 kDa. Furthermore, we have obtained gram quantities of electrophoretically homogeneous [> 99.9%(w/w)] HEWL by single-step semi-preparative scale cation-exchange FPLC with a yield of about 50%. Parallel studies of crystal growth kinetics, salt repartitioning and crystal perfection with this highly purified material showed fourfold increases in the growth-step velocities and significant enhancement in the structural homogeneity of HEWL crystals.

  13. Kinetics of agonist-induced intrinsic fluorescence changes in the Torpedo acetylcholine receptor.

    Science.gov (United States)

    Kawai, Hideki; Raftery, Michael A

    2010-05-01

    The nicotinic acetylcholine receptor from Torpedo electric organs is a ligand-gated ion channel that undergoes conformational transitions for activation and/or desensitization. Earlier work suggested that intrinsic fluorescence changes of the receptor monitors kinetic transitions toward the high-affinity, desensitized state. Here, using highly purified membrane preparations to minimize contaminating fluorescence, we examined kinetic mechanisms of the receptor as monitored by its intrinsic fluorescence. Fluorescence changes were specific to the receptor as they were blocked by alpha-bungarotoxin and were induced by agonists, but not by the antagonist hexamethonium. Acetylcholine, carbamylcholine and suberyldicholine showed only one kinetic phase with relatively fast rates (t(1/2) = 0.2-1.2 s). Effective dissociation constants were at least an order of magnitude higher than the high affinity, equilibrium binding constants for these agonists. A semirigid agonist isoarecolone-methiodide, whose activation constant was approximately 3-fold lower than acetylcholine, induced an additional slow phase (t(1/2) = 4.5-9 s) with apparent rates that increased and then decreased in a concentration dependent manner, revealing a branched mechanism for conformational transitions. We propose that the intrinsic fluorescence changes of the receptor describe a process(es) toward a fast desensitization state prior to the formation of the high affinity state.

  14. Dissecting the interactions of SERRATE with RNA and DICER-LIKE 1 in Arabidopsis microRNA precursor processing

    KAUST Repository

    Iwata, Yuji

    2013-08-05

    Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing. 2013 The Author(s) 2013.

  15. Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Peter Rohloff

    Full Text Available Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+/H(+ countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

  16. A specific and sensitive radioimmunoassay for human choriogonadotropin

    International Nuclear Information System (INIS)

    Muralidhar, K.; Chaudhuri, G.; Lippes, J.; Bahl, O.P.

    1983-01-01

    A specific and sensitive radioimmunoassay for human choriogonadotropin (hCG) has been developed using rabbit antiserum to chemical analogs of beta subunit of human chorionic gonadotropin prepared by controlled reduction and S-alkylation of its disulfied linkages. The assay was highly specific for hCG as the binding of [ 125 1]-hCG to the antibody was not affected by standard human lutropin, by human male serum, postpartum serum from women, serum from post-menopausal women and human menopausal gonadotropin (Pergonal). The assay was highly sensitive, the minimal detection limit in terms of highly purified hCG (L-129, 12.500 IU/mg) being 1 ng/ml or 0.2 ng/tube (or 12 mlU/ml in terms of WHO 2nd international reference preparation of hCG). Using this assay we were unable to detect any immunoreactive hCG in human tissues like lung, liver and colon. The high specificity, sensitivity, accuracy and reproducibility of the assay make this a highly desirable radioimmunoassay for human choriogonadotropin. (orig.)

  17. Mesenchymal Inflammation Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Disease Evolution in Human Pre-leukemia.

    Science.gov (United States)

    Zambetti, Noemi A; Ping, Zhen; Chen, Si; Kenswil, Keane J G; Mylona, Maria A; Sanders, Mathijs A; Hoogenboezem, Remco M; Bindels, Eric M J; Adisty, Maria N; Van Strien, Paulina M H; van der Leije, Cindy S; Westers, Theresia M; Cremers, Eline M P; Milanese, Chiara; Mastroberardino, Pier G; van Leeuwen, Johannes P T M; van der Eerden, Bram C J; Touw, Ivo P; Kuijpers, Taco W; Kanaar, Roland; van de Loosdrecht, Arjan A; Vogl, Thomas; Raaijmakers, Marc H G P

    2016-11-03

    Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Tandem SUMO fusion vectors for improving soluble protein expression and purification.

    Science.gov (United States)

    Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

    2015-12-01

    Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Sulodexide: implicazioni cliniche ed economiche

    Directory of Open Access Journals (Sweden)

    Orietta Zaniolo

    2005-06-01

    Full Text Available Sulodexide is a highly purified glycosaminoglycan approved for leg ulcers treatment. It contains two principal components: heparan sulfate, a fast-moving heparin fraction, (80% and dermatan sulfate (20%. Sulodexide is available as an oral agent and as an injectable preparation. Its pharmacological action is obtained by dose-dependent coagulation factors inhibition: dermatan sulfate upgrades the physiological action of a selective thrombin inhibitor, heparin cofactor II, and heparan sulfate depresses activated factor X, via an increase of antithrombin III action. The antithrombotic action is enhanced by platelet aggregation inhibition and by the activation of the fibrinolytic system. This paper summarizes the results of some of the main trials that evaluated sulodexide in the treatment of peripheral occlusive arterial disease and venous leg ulcers; a trial on prevention of recurrent deep venous thrombosis with sulodexide is also reviewed. We analyzed data about the clinical and economical impact of chronic venous insufficiency with a particular attention to the cost of medication, hospitalization and management of leg ulcers. The hypothetical savings correlated to the reduction of leg ulcers incidence and healing time attainable with sulodexide have been estimated. A comparison between the different acquisition costs of the drugs frequently used to treat leg ulcers is also provided. Finally we reviewed some quality of life trials in which the psychological and sociological influence of the disease and its treatments on the patient are assessed.

  20. Near-ultraviolet mutation of transforming DNA irradiated in vivo

    International Nuclear Information System (INIS)

    Cabrera-Juarez, E.; Setlow, J.K.

    1981-01-01

    Our previous work has demonstrated that whereas near-UV radiation is not a mutagen for Haemophilus influenzae cells, it does induce mutations in purified transforming DNA. In order to test various hypotheses concerning this difference. We have irradiated cells at 334 and 365 nm, then lysed them and assayed the DNA for induced mutations and for inactivation of transforming ability. The inactivation was only a little lower than observed with highly purified transforming DNA. The DNA irradiated in vivo was mutated at both wavelengths, but with considerably lower efficiency than was purified DNA. Neither incubation of the cells after irradiation and before lysis nor freezing and thawing the cells significantly changed the amount of mutation. It is concluded that there is some protection of the DNA against premutational lesions by the in vivo environment, but that it is not enough to account for the total lack of mutation of the cells. A probable explanation of this lack of cell mutation is that lethal lesions in the cells are induced much more readily than premutational lesions. (orig.)

  1. Prescription n-3 fatty acids, but not eicosapentaenoic acid alone, improve reference memory-related learning ability by increasing brain-derived neurotrophic factor levels in SHR.Cg-Lepr(cp)/NDmcr rats, a metabolic syndrome model.

    Science.gov (United States)

    Hashimoto, Michio; Inoue, Takayuki; Katakura, Masanori; Tanabe, Yoko; Hossain, Shahdat; Tsuchikura, Satoru; Shido, Osamu

    2013-10-01

    Metabolic syndrome is implicated in the decline of cognitive ability. We investigated whether the prescription n-3 fatty acid administration improves cognitive learning ability in SHR.Cg-Lepr(cp)/NDmcr (SHR-cp) rats, a metabolic syndrome model, in comparison with administration of eicosapentaenoic acid (EPA, C20:5, n-3) alone. Administration of TAK-085 [highly purified and concentrated n-3 fatty acid formulation containing EPA ethyl ester and docosahexaenoic acid (DHA, C22:6, n-3) ethyl ester] at 300 mg/kg body weight per day for 13 weeks reduced the number of reference memory-related errors in SHR-cp rats, but EPA alone had no effect, suggesting that long-term TAK-085 administration improves cognitive learning ability in a rat model of metabolic syndrome. However, the working memory-related errors were not affected in either of the rat groups. TAK-085 and EPA administration increased plasma EPA and DHA levels of SHR-cp rats, associating with an increase in EPA and DHA in the cerebral cortex. The TAK-085 administration decreased the lipid peroxide levels and reactive oxygen species in the cerebral cortex and hippocampus of SHR-cp rats, suggesting that TAK-085 increases antioxidative defenses. Its administration also increased the brain-derived neurotrophic factor levels in the cortical and hippocampal tissues of TAK-085-administered rats. The present study suggests that long-term TAK-085 administration is a possible therapeutic strategy for protecting against metabolic syndrome-induced learning decline.

  2. Impact of short-chain galactooligosaccharides on the gut microbiome of lactose-intolerant individuals.

    Science.gov (United States)

    Azcarate-Peril, M Andrea; Ritter, Andrew J; Savaiano, Dennis; Monteagudo-Mera, Andrea; Anderson, Carlton; Magness, Scott T; Klaenhammer, Todd R

    2017-01-17

    Directed modulation of the colonic bacteria to metabolize lactose effectively is a potentially useful approach to improve lactose digestion and tolerance. A randomized, double-blind, multisite placebo-controlled trial conducted in human subjects demonstrated that administration of a highly purified (>95%) short-chain galactooligosaccharide (GOS), designated "RP-G28," significantly improved clinical outcomes for lactose digestion and tolerance. In these individuals, stool samples were collected pretreatment (day 0), after GOS treatment (day 36), and 30 d after GOS feeding stopped and consumption of dairy products was encouraged (day 66). In this study, changes in the fecal microbiome were investigated using 16S rRNA amplicon pyrosequencing and high-throughput quantitative PCR. At day 36, bifidobacterial populations were increased in 27 of 30 of GOS subjects (90%), demonstrating a bifidogenic response in vivo. Relative abundance of lactose-fermenting Bifidobacterium, Faecalibacterium, and Lactobacillus were significantly increased in response to GOS. When dairy was introduced into the diet, lactose-fermenting Roseburia species increased from day 36 to day 66. The results indicated a definitive change in the fecal microbiome of lactose-intolerant individuals, increasing the abundance of lactose-metabolizing bacteria that were responsive to dietary adaptation to GOS. This change correlated with clinical outcomes of improved lactose tolerance.

  3. Smart polymeric materials in forms of fiber and film

    International Nuclear Information System (INIS)

    Sugo, Takanobu

    1998-01-01

    Chemical grafting: graft polymerization is a powerful technology to append novel functionality to base fibers, clothes, felts, films and others, while maintaining their original properties. As shown in Figure 1, while a gardener may use a pair of shears to cut the branch, to cut the molecular branch of a polymeric material, one can utilize the radiation energy. Effective utilization of the radiation energy can proceed to a novel reaction that is impossible for other conventional methods and develop a new material bearing outstanding functions. This technology is named radiation-induced graft polymerization (RIGP). In this article, the present research and development of novel functional polymeric materials by radiation-induced graft polymerization is described. The felt of intertwined fibers has been widely used as a filter to remove particles from air but not toxic gaseous compounds. However, by RIGP, one can transform the felt into a high functional filter that will absorb the toxic gaseous compounds while removing particles simultaneously. As a result, the RIGP technology, which is impossible by conventional technology, has enabled the development of a novel functional material that produce highly pure air. Commercialization of this filter for applications in a semiconductor manufacturing facility and as an air purifier is under process. Moreover, this filter can also be used to produce highly purified water by removing toxic heavy metals. Commercially available polyethylene films are also been transform into conductive separators by RIGP to increase the lifetime of a battery by more than five-fold. (J.P.N)

  4. Acute recurrent diverticulitis is prevented by oral administration of a polybacterial lysate suspension.

    Science.gov (United States)

    Dughera, L; Serra, A M; Battaglia, E; Tibaudi, D; Navino, M; Emanuelli, G

    2004-06-01

    The main cause of acute diverticulitis is the abnormal accumulation of fecal bacteria within the diverticular lumen, leading to a balancing between normal probiotic microflora and pathogenic species; Gram negative Entero-bacteriaceae, mainly Escherichia coli and Proteus spp, are the genders that usually cause the disease-related symptoms, due to their ability to adhere to intestinal mucosa. The intestine is well known as the largest human lymphoepithelial organ and daily produces more antibodies, mainly secretory IgAs, than do all other lymphoid tissues. IgAs have different immune and anti-inflammatory properties. The aim of this study was to verify the efficacy of an oral immunostimulant highly-purified, polymicrobial lysate in the prevention of recurrent attacks of diverticulitis and in the improvement of symptoms. The study was carried out on 83 consecutive patients suffering from recurrent symptomatic acute diverticulitis and with at least 2 attacks in the previous year; patients were randomly assigned to receive (group A) an oral polybacterial lysate suspension or to a no-treatment clinical follow-up as controls (group B). A total of 76 patients (41 in group A and 35 in group B) terminated the study period. the sums of the scores for symptoms, reported on day schedules, were calculated and examined by means of ANOVA statistical analysis. Statistical differences between group A vs group B were recorded after 1 month (precurrent diverticulitis is effective and well tolerated, probably due to a direct stimulation of IgA-mediated mucosal defences.

  5. DNA binding of sunitinib: Spectroscopic evidence via circular dichroism and nuclear magnetic resonance.

    Science.gov (United States)

    Kiss, Eszter; Mirzahosseini, Arash; Hubert, Ágnes; Ambrus, Attila; Őrfi, László; Horváth, Péter

    2018-02-20

    Sunitinib is a non-selective tyrosine kinase inhibitor, but in its chemical structure there can be discovered certain features, which suggest the ability to bind to DNA. These elements are the planar aromatic system and the tertiary amine function, which is protonated at the pH of the organism. In this study, the binding of the drug sunitinib to DNA was investigated using circular dichroism (CD), 1 H NMR and UV spectroscopies, along with CD melting. For these studies DNA was isolated from calf thymus (CT), salmon fish sperm (SS), and chicken erythrocyte (CE), however for our purposes an artificially constructed and highly purified plasmid DNA (pUC18) preparation proved to be the most suitable. DNA binding of the drug was confirmed by shifts in the characteristic CD bands of the DNA, the appearance of an induced CD (ICD) signal in the upper absorption region of sunitinib (300 nm-500 nm), and the evidence from CD melting studies and the NMR. Based on the CD and NMR measurements, it can be assumed that sunitinib has a multiple-step binding mechanism. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Recombinant follitropin alfa/lutropin alfa in fertility treatment

    Directory of Open Access Journals (Sweden)

    Ahmed Gibreel

    2009-12-01

    Full Text Available Ahmed Gibreel1, Siladitya Bhattacharya21School of Medicine and Dentistry, University of Aberdeen; 2Aberdeen Maternity Hospital, Aberdeen, UKAbstract: Recombinant human follicle stimulating hormone (rFSH and luteinizing hormone (LH, also known as follitropin alpha and lutropin alpha, are manufactured by genetic engineering techniques which ensure high quality and batch to batch consistency. Follitropin alpha can be used for controlled ovarian hyperstimulation in assisted reproduction, ovulation induction for WHO group I and II anovulatory infertility and in men with hypogonadotrophic hypogonadism (HH or idiopathic oligo-asthenospermia. Current evidence suggests superiority of urinary human menopausal gonadotropin (HMG over follitropin alpha in controlled ovarian hyperstimulation for IVF in terms of live birth rate per couple. Addition of lutropin to follitropin alpha in an unselected IVF population does not appear to confer any benefit; however, it may have a role in ovulation induction in women with hypothalamic hypogonadism. Urinary HMG preparations (especially currently available highly purified preparations are more cost effective than rFSH in terms of cost per ongoing pregnancy. However, women using rFSH injection pen devices have higher levels of satisfaction as compared to those using urinary HMG by means of conventional syringes.Keywords: infertility, follicle stimulating hormone, luteinizing hormone, follitropin alpha, lutropin alpha, in-vitro fertilization, urinary gonadotrophins

  7. Ammonia recovery from landfill leachate using hydrophobic membrane contactors.

    Science.gov (United States)

    Amaral, Míriam C S; Magalhães, Nátalie C; Moravia, Wagner G; Ferreira, Carolina D

    2016-11-01

    This article aims to evaluate membrane contactors capability to remove and recover ammonia from landfill leachate (LFL). A hydrophobic hollow fiber membrane module was used to achieve such purpose. A sulfuric acid diluted solution was used as extraction solution to speed up ammonia content removal. Several factors that have influence on ammonia removal and recovery capability such as ammonia solution pH, concentration of sulfuric acid solutions and flow rate of liquid phases have been examined. Microfiltration was the method used as pretreatment. The results have shown that membrane contactor operated with LFL (pH 10), 0.1 M acid solution and liquid flow rate up to 0.5 L min -1 achieved 99.9% of ammonia removal, which corresponds to 79.1% of ammonia recovery from the extraction solution, and it is capable to produce highly purified ammonium sulfate solutions (41.2%, wt wt -1 ) to be used as fertilizer. The concentration of total ammonia nitrogen (TAN) in the residual LFL complies with Brazilian law requirements of 20.0 mg L -1 of TAN, regarding the disposal of effluents.

  8. Phytochrome pelletability induced by irradiation in vivo: test for in vitro binding of added [35S]phytochrome

    International Nuclear Information System (INIS)

    Pratt, L.H.

    1980-01-01

    Undegraded, highly purified [ 35 S]phytochrome was immunoaffinity-purified either from dark control oat (cv. Garry) shoots or from etiolated oat shoots that were previously irradiated first with red and then with far-red light so that, if proper extraction conditions had been utilized, about 60% of the total phytochrome would have been pelletable. When [ 35 S]phytochrome was added to extraction buffer immediately prior to homogenization of etiolated oat shoots, pelletability assays indicated that there was no preferential binding of [ 35 S]phytochrome regardless of (a) whether it was purified from dark control or irradiated shoots, (b) whether it was added as phytochrome-red-absorbing form or phytochrome-far-red-absorbing form, or (c) whether it was added to dark control or red-irradiated shoots. Similarly, binding of [ 35 S]phytochrome to resuspended pellets obtained from crude oat extracts was not specific for the source of [ 35 S]phytochrome, for its form, or for the irradiation treatment given to intact shoots used to prepare the resuspended pellets. No evidence was obtained to support the hypothesis that phytochrome binds with specificity to particulate material in vitro under conditions used to assay for light-enhanced, in vivo-induced phytochrome pelletability

  9. Corifollitropin alfa compared to daily rFSH or HP-HMG in GnRH antagonist controlled ovarian stimulation protocol for patients undergoing assisted reproduction.

    Science.gov (United States)

    Souza, Priscila Morais Galvão; Carvalho, Bruno Ramalho de; Nakagawa, Hitomi Miura; Rassi, Thalita Reis Esselin; Barbosa, Antônio César Paes; Silva, Adelino Amaral

    2017-06-01

    This study aimed to compare the outcomes of controlled ovarian stimulation (COS) with corifollitropin alfa versus daily recombinant follicle-stimulating hormone (rRFSH) or highly purified human menopausal gonadotropin (HP-HMG) in patients undergoing in vitro fertilization (IVF) cycles based on gonadotropin-releasing hormone (GnRH) antagonist protocols. The primary endpoints were total number of oocytes and mature oocytes. This retrospective study looked into 132 controlled ovarian stimulation cycles from IVF or oocyte cryopreservation performed in a private human reproduction center between January 1 and December 31, 2014. Enrollment criteria: women aged 0.05). There were no significant differences in fertilization (76.9% vs. 76.8%, p=1.0), biochemical pregnancy (66.7% vs. 47.2%, p=0.1561) or embryo implantation rates (68.7% vs. 50%, p=0.2588) between the groups using corifollitropin alfa and rFSH or HMG, respectively. Corifollitropin alfa seems to be as effective as rFSH or HP-HMG when used in the first seven days of ovulation induction for patients undergoing assisted reproduction in GnRH antagonist protocols.

  10. Production of specific antisera for radioimmunoassay of human luteinizing hormone (LH) in the presence of human chorionic gonadotropin (hCG)

    International Nuclear Information System (INIS)

    Thorell, J.I.; Jeppsson, S.; Holmstrom, B.

    1976-01-01

    A specific radioimmunoassay for LH, which measures plasma LH in the presence of human chorionic gonadotropin (hCG) is described. Rabbits were immunized with highly purified native LH. One of the antisera with a difference in its reactivity against LH and hCG was further purified by affinity chromatography on a column with hCG coupled to Sepharose 4B. The adsorbed antiserum and 125 I-LH was used in a double antibody assay. The LH standard (MRC/68/40) efficiently inhibited the binding of 125 I-LH, and the standard curve showed a sensitivity of 0.5 ng/ml in the sample. hCG up to 10,000 ng/ml did not inhibit the binding of 125 I-LH. The plasma level of LH in pregnant women in the first trimester was low (1.3 +- 0.1 ng/ml). When LH was measured in fertile or menopausal women with or without stimulation with LH/FSH releasing hormone (LH-RH)/sup x/ the results agreed to those found with our conventional LH-assay based on antiserum against hCG

  11. Production and Characterization of HCG Poly clonal Antibodies

    International Nuclear Information System (INIS)

    Moustafa, K.A.

    2009-01-01

    To prepare a radioimmunoassay system for measuring hcg hormone, the anti-hcg polyclonal antibodies must be firstly prepared. The present study was aimed to produce and characterize the anti-hcg polyclonal antibodies. The anti hcg polyclonal Antibes which were produced by immunizing five Balb/c mice with a highly purified β-hcg hormone. A complete Freunds adjuvant was used for the first injection while incomplete Freunds adjuvant was used for the following boosters. Blood spots were obtained from the immunized mice and tested for the presence of antibodies. At the end of the immunization schedule, the mice was exposed to anesthesia using ether then dissected and the blood samples were collected from the heart. The serum (antisera) was separated from the blood containing anti-hcg polyclonal antibodies. The produced antibodies were purified then immobilized onto the surface of magnetic particles to prepare the solid phase anti-hcg magnetic particles. The characterization parameters include accuracy, parallelism, sensitivity, specificity and comparison studies between the prepared system and the commercial used one, were performed. Also, the labeling of hcg hormone with 1251 was performed using chloramine-T method. The obtained results indicated that the produced solid phase of polyclonal anti-hcg for measuring the hcg hormone was good, accurate, sensitive, specific and highly correlated with that of the commercial method

  12. Frictional characteristics of stainless steel 440C lubricated with water at pressurized high temperature

    International Nuclear Information System (INIS)

    Kim, E. H.; Lee, J. S.; Kim, J. H.; Kim, J. I.

    2001-01-01

    The fatigue life of stainless steel bearings is one of the most critical factors to determine the performance of the driving system. Because the bearings which are installed on the driving mechanism in the nuclear reactor are operated at high temperature and high pressure and especially lubricated with water with low viscosity, the friction and wear characteristics of the bearing material should be investigated thoroughly. In many control element drive mechanisms in the nuclear reactor the support bearings are made of the stainless steel and the sliding bearing ceramic material mainly. This study is focused on the characteristics of support bearing which may be used in the SMART. The ball bearings are made of standardized 440C stainless steel, and it supports thrust load including the weight of the driving system and external force. The friction and wear characteristics of this material operating under severe lubrication condition are not well known yet, however it will be changed with respect to temperature and boundary pressure. In this paper the friction characteristics are investigated experimentally using the reciprocating tribometer which can simulate the SMART operating conditions. Highly purified water is used as lubricant, and the water is warmed up and pressurized. Friction forces on the reciprocating specimens are measured insitu strain gages

  13. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor-binding domain of botulinum neurotoxin serotype D

    International Nuclear Information System (INIS)

    Zhang, Yanfeng; Gao, Xiaoli; Qin, Ling; Buchko, Garry W.; Robinson, Howard; Varnum, Susan M.

    2010-01-01

    The receptor-binding domain of botulinum neurotoxin serotype D was expressed in E. coli using a codon-optimized cDNA. The highly purified protein crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 Å, and the crystals diffracted to 1.65 Å resolution. Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D-HCR was expressed at a high level (150–200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D-HCR was obtained. The recombinant BoNT/D-HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 Å. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit

  14. Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings.

    Science.gov (United States)

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet T; Meents, Miranda J; Lao, Jeemeng; González Fernández-Niño, Susana M; Petzold, Christopher J; Frommer, Wolf B; Samuels, A Lacey; Heazlewood, Joshua L

    2016-03-04

    The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

  15. Transparent, Flexible Strain Sensor Based on a Solution-Processed Carbon Nanotube Network.

    Science.gov (United States)

    Lee, Jieun; Lim, Meehyun; Yoon, Jinsu; Kim, Min Seong; Choi, Bongsik; Kim, Dong Myong; Kim, Dae Hwan; Park, Inkyu; Choi, Sung-Jin

    2017-08-09

    The demands for transparent, flexible electronic devices are continuously increasing due to their potential applications to the human body. In particular, skin-like, transparent, flexible strain sensors have been developed to realize multifunctional human-machine interfaces. Here, we report a sandwich-like structured strain sensor with excellent optical transparency based on highly purified, solution-processed, 99% metallic CNT-polydimethylsiloxane (PDMS) composite thin films. Our CNT-PDMS composite strain sensors are mechanically compliant, physically robust, and easily fabricated. The fabricated strain sensors exhibit a high optical transparency of over 92% in the visible range with acceptable sensing performances in terms of sensitivity, hysteresis, linearity, and drift. We also found that the sensitivity and linearity of the strain sensors can be controlled by the number of CNT sprays; hence, our sensor can be applied and controlled based on the need of individual applications. Finally, we investigated the detections of human activities and emotions by mounting our transparent strain sensor on various spots of human skins.

  16. Vaccination of calves with EspA, a key colonisation factor of Escherichia coli O157:H7, induces antigen-specific humoral responses but does not confer protection against intestinal colonisation.

    Science.gov (United States)

    Dziva, Francis; Vlisidou, Isabella; Crepin, Valérie F; Wallis, Timothy S; Frankel, Gad; Stevens, Mark P

    2007-07-20

    Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are frequently associated with direct or indirect contact with ruminant faeces and may result in haemorrhagic colitis and severe renal and neurological sequelae. Broadly cross-protective vaccines for control of EHEC do not yet exist and the molecular mechanisms that influence bacterial persistence in the intestines of ruminants are incompletely understood. We sought to determine the role in colonisation and protective efficacy of EspA, which forms a filamentous extension of the locus of enterocyte effacement-encoded type III secretion system that injects EHEC proteins into enterocytes. A non-polar deletion of espA severely impaired the ability of E. coli O157:H7 to colonise the intestines of calves. Vaccination of calves with highly purified recombinant EspA induced high-titre antigen-specific IgG1 (also reactive to native EspA) and salivary IgA responses, however these responses did not protect calves against intestinal colonisation by E. coli O157:H7 upon experimental infection.

  17. Response surface modeling for hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores

    Science.gov (United States)

    2014-01-01

    Response surface methodology using a face-centered cube design was used to describe and predict spore inactivation of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure of six spore-contaminated materials to hot, humid air. For each strain/material pair, an attempt was made to fit a first or second order model. All three independent predictor variables (temperature, relative humidity, and time) were significant in the models except that time was not significant for B. thuringiensis Al Hakam on nylon. Modeling was unsuccessful for wiring insulation and wet spores because there was complete spore inactivation in the majority of the experimental space. In cases where a predictive equation could be fit, response surface plots with time set to four days were generated. The survival of highly purified Bacillus spores can be predicted for most materials tested when given the settings for temperature, relative humidity, and time. These predictions were cross-checked with spore inactivation measurements. PMID:24949256

  18. Separation of Beating Cardiac Myocytes from Suspensions of Heart Cells

    Science.gov (United States)

    Pretlow, Thomas G.; Glick, Melvin R.; Reddy, William J.

    1972-01-01

    Heart cells were obtained in suspension after incubation with collagenase and hyaluronidase in Saline A. Cardiac myocytes were separated by isopycnic centrifugation in 88.6 to 92.4% purity from other heart cells with different densities, and by velocity or rate-zonal sedimentation, in 92.8 to 97.4% purity from heart cells with different diameters. A previously described computer integration of the differential sedimentation equation was used to determine the centrifugal force, duration of centrifugation and gradient design, which would permit the separation of cardiac myocytes from other heart cells by velocity sedimentation. The myocytes continued to contract rhythmically after being recovered from the density gradients. Velocity sedimentation was superior to isopycnic sedimentation for the separation of cardiac myocytes from heart cell suspensions because it gave the most highly purified myocytes, resulted in recovery of the largest proportion of myocytes in purified fractions from the gradient and required lower centrifugal forces for shorter periods of time. The potential significance of the availability of pure cardiac myocytes is discsused. ImagesFig 2Fig 1 PMID:4336547

  19. Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

    Science.gov (United States)

    Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

    2015-12-19

    The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

  20. Differential in vitro response of microsomal subfractions to auxins

    International Nuclear Information System (INIS)

    Wilkinson, E.E.; Brightman, A.O.; Zhu, X.-Z.; Morre, J.

    1990-01-01

    Soybean microsomes incubated with or without 1 μM 2,4-D loose phosphatidylcholine, phosphatidylinositol and other phospholipids. Vesicles labeled in vivo with [ 14 C]choline prior to isolation and incubation exhibit rapid 2,4-D-induced hydrolysis of phosphatidylcholine to a plateau within 15 min. As much as 50% of the radioactivity of the membrane appeared in the soluble supernatant. However, when separated into plasma membrane (PM)-enriched and PM-depleted fractions, the PM-enriched fraction was unresponsive to auxin. Similarly, highly purified pM fractions from soybean microsomes exhibited no auxin-stimulated breakdown of phosphoinosotides, and no stimulations of phospholipase C or of phospholipase A 2 . Instead, PM exhibited an auxin-stimulated NADH oxidase activity not shown by microsomes depleted of PM. The results suggest that in vitro auxin effects on phospholipid metabolism involve components of the microsomal fraction other than PM such as the endoplasmic reticulum or tonoplast, whereas PM responds by enhanced NADH oxidase activity

  1. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

    Science.gov (United States)

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-01-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix. PMID:23105972

  2. Monolithic Chip for High-throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells.

    Science.gov (United States)

    Fachin, Fabio; Spuhler, Philipp; Martel-Foley, Joseph M; Edd, Jon F; Barber, Thomas A; Walsh, John; Karabacak, Murat; Pai, Vincent; Yu, Melissa; Smith, Kyle; Hwang, Henry; Yang, Jennifer; Shah, Sahil; Yarmush, Ruby; Sequist, Lecia V; Stott, Shannon L; Maheswaran, Shyamala; Haber, Daniel A; Kapur, Ravi; Toner, Mehmet

    2017-09-07

    Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm-50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15-20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.

  3. Gene Expression Commons: an open platform for absolute gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Jun Seita

    Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  4. Towards electrochemical purification of chemically reduced graphene oxide from redox accessible impurities.

    Science.gov (United States)

    Tan, Shu Min; Ambrosi, Adriano; Khezri, Bahareh; Webster, Richard D; Pumera, Martin

    2014-04-21

    The electrochemical properties of graphene are highly sensitive to residual metallic impurities that persist despite various purification efforts. To accurately evaluate the electrochemical performance of graphene, highly purified materials free of metallic impurities are required. In this study, the partial purification of chemically reduced graphene oxides prepared via Hummers (CRGO-HU) and Staudenmaier (CRGO-ST) oxidation methods was performed through cyclic voltammetric (CV) scans executed in nitric acid, followed by CV measurements of cumene hydroperoxide (CHP). The purification of graphene was monitored by the changes in the peak current and potential of CHP which is sensitive to iron impurities. The CRGOs were characterised by inductively coupled plasma-mass spectrometry (ICP-MS), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and CV. The micrographs revealed CRGOs of similar morphologies, but with greater defects in CRGO-HU. The dependencies of CHP peak current and peak potential on the number of purification cycles exhibit greater efficiency of removing iron impurities from CRGO-HU than CRGO-ST. This can be attributed to the oxidative method that is used in CRGO-HU production, which exposes more defect sites for iron impurities to reside in. This facile electrochemical purification of graphenes can be utilised as a routine preparation and cleaning method of graphene before electrochemical measurements for analytes that show exceptional sensitivity towards electrocatalytic metallic impurities in sp(2) nanocarbon materials.

  5. Purification of Yeast Native Reagents for the Analysis of Chromatin Function-II: Multiprotein Complexes and Biochemical Assays.

    Science.gov (United States)

    Lacoste, Nicolas; Bhat, Wajid; Côté, Jacques

    2017-01-01

    Post-translational modifications of histones play essential roles in regulating chromatin structure and function. These are tightly regulated in vivo and there is an intricate cross-talk between different marks as they are recognized by specific reader modules present in a large number of nuclear factors. In order to precisely dissect these processes in vitro native reagents like purified chromatin and histone modifying/remodeling enzymes are required to more accurately reproduce physiological conditions. The vast majority of these enzymes need to be part of stable multiprotein complexes with cofactors enabling them to act on chromatin substrates and/or read specific histone marks. In the accompanying chapter, we have described the protocol for purification of native chromatin from yeast cells (Chapter 3 ). Here, we present the methods to obtain highly purified native chromatin modifying complexes from Saccharomyces cerevisiae, based on Tandem Affinity Purification (TAP). We also present possible applications and useful functional assays that can be performed using these yeast native reagents.

  6. Immunolocalization and Distribution of Rubella Antigen in Fatal Congenital Rubella Syndrome

    Science.gov (United States)

    Lazar, Mihaela; Perelygina, Ludmila; Martines, Roosecelis; Greer, Patricia; Paddock, Christopher D.; Peltecu, Gheorghe; Lupulescu, Emilia; Icenogle, Joseph; Zaki, Sherif R.

    2015-01-01

    Background An estimated 100,000 cases of congenital rubella syndrome (CRS) occur worldwide each year. The reported mortality rate for infants with CRS is up to 33%. The cellular mechanisms responsible for the multiple congenital defects in CRS are presently unknown. Here we identify cell types positive for rubella virus (RV) in CRS infants. Methods Cells and organs involved in RV replication were identified in paraffin-embedded autopsy tissues from three fatal case-patients by histopathologic examination and immunohistochemical (IHC) staining using a rabbit polyclonal RV antibody. Normal rabbit antisera and RV antisera preabsorbed with highly purified RV served as negative controls. Results RV antigen was found in interstitial fibroblasts in the heart, adventitial fibroblasts of large blood vessels, alveolar macrophages, progenitor cells of the outer granular layer of the brain, and in capillary endothelium and basal plate in the placenta. The antibody specificity was verified by IHC staining of multiple tissue sections from other infectious disease cases. RV infection of each cell type is consistent with abnormalities which have been identified in patients with CRS, in the heart, large blood vessels, and brain. Antigen distribution was consistent with inflammatory response to vascular injury and systemic spread of RV. Conclusions The identification of RV positive cell types in CRS is important to better understand the pathology and pathogenesis of CRS. PMID:26870820

  7. Methemoglobin Is an Endogenous Toll-Like Receptor 4 Ligand—Relevance to Subarachnoid Hemorrhage

    Directory of Open Access Journals (Sweden)

    Min Seong Kwon

    2015-03-01

    Full Text Available Neuroinflammation is a well-recognized consequence of subarachnoid hemorrhage (SAH, and may be responsible for important complications of SAH. Signaling by Toll-like receptor 4 (TLR4-mediated nuclear factor κB (NFκB in microglia plays a critical role in neuronal damage after SAH. Three molecules derived from erythrocyte breakdown have been postulated to be endogenous TLR4 ligands: methemoglobin (metHgb, heme and hemin. However, poor water solubility of heme and hemin, and lipopolysaccharide (LPS contamination have confounded our understanding of these molecules as endogenous TLR4 ligands. We used a 5-step process to obtain highly purified LPS-free metHgb, as confirmed by Fourier Transform Ion Cyclotron Resonance mass spectrometry and by the Limulus amebocyte lysate assay. Using this preparation, we show that metHgb is a TLR4 ligand at physiologically relevant concentrations. metHgb caused time- and dose-dependent secretion of the proinflammatory cytokine, tumor necrosis factor α (TNFα, from microglial and macrophage cell lines, with secretion inhibited by siRNA directed against TLR4, by the TLR4-specific inhibitors, Rs-LPS and TAK-242, and by anti-CD14 antibodies. Injection of purified LPS-free metHgb into the rat subarachnoid space induced microglial activation and TNFα upregulation. Together, our findings support the hypothesis that, following SAH, metHgb in the subarachnoid space can promote widespread TLR4-mediated neuroinflammation.

  8. Anticancer Effect and Apoptosis Induction of Cymbopogon citratus Plant on Head and Neck HTB43 Cancer Cell Lines

    International Nuclear Information System (INIS)

    Yen, N.; Zainah Adam; Arapoc, D.J.; Mohamed Zaffar Ali Mohamed Amiroudine; Shafii Khamis

    2016-01-01

    Lemongrass (Cymbopogon citratus) is a major crop in Asia, South and Central America, Africa and other tropical countries. It is commonly used as a culinary herb in Asian cuisine and also as medicinal herb in India. The objective of the present study was to investigate the anticancer effect of Cymbopogon citratus essential oil on human head and neck cancer cell lines. Lemongrass essential oil was obtained from fresh lemongrass leaves using the microwave extractor. The yield of the highly purified oil obtained was 0.62 %. The oil was investigated for its in-vitro cytotoxicity and apoptosis induction against human head and neck HTB43 cancer cell lines. The results showed promising cytotoxic effects with IC 50 value of 15.42 ± 6.10 μg/ ml (HTB43) respectively. In addition, the essential oil has found inducing apoptotic effect within HTB43 cells (25.71 ± 1.43 %). These preliminary results indicated that the oil had apoptosis-based cytotoxicity against head and neck cancer cells. However, further investigations need to be carried out in order to elucidate the anti-cancer properties of the oil. The phytochemicals properties of the active compounds found in the oil also need to be studied. (author)

  9. Functions of replication factor C and proliferating-cell nuclear antigen: Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4

    International Nuclear Information System (INIS)

    Tsurimoto, Toshiki; Stillman, B.

    1990-01-01

    The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase δ on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4

  10. Secretin-radioimmunoassay, physiology and pathophysiology in man

    International Nuclear Information System (INIS)

    Haecki, W.H.

    1978-01-01

    Production of antibodies to secretin for radioimmunoassay is straightforward. Secretin is iodinated by weak oxydation with lactoperoxydase and subsequent purification by ionexchange chromatography (Sephadex C25). The specific activity of fresh label is between 650 and 900 mCi x mol -6 . The label is highly purified and may be used in radioimmunoassay for several months. In order to eliminate plasma interference sepharose-beads with covalently coupled secretin antibodies are used to produce secretin-free standard plasma samples. Delay in the separation of plasma from fresh blood samples can lead to erronous results, even to falsely elevated secretin levels. - Duo-denal acidification only leads to physiological increases of secretin plasma levels. This may happen by intraduodenal instillation of acid, or by an acidic oral drink, or to a lesser extent after a meal. Secretin is distributed throughout the plasma volume and has a short halflife of around 3 minutes. Impaired release of secretin is found in children with coeliac disease. The role of secretin in peptic ulcer however is not clear. Chronic pancreatitis and renal insufficiency are without effect on plasma secretin levels. (orig.) [de

  11. Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment

    Directory of Open Access Journals (Sweden)

    J. Fraser Wright

    2014-03-01

    Full Text Available Adeno-associated virus (AAV-based vectors expressing therapeutic genes continue to demonstrate great promise for the treatment of a wide variety of diseases and together with other gene transfer vectors represent an emerging new therapeutic paradigm comparable in potential impact on human health to that achieved by recombinant proteins and vaccines. A challenge for the current pipeline of AAV-based investigational products as they advance through clinical development is the identification, characterization and lot-to-lot control of the process- and product-related impurities present in even highly purified preparations. Especially challenging are AAV vector product-related impurities that closely resemble the vector itself and are, in some cases, without clear precedent in established biotherapeutic products. The determination of acceptable levels of these impurities in vectors prepared for human clinical product development, with the goal of new product licensure, requires careful risk and feasibility assessment. This review focuses primarily on the AAV product-related impurities that have been described in vectors prepared for clinical development.

  12. Plasma insulin disturbances in primary hyperparathyroidism

    Science.gov (United States)

    Kim, Hakjoong; Kalkhoff, Ronald K.; Costrini, Nicholas V.; Cerletty, James M.; Jacobson, Mitchell

    1971-01-01

    Plasma insulin dynamics were evaluated in 10 patients with primary hyperparathyroidism before and after parathyroidectomy and correction of hypercalcemia. Before surgery fasting plasma insulin concentrations and insulin responses to administered glucose, tolbutamide, and glucagon were significantly greater than postoperative values. Hyperinsulinemia was not associated with altered glucose curves during glucose or glucagon tolerance tests, but a relatively greater insulin response to tolbutamide resulted in an increased hypoglycemic effect following its administration. The glucose-lowering action of intravenous insulin was slightly impaired before treatment. Intramuscular injections of parathormone to six normal men for 8 days induced mild hypercalcemia and hypophosphatemia and reproduced augmented plasma insulin responses to oral glucose and intravenous tolbutamide. 4-hr intravenous infusions of calcium to another group of six normal men raised serum calcium concentrations above 11 mg/100 ml. This did not alter glucose or insulin curves during oral glucose tolerance but markedly accentuated insulin responses to tolbutamide and potentiated its hypoglycemic effect. When highly purified parathormone was incubated with isolated pancreatic islets of male rats, glucose-stimulated insulin secretion was unaffected. These findings suggest that chronic hypercalcemia of hyperparathyroidism sustains a form of endogenous insulin resistance that necessitates augmented insulin secretion to maintain plasma glucose homeostasis. This state is insufficient to oppose tolbutamide-induced hypoglycemia because of an additional direct, selective enhancement of hypercalcemia on pancreatic beta cell responsiveness to the sulfonylurea. The possible direct role of parathormone in these events has not been established. PMID:5129311

  13. GMP-compliant, large-scale expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Okjae Lim

    Full Text Available Ex vivo-expanded, allogeneic natural killer (NK cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP conditions. After a single step of magnetic depletion of CD3(+ T cells, the depleted peripheral blood mononuclear cells (PBMCs were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3(-CD16(+CD56(+ NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.

  14. Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

    Science.gov (United States)

    Zanon, Veronica; Pilipow, Karolina; Scamardella, Eloise; De Paoli, Federica; De Simone, Gabriele; Price, David A; Martinez Usatorre, Amaia; Romero, Pedro; Mavilio, Domenico; Roberto, Alessandra; Lugli, Enrico

    2017-09-01

    Human T memory stem (T SCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8 + T-cell differentiation and allows the generation of CD45RO - CD45RA + CCR7 + CD27 + CD95 + -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human T SCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim.

  15. Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium

    Directory of Open Access Journals (Sweden)

    Nie Weijia

    2008-11-01

    Full Text Available Abstract Background Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce. Results The toxin genes tcdA and tcdB were amplified by PCR using chromosomal DNA from a toxigenic strain as a template, and cloned into a shuttle vector pHis1522. The sequences of both tcdA and tcdB genes in the vector have been verified by DNA sequencing. The constructs were transformed into B. megaterium protoplasts and the protein expression was controlled under a xylose promoter. The recombinant toxins (rTcdA and rTcdB were purified from bacterial crude extracts. Approximately 5 – 10 mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. The resulting rTcdA and rTcdB had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination. Conclusion We have generated the full length and active recombinant TcdA and TcdB in Bacillus megaterium.

  16. PHYSICOCHEMICAL STUDIES OF THE CARCINOEMBRYONIC ANTIGENS OF THE HUMAN DIGESTIVE SYSTEM

    Science.gov (United States)

    Krupey, John; Gold, Phil; Freedman, Samuel O.

    1968-01-01

    A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria. The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation. Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues. PMID:4299103

  17. Isolation and characterization of hair follicle stem cells from Arbas Cashmere goat.

    Science.gov (United States)

    He, Nimantana; Dong, Zhenguo; Tao, Li; Zhao, Sirguleng; Bou, Shorgan; Liu, Dongjun

    2016-12-01

    In this study, highly purified hair follicle stem cells from Arbas Cashmere goat (gHFSCs) were isolated using enzyme digestion and adhesion to type IV collagen. The biological characteristics of the gHFSCs were identified by morphological observation, growth curve, markers assay and differentiation in vitro. The gHFSCs were in small cell size with typical cobblestone morphology, good adhesion and high refractive index. Immunocytochemistry staining showed the cells were expressing Krt15, Krt19, CD34, Itgβ1 and Krt14. Cell growth curve indicated that cultured gHFSCs had strong proliferation ability. Krt14 and CD34 were high expressed at the mRNA level, respectively, 39.68 and 24.37 times of the Cashmere goat keratinocytes, and krt15 expression was 5.62 times and itgβ1 expression was 1.81 times higher (p < 0.01). Western blot detected the expression of all the above markers. After osteogenic induction, the cells were positive for Von Kossa staining and expressed Osteocalcin. Sulfated proteoglycans in cartilaginous matrices were positively stained by Alcian blue after chondrogenic induction and COL2A1 was expressed. In myogenic induction, Hoechst 33342 staining evidenced cytoplasm fusion and positive expression of MyoG was detected by immunocytochemistry.

  18. Antiproliferative effects of galectin-1 from Rana catesbeiana eggs on human leukemia cells and its binding proteins in human cells.

    Science.gov (United States)

    Yasumitsu, Hidetaro; Mochida, Keiichi; Yasuda, Chie; Isobe, Masaharu; Kawsar, Sarkar M A; Fujii, Yuki; Matsumoto, Ryo; Kanaly, Robert A; Ozeki, Yasuhiro

    2011-12-01

    Galectin-1 from American bullfrog, RCG1, was isolated to high purity, and its growth inhibitory properties against human cells were examined. The results demonstrated that highly purified RCG1 induced large cell aggregates and revealed cell-type-specific growth inhibition. It significantly inhibited all human leukemia cell lines tested such as HL-60, U937, and K562 cells but did not inhibit human colon cancer cell line, Colo 201, or mouse mammary tumor cell line FM3A cells. Although most of the galectin-induced growth inhibitions are known to be apoptic, RCG1 induced growth arrest and neither apoptosis nor necrosis. RCG1-mediated growth inhibition was specifically suppressed by the corresponding sugar, lactose, but not by sucrose or even the structurally similar sugar, melibiose. Several studies have reported that galectin-mediated biological functions were modulated by charge modification. Since the high purity of RCG1 was demonstrated but a moderate degree of growth inhibition occurred, it is possible protein charge modification was examined by isoelectric focusing, and it was found to be highly heterogeneous in charge. RCG1 binding proteins in human cells were analyzed by lectin blotting using biotinylated RCG1, and lectin blotting revealed that in human cell extracts the specific proteins at molecular weight 37 and 50 kDa possessed the responsive features of RCG1 binding and lactose competition.

  19. Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

    DEFF Research Database (Denmark)

    Jensen, Lena Vinther; Lademann, Ulrik Axel; Andersen, Elisabeth Veyhe

    2014-01-01

    as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N......-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1......), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1...

  20. Chitinolytic enzymes from Clostridium aminovalericum: activity screening and purification.

    Science.gov (United States)

    Simůnek, J; Tishchenko, G; Rozhetsky, K; Bartonová, H; Kopecný, J; Hodrová, B

    2004-01-01

    A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.

  1. Oxindole alkaloids from Uncaria tomentosa induce apoptosis in proliferating, G0/G1-arrested and bcl-2-expressing acute lymphoblastic leukaemia cells.

    Science.gov (United States)

    Bacher, Nicole; Tiefenthaler, Martin; Sturm, Sonja; Stuppner, Hermann; Ausserlechner, Michael J; Kofler, Reinhard; Konwalinka, Günther

    2006-03-01

    Natural products are still an untapped source of promising lead compounds for the generation of antineoplastic drugs. Here, we investigated for the first time the antiproliferative and apoptotic effects of highly purified oxindole alkaloids, namely isopteropodine (A1), pteropodine (A2), isomitraphylline (A3), uncarine F (A4) and mitraphylline (A5) obtained from Uncaria tomentosa, a South American Rubiaceae, on human lymphoblastic leukaemia T cells (CCRF-CEM-C7H2). Four of the five tested alkaloids inhibited proliferation of acute lymphoblastic leukaemia cells. Furthermore, the antiproliferative effect of the most potent alkaloids pteropodine (A2) and uncarine F (A4) correlated with induction of apoptosis. After 48 h, 100 micromol/l A2 or A4 increased apoptotic cells by 57%. CEM-C7H2 sublines with tetracycline-regulated expression of bcl-2, p16ink4A or constitutively expressing the cowpox virus protein crm-A were used for further studies of the apoptosis-inducing properties of these alkaloids. Neither overexpression of bcl-2 or crm-A nor cell-cycle arrest in G0/G1 phase by tetracycline-regulated expression of p16INK4A could prevent alkaloid-induced apoptosis. Our results show the strong apoptotic effects of pteropodine and uncarine F on acute leukaemic lymphoblasts and recommend the alkaloids for further studies in xenograft models.

  2. Vaccine Adjuvants: from 1920 to 2015 and Beyond

    Science.gov (United States)

    Di Pasquale, Alberta; Preiss, Scott; Tavares Da Silva, Fernanda; Garçon, Nathalie

    2015-01-01

    The concept of stimulating the body’s immune response is the basis underlying vaccination. Vaccines act by initiating the innate immune response and activating antigen presenting cells (APCs), thereby inducing a protective adaptive immune response to a pathogen antigen. Adjuvants are substances added to vaccines to enhance the immunogenicity of highly purified antigens that have insufficient immunostimulatory capabilities, and have been used in human vaccines for more than 90 years. While early adjuvants (aluminum, oil-in-water emulsions) were used empirically, rapidly increasing knowledge on how the immune system interacts with pathogens means that there is increased understanding of the role of adjuvants and how the formulation of modern vaccines can be better tailored towards the desired clinical benefit. Continuing safety evaluation of licensed vaccines containing adjuvants/adjuvant systems suggests that their individual benefit-risk profile remains favorable. Adjuvants contribute to the initiation of the innate immune response induced by antigens; exemplified by inflammatory responses at the injection site, with mostly localized and short-lived effects. Activated effectors (such as APCs) then move to draining lymph nodes where they direct the type, magnitude and quality of the adaptive immune response. Thus, the right match of antigens and adjuvants can potentiate downstream adaptive immune responses, enabling the development of new efficacious vaccines. Many infectious diseases of worldwide significance are not currently preventable by vaccination. Adjuvants are the most advanced new technology in the search for new vaccines against challenging pathogens and for vulnerable populations that respond poorly to traditional vaccines. PMID:26343190

  3. High-quality total RNA isolation from melon (Cucumis melo L. fruits rich in polysaccharides

    Directory of Open Access Journals (Sweden)

    Gabrielle Silveira de Campos

    2017-08-01

    Full Text Available Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazil’s main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1 guanidine thiocyanate/phenol/chloroform; T2 sodium azide/?-mercaptoethanol; T3 phenol/guanidine thiocyanate; T4 CTAB/PVP/?-mercaptoethanol; T5 SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6 sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.

  4. The radioimmunoassay of human serum inhibin

    International Nuclear Information System (INIS)

    McLachlan, R.; Robertson, D.M.; Healy, D.L.; Findlay, J.K.; De Kretser, D.M.; Burger, H.G.

    1987-01-01

    The measurement of circulating inhibin is essential to the advancement of our understanding of its role in the control of FSH secretion. Recently, sensitive in vitro bioassay systems have been described which have allowed the measurement of inhibin bioactivity in the peripheral serum of immature female rats, sheep and women (C. Tsonis, personal communication) following ovarian hyperstimulation with exogenous gonadotrophins. Inhibin bioactivity has also been shown to rise in ovarian venous blood in the late follicular phase of women and during FSHG administration to primates. Such studies have some important limitations, in particular, the bioassay systems employed lack the sensitivity required for the study of normal physiology, and questions regarding the specificity of in vitro bioassay in application to serum samples have arisen following the reports of FSH releasing factors (transforming growth factor-beta inhibin B subunit dimers) that may confound the accurate assessment of inhibin bioactivity. This paper reports on the development of sensitive and specific radioimmunoassay (RIA) systems applicable to human serum utilizing highly purified preparations of 58kDa and 31kDa bovine follicular fluid (bFF) inhibin. The development of two RIA systems utilizing antisera raised to either or 31kDa bFF inhibin is detailed and their application to the study of inhibin physiology in the human female during ovulation inducting, the normal menstrual cycle and gestation described

  5. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Lindolfo da Silva Meirelles

    2016-03-01

    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

  6. Calmodulin-activated cyclic nucleotide phosphodiesterase from brain. Relationship of subunit structure to activity assessed by radiation inactivation

    International Nuclear Information System (INIS)

    Kincaid, R.L.; Kemdner, E.; Manganiello, V.C.; Osborne, J.C.; Vaughan, M.

    1981-01-01

    The apparent target sizes of the basal and calmodulin-dependent activities of calmodulin-activated phosphodiesterase from bovine brain were estimated using target theory analysis of data from radiation inactivation experiments. Whether crude or highly purified samples were irradiated, the following results were obtained. Low doses of radiation caused a 10 to 15% increase in basal activity, which, with further irradiation, decayed with an apparent target size of approx.60,000 daltons. Calmodulin-dependent activity decayed with an apparent target size of approx.105,000 daltons. The percentage stimulation of enzyme activity by calmodulin decreased markedly as a function of radiation dosage. These observations are consistent with results predicted by computer-assisted modeling based on the assumptions that: 1) the calmodulin-activated phosphodiesterase exists as a mixture of monomers which are fully active in the absence of calmodulin and dimers which are inactive in the absence of calmodulin; 2) in the presence of calmodulin, a dimer exhibits activity equal to that of two monomers; 3) on radiation destruction of a dimer, an active monomer is generated. This monomer-dimer hypothesis provides a plausible explanation for and definition of basal and calmodulin-dependent phosphodiesterase activity

  7. Overexpression of DR-nm23, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells.

    Science.gov (United States)

    Venturelli, D; Martinez, R; Melotti, P; Casella, I; Peschle, C; Cucco, C; Spampinato, G; Darzynkiewicz, Z; Calabretta, B

    1995-01-01

    Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:7638209

  8. Microbial removal of the pharmaceutical compounds Ibuprofen and diclofenac from wastewater.

    Science.gov (United States)

    Langenhoff, Alette; Inderfurth, Nadia; Veuskens, Teun; Schraa, Gosse; Blokland, Marco; Kujawa-Roeleveld, Katarzyna; Rijnaarts, Huub

    2013-01-01

    Studies on the occurrence of pharmaceuticals show that the widely used pharmaceuticals ibuprofen and diclofenac are present in relevant concentrations in the environment. A pilot plant treating hospital wastewater with relevant concentrations of these pharmaceuticals was evaluated for its performance to reduce the concentration of the pharmaceuticals. Ibuprofen was completely removed, whereas diclofenac yielded a residual concentration, showing the necessity of posttreatment to remove diclofenac, for example, activated carbon. Successively, detailed laboratory experiments with activated sludge from the same wastewater treatment plant showed bioremediation potential in the treatment plant. The biological degradation pathway was studied and showed a mineralisation of ibuprofen and degradation of diclofenac. The present microbes were further studied in laboratory experiments, and DGGE analyses showed the enrichment and isolation of highly purified cultures that degraded either ibuprofen or diclofenac. This research illuminates the importance of the involved bacteria for the effectiveness of the removal of pharmaceuticals in a wastewater treatment plant. A complete removal of pharmaceuticals from wastewater will stimulate water reuse, addressing the worldwide increasing demand for clean and safe fresh water.

  9. Microbial Removal of the Pharmaceutical Compounds Ibuprofen and Diclofenac from Wastewater

    Directory of Open Access Journals (Sweden)

    Alette Langenhoff

    2013-01-01

    Full Text Available Studies on the occurrence of pharmaceuticals show that the widely used pharmaceuticals ibuprofen and diclofenac are present in relevant concentrations in the environment. A pilot plant treating hospital wastewater with relevant concentrations of these pharmaceuticals was evaluated for its performance to reduce the concentration of the pharmaceuticals. Ibuprofen was completely removed, whereas diclofenac yielded a residual concentration, showing the necessity of posttreatment to remove diclofenac, for example, activated carbon. Successively, detailed laboratory experiments with activated sludge from the same wastewater treatment plant showed bioremediation potential in the treatment plant. The biological degradation pathway was studied and showed a mineralisation of ibuprofen and degradation of diclofenac. The present microbes were further studied in laboratory experiments, and DGGE analyses showed the enrichment and isolation of highly purified cultures that degraded either ibuprofen or diclofenac. This research illuminates the importance of the involved bacteria for the effectiveness of the removal of pharmaceuticals in a wastewater treatment plant. A complete removal of pharmaceuticals from wastewater will stimulate water reuse, addressing the worldwide increasing demand for clean and safe fresh water.

  10. Characterization of heparan sulfate N-deacetylase/N-sulfotransferase isoform 4 using synthetic oligosaccharide substrates.

    Science.gov (United States)

    Li, Yi-Jun; Yin, Feng-Xin; Zhang, Xin-Ke; Yu, Jie; Zheng, Shuang; Song, Xin-Lei; Wang, Feng-Shan; Sheng, Ju-Zheng

    2018-03-01

    The final structure of heparan sulfate chains is strictly regulated in vivo, though the biosynthesis is not guided by a template process. N-deacetylase/N-sulfotransferase (NDST) is the first modification enzyme in the HS biosynthetic pathway. The N-sulfo groups introduced by NDST are reportedly involved in determination of the susceptibility to subsequent processes catalyzed by C 5 -epimerse and 3-O-sulfotransferases. Understanding the substrate specificities of the four human NDST isoforms has become central to uncovering the regulatory mechanism of HS biosynthesis. Highly-purified recombinant NDST-4 (rNDST-4) and a selective library of structurally-defined oligosaccharides were employed to determine the substrate specificity of rNDST-4. Full-length rNDST-4 lacks obvious N-deacetylase activity, and displays only N-sulfotransferase activity. Unlike NDST-1, NDST-4 did not show directional N-sulfotransferase activity while the N-deacetylase domain was inactive. Individual NDST-4 could not effectively assume the key role in the distribution of N-S domains and N-Ac domains in HS biosynthesis in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Research and Development of Some Advanced High Temperature Titanium Alloys for Aero-engine

    Directory of Open Access Journals (Sweden)

    CAI Jian-ming

    2016-08-01

    Full Text Available Some advanced high temperature titanium alloys are usually selected to be manufactured into blade, disc, case, blisk and bling under high temperature environment in compressor and turbine system of a new generation high thrust-mass ratio aero-engine. The latest research progress of 600℃ high temperature titanium alloy, fireproof titanium alloy, TiAl alloy, continuous SiC fiber reinforced titanium matrix composite and their application technology in recent years in China were reviewed in this paper. The key technologies need to be broken through in design, processing and application of new material and component are put forward, including industrial ingot composition of high purified and homogeneous control technology, preparation technology of the large size bar and special forgings, machining technology of blisk and bling parts, material property evaluation and application design technique. The future with the continuous application of advanced high temperature titanium alloys, will be a strong impetus to the development of China's aero-engine technology.

  12. Monocyte IL-10 produced in response to lipopolysaccharide modulates thrombin generation by inhibiting tissue factor expression and release of active tissue factor-bound microparticles.

    Science.gov (United States)

    Poitevin, Stéphane; Cochery-Nouvellon, Eva; Dupont, Annick; Nguyen, Philippe

    2007-04-01

    Lipopolysaccharide (LPS)-stimulated monocytes are known to have a procoagulant effect. This property is currently explained by the fact that monocytes, in response to LPS, can express tissue factor (TF) and undergo a process of membrane microvesiculation. Interleukin-10 (IL-10) has been shown to downregulate TF expression and inhibit procoagulant activity (PCA). In order to further characterize the inhibitory effect of IL-10 on LPS-induced PCA, we used the integrated system of analysis of kinetics of thrombin generation in normal plasma (thrombinography). For this, we developed an original method of elutriation allowing to obtain a highly purified monocyte preparation, under endotoxin-free conditions. Thrombin generation was measured using a highly sensitive and specific fluorogenic method which we adapted to inhibit the contact factor pathway. Results show that recombinant human IL-10 decreased the kinetics of thrombin generation in a dose-dependent manner. Furthermore, the inhibition of endogenous IL-10 released by monocytes in response to LPS is associated with an increase in the kinetics of thrombin generation. We demonstrated that this effect was a consequence of the up-regulation of TF expression and TF-bound microparticle release. In conclusion, we report that IL-10 can regulate thrombin generation in conditions close to physiology as allowed by thrombinography, and that endogenous IL-10 regulates TF expression and release of active TF-bound microparticles by a negative feed back loop through IL-10 receptor alpha.

  13. Development of a millimetrically scaled biodiesel transesterification device that relies on droplet-based co-axial fluidics

    Science.gov (United States)

    Yeh, S. I.; Huang, Y. C.; Cheng, C. H.; Cheng, C. M.; Yang, J. T.

    2016-07-01

    In this study, we investigated a fluidic system that adheres to new concepts of energy production. To improve efficiency, cost, and ease of manufacture, a millimetrically scaled device that employs a droplet-based co-axial fluidic system was devised to complete alkali-catalyzed transesterification for biodiesel production. The large surface-to-volume ratio of the droplet-based system, and the internal circulation induced inside the moving droplets, significantly enhanced the reaction rate of immiscible liquids used here - soybean oil and methanol. This device also decreased the molar ratio between methanol and oil to near the stoichiometric coefficients of a balanced chemical equation, which enhanced the total biodiesel volume produced, and decreased the costs of purification and recovery of excess methanol. In this work, the droplet-based co-axial fluidic system performed better than other methods of continuous-flow production. We achieved an efficiency that is much greater than that of reported systems. This study demonstrated the high potential of droplet-based fluidic chips for energy production. The small energy consumption and low cost of the highly purified biodiesel transesterification system described conforms to the requirements of distributed energy (inexpensive production on a moderate scale) in the world.

  14. A novel antigen-toxin chimeric protein: myelin basic protein-pseudomonas exotoxin (MBP-PE 40) for treatment of experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Brenner, T; Steinberger, I; Soffer, D; Beraud, E; Ben-Nun, A; Lorberboum-Galski, H

    1999-06-01

    Myelin basic protein (MBP), is a major component of the central nervous system (CNS) myelin. MBP can stimulate T cells that migrate into the CNS, initiating a cascade of events that result in perivascular infiltration and demyelination. EAE is an inflammatory and demyelinating autoimmune disease of the CNS that serves as a model for the human disease Multiple Sclerosis (MS). Taking advantage of the fact that EAE can be mediated by T cells, able to recognize MBP or its peptides, we developed a new approach to target anti-MBP T cells by fusing an MBP-sequence to a toxin. In the new chimeric protein, an oligonucleotide coding for the guinea pig MBP encephalitogenic moiety (residues 68-88) was fused to a cDNA encoding a truncated form of the PE gene (PE40). The chimeric gene termed MBP-PE was expressed in E. coli and highly purified. MBP-PE chimeric protein was cytotoxic to various anti-MBP T cells. Moreover, treatment with the novel MBP-toxin blocked the clinical signs of EAE as well as CNS inflammation and demyelination. A chimeric protein such as MBP-PE40 presents a novel prototype of chimeric proteins, composed of antigen/peptide-toxin, that could prove to be an efficient and specific immunotherapeutic agent for autoimmune diseases in which a known antigen is involved.

  15. Purification and partial characterization of xanthine oxidase from human milk.

    Science.gov (United States)

    Abadeh, S; Killacky, J; Benboubetra, M; Harrison, R

    1992-07-21

    Xanthine oxidase was purified from human milk in yields comparable with those obtained from bovine milk. The freshly purified enzyme appeared homogeneous in gel permeation FPLC and SDS-PAGE, consistent with its being a homodimer with total M(r) 290,000 +/- 6000. The ultraviolet/visible absorption spectrum differed only slightly from that of bovine milk enzyme and showed an A280/A450 ratio of 5.13 +/- 0.29, indicating a high degree of purity. Xanthine oxidase activities of purified enzyme varied with batches of milk, ranging between 3 and 46 mU/mg protein; values that are some two to three orders of magnitude smaller than those shown by the most highly purified samples of bovine milk enzyme. Direct comparison with commercially-available bovine milk enzyme showed that activities involving xanthine as reducing substrate were 1-6% that of the bovine enzyme, whereas those involving NADH, in contrast, were of the same order for the two enzymes. Anaerobic bleaching experiments indicated that less than 2% of the human enzyme was present as a form active with xanthine. These findings, together with the activity data, are consistent with a very high content, possibly greater than 98%, of demolybdo- and/or desulpho-forms of human enzyme, both of which occur, to a lesser extent, in bovine xanthine oxidase. Molybdenum assay indicated that demolybdo-enzyme could only account for some 26% of this inactive component, suggesting that desulpho-enzyme may account for the remainder.

  16. Omega-3 Fatty Acids Protect Renal Functions by Increasing Docosahexaenoic Acid-Derived Metabolite Levels in SHR.Cg-Leprcp/NDmcr Rats, a Metabolic Syndrome Model

    Directory of Open Access Journals (Sweden)

    Masanori Katakura

    2014-03-01

    Full Text Available The omega-3 polyunsaturated fatty acids (ω-3 PUFAs docosahexaenoic acid (DHA and/or eicosapentaenoic acid (EPA protect against diabetic nephropathy by inhibiting inflammation. The aim of this study was to assess the effects of highly purified DHA and EPA or EPA only administration on renal function and renal eicosanoid and docosanoid levels in an animal model of metabolic syndrome, SHR.Cg-Leprcp/NDmcr (SHRcp rats. Male SHRcp rats were divided into 3 groups. Control (5% arabic gum, TAK-085 (300 mg/kg/day, containing 467 mg/g EPA and 365 mg/g DHA, or EPA (300 mg/kg/day was orally administered for 20 weeks. The urinary albumin to creatinine ratio in the TAK-085-administered group was significantly lower than that in other groups. The glomerular sclerosis score in the TAK-085-administered group was significantly lower than that in the other groups. Although DHA levels were increased in total kidney fatty acids, the levels of nonesterified DHA were not significantly different among the 3 groups, whereas the levels of protectin D1, resolvin D1, and resolvin D2 were significantly increased in the TAK-085-administered group. The results show that the use of combination therapy with DHA and EPA in SHRcp rats improved or prevented renal failure associate with metabolic syndrome with decreasing triglyceride levels and increasing ω-3 PUFA lipid mediators.

  17. Fetuin-A (α2HS-glycoprotein) is a serum chemo-attractant that also promotes invasion of tumor cells through Matrigel.

    Science.gov (United States)

    Nangami, Gladys N; Watson, Kurt; Parker-Johnson, Kitani; Okereke, Kelechi O; Sakwe, Amos; Thompson, Pamela; Frimpong, Nanna; Ochieng, Josiah

    2013-09-06

    The present study was conducted to determine whether fetuin-A, a dominant serum protein plays a role in chemo-attraction and chemo-invasion of carcinoma cells in vitro. Serum is normally used as positive chemotaxis control in Boyden chamber motility assays, prompting the need to identify the factor/s in serum that contributes the bulk of chemo-taxis and invasion. Serum has a plethora of chemotactic factors including stromal derived factor 1 also known as CXCL12. Using highly purified fetuin-A, we compared its chemo-attraction potential to culture medium containing 10% fetal bovine serum. We also investigated its ability to attract tumor cells through a bed of Matrigel (invasion assay). We demonstrated, using similar concentration range of fetuin-A found in blood, that it robustly supports both directed chemo-attraction and invasion of breast tumor cells. More importantly, we showed that at low concentrations (fetuin-A coated wells) itinteracts synergistically with CXCL12 to promote chemotaxis. The presence of plasminogen (PL) blunted the fetuin-A mediated chemotaxis. Taken together, the data suggest an in vivo chemotaxis/invasion role for fetuin-A. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Potential of hot water extraction of birch wood to produce high-purity dissolving pulp after alkaline pulping.

    Science.gov (United States)

    Borrega, Marc; Tolonen, Lasse K; Bardot, Fanny; Testova, Lidia; Sixta, Herbert

    2013-05-01

    The potential of hot water extraction of birch wood to produce highly purified dissolving pulp in a subsequent soda-anthraquinone pulping process was evaluated. After intermediate extraction intensities, pulps with low xylan content (3-5%) and high cellulose yield were successfully produced. Increasing extraction intensity further decreased the xylan content in pulp. However, below a xylan content of 3%, the cellulose yield dramatically decreased. This is believed to be due to cleavage of glycosidic bonds in cellulose during severe hot water extractions, followed by peeling reactions during alkaline pulping. Addition of sodium borohydride as well as increased anthraquinone concentration in the pulping liquor increased the cellulose yield, but had no clear effects on pulp purity and viscosity. The low intrinsic viscosity of pulps produced after severe extraction intensities and soda-anthraquinone pulping corresponded to the viscosity at the leveling-off degree of polymerization, suggesting that nearly all amorphous cellulose had been degraded. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Biological adsorptive process for removal of organics from raw water as pre-treatment of a demineralisation plant; Biologisch adsorptives Verfahren zur Entfernung von Organika aus Rohwaessern als Vorstufe fuer Vollentsalzungsanlagen

    Energy Technology Data Exchange (ETDEWEB)

    Schoenfelder, T. [KNG Kraftwerks- und Netzgesellschaft mbH, Kraftwerk Rostock, Rostock (Germany); Keil, U. [STEAG encotec, Essen (Germany)

    2002-07-01

    The production of highly-purified demineralised water as feedwater for boilers with values of <0.2 {mu}S/cm conductivity and <200 ppb TOC is essential for the quality of steam within the steamwater cycle. Especially for the combined mode of operation with additional dosing of oxygen it is necessary not only to stay within these values but to remain below the requirements. Nowadays it is no problem to produce demineralized water with corresponding low conductivity. The sufficient reduction of TOC has become the main problem. The Rostock Power Plant, in co-operation with Steag encotec GmbH, Essen, and the University Hamburg Harburg, developed a new treatment and tested it for more than two years with accompanying analysis. (orig.) [German] Um die VGB-Richtwerte fuer das Kesselspeisewasser im Kraftwerk Rostock zu erreichen, wurde eine Vollentsalzungsanlage einschliesslich Vorreinigungsstufe errichtet. Sie bestand aus nur einem Aktivkohlefilter zur Adsorption der im Trinkwasser enthaltenen geloesten organischen Substanzen. Hohe Aluminiumgehalte im Trinkwasser fuehrten bald zu einer Blockierung der Aktivkohle, wodurch die Adsorbtion der Organika an der Aktivkohle erheblich gestoert wurde. Daraufhin erfolgte die Nachruestung eines zweiten Adsorberfilters, der vor den Aktivkohlefilter geschaltet wurde. Die erhoffte Reduzierung der Organika durch die neu errichtete zweistufige Ozonierung trat jedoch nicht ein. Im Gegenteil, die teilweise nur angespaltenen Organika wurden vom Adsorberharz der Vorreinigung teilweise irreversibel adsorbiert. Ueber Abhilfemassnahmen wird berichtet. (orig.)

  20. Purity of graphene oxide determines its antibacterial activity

    Science.gov (United States)

    Barbolina, I.; Woods, C. R.; Lozano, N.; Kostarelos, K.; Novoselov, K. S.; Roberts, I. S.

    2016-06-01

    Nanomaterials based on two-dimensional (2D) atomic crystals are considered to be very promising for various life-science and medical applications, from drug delivery to tissue modification. One of the most suitable materials for these purposes is graphene oxide (GO), thanks to a well-developed methods of production and water solubility. At the same time, its biological effect is still debated. Here we demonstrate that highly purified and thoroughly washed GO neither inhibited nor stimulated the growth of E.coli, ATCC25922; E.coli NCIMB11943 and S.aureus ATCC25923 at concentrations of up to 1 mg ml-1. Moreover, transmission electron microscopy (TEM) of GO exposed bacteria did not reveal any differences between GO exposed and not exposed populations. In contrast, a suspension of insufficiently purified GO behaved as an antibacterial material due to the presence of soluble acidic impurities, that could be removed by extended purification or neutralisation by alkaline substrates. A standardised protocol is proposed for the generation of clean GO, so it becomes suitable for biological experiments. Our findings emphasise the importance of GO purification status when dealing with biological systems as the true effect of material can be masked by the impact of impurities.

  1. Isolation of a Ca++-dependent phospholipase A2 from human platelets

    International Nuclear Information System (INIS)

    Raghupathi, R.; Franson, R.C.

    1987-01-01

    Phospholipases A 2 mobilize arachidonic acid from cellular phospholipids. A Ca ++ -dependent phospholipase A 2 (PLA 2 ) from human platelets was purified 1900-fold over homogenate by acid extraction with 0.36N H 2 SO 4 , heat treatment at 100 0 C and ion-exchange chromatography. The purified enzyme hydrolyzed [1- 14 C]oleate-labelled autoclaved E. coli with a specific activity of 152 nmoles.min. -1 mg protein -1 . The enzyme was active between pH 6.0 and pH 8.5, with maximal activity at pH 7.0; EDTA completely inhibited; the enzyme, which requires 5mM CaCl 2 for optimal activity. Increasing concentrations of NaCl stimulated activity to a maximum of 8-fold over control at 150mM; higher concentrations were inhibitory. Molecular sizing estimates by Sephadex G-75 gel filtration and SDS-PAGE correspond to reported values for PLA 2 from other sources. Non-denaturing electrophoresis at pH 4.5 resulted in five protein bands. These data suggest that this highly purified preparation is contaminated by proteins of similar molecular size or indicates the existence of multiple forms of the enzyme which has reported for sheep and human platelet phospholipases A 2

  2. Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

    International Nuclear Information System (INIS)

    Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1986-01-01

    A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn 2+ and [γ- 32 P]ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes

  3. Designing of luminescent GdPO4:Eu@LaPO4@SiO2 core/shell nanorods: Synthesis, structural and luminescence properties

    Science.gov (United States)

    Ansari, Anees A.; Labis, Joselito P.; Aslam Manthrammel, M.

    2017-09-01

    GdPO4:Eu3+ (core) and GdPO4:Eu@LaPO4 (core/shell) nanorods (NRs) were successfully prepared by urea based co-precipitation process at ambient conditions which was followed by coating with amorphous silica shell via the sol-gel chemical route. The role of surface coating on the crystal structure, crystallinity, morphology, solubility, surface chemistry and luminescence properties were well investigated by means of X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray (EDX) analysis, Fourier Transform Infrared (FTIR), UV-Vis, and photoluminescence spectroscopy. XRD pattern revealed highly purified, well-crystalline, single phase-hexagonal-rhabdophane structure of GdPO4 crystal. The TEM micrographs exhibited highly crystalline and narrow size distributed rod-shaped GdPO4:Eu3+ nanostructures with average width 14-16 nm and typical length 190-220 nm. FTIR spectra revealed characteristic infrared absorption bands of amorphous silica. High absorbance in a visible region of silica modified core/shell/Si NRs in aqueous environment suggests the high solubility along with colloidal stability. The photoluminescence properties were remarkably enhanced after growth of undoped LaPO4 layers due to the reduction of nonradiative transition rate. The advantages of presented high emission intensity and high solubility of core/shell and core/shell/Si NRs indicated the potential applications in monitoring biological events.

  4. Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate.

    Science.gov (United States)

    Elustondo, P A; Nichols, M; Negoda, A; Thirumaran, A; Zakharian, E; Robertson, G S; Pavlov, E V

    Mitochondrial permeability transition pore (mPTP) opening allows free movement of ions and small molecules leading to mitochondrial membrane depolarization and ATP depletion that triggers cell death. A multi-protein complex of the mitochondrial ATP synthase has an essential role in mPTP. However, the molecular identity of the central 'pore' part of mPTP complex is not known. A highly purified fraction of mammalian mitochondria containing C-subunit of ATPase (C-subunit), calcium, inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB) forms ion channels with properties that resemble the native mPTP. We demonstrate here that amount of this channel-forming complex dramatically increases in intact mitochondria during mPTP activation. This increase is inhibited by both Cyclosporine A, an inhibitor of mPTP and Ruthenium Red, an inhibitor of the Mitochondrial Calcium Uniporter. Similar increases in the amount of complex formation occurs in areas of mouse brain damaged by ischemia-reperfusion injury. These findings suggest that calcium-induced mPTP is associated with de novo assembly of a channel comprising C-subunit, polyP and PHB.

  5. Development of homologous radioimmunoassays for equine growth hormone and equine prolactin and their application to the detection of circulating levels of hormone in horse plasma

    International Nuclear Information System (INIS)

    Cahill, C.M.; Hayden, T.J.; Ven der Kolk, H.; Goode, J.A.

    1994-01-01

    Highly purified and well-characterized preparations of equine prolactin and growth hormone from equine pituitary glands were employed to set up highly sensitive and specific homologous radioimmunoassays (RIA) for the measurement of hormone in horse plasma. The limit of sensitivity of the GH RIA was 1.2 ng/ml with mean intra -and inter- assay coefficients of variation (CV) of 6.6 and 10%, respectively. The sensitivity of the equine prolactin (ePRL) RIA was 0.5 ng/ml with mean intra and inter-assay CV of 9.1 and 15.6%, respectively. Dose-response curves of a crude pituitary gland extract and plasma samples collected from a mare and foal were parallel to the standards and the PRL RIA was clinically validated by administration of thyrotropin-releasing hormone (TRH). Plasma samples taken at 15 min intervals over 24 h from lactating mares gave 24 h mean GH values in the range 5.5 to 7.95 ng/ml. Large intermittent elevations of GH activity were detected. The mean 24 h PRL concentrations were between 3.2-10.4 ng/ml in the lactating animals, with higher concentrations earlier in lactation. Long episodic bursts of PRL were detected. (authors). 48 refs., 9 figs

  6. Catalase-dependent H2O2 consumption by cardiac mitochondria and redox-mediated loss in insulin signaling.

    Science.gov (United States)

    Rindler, Paul M; Cacciola, Angela; Kinter, Michael; Szweda, Luke I

    2016-11-01

    We have recently demonstrated that catalase content in mouse cardiac mitochondria is selectively elevated in response to high dietary fat, a nutritional state associated with oxidative stress and loss in insulin signaling. Catalase and various isoforms of glutathione peroxidase and peroxiredoxin each catalyze the consumption of H 2 O 2 Catalase, located primarily within peroxisomes and to a lesser extent mitochondria, has a low binding affinity for H 2 O 2 relative to glutathione peroxidase and peroxiredoxin. As such, the contribution of catalase to mitochondrial H 2 O 2 consumption is not well understood. In the current study, using highly purified cardiac mitochondria challenged with micromolar concentrations of H 2 O 2 , we found that catalase contributes significantly to mitochondrial H 2 O 2 consumption. In addition, catalase is solely responsible for removal of H 2 O 2 in nonrespiring or structurally disrupted mitochondria. Finally, in mice fed a high-fat diet, mitochondrial-derived H 2 O 2 is responsible for diminished insulin signaling in the heart as evidenced by reduced insulin-stimulated Akt phosphorylation. While elevated mitochondrial catalase content (∼50%) enhanced the capacity of mitochondria to consume H 2 O 2 in response to high dietary fat, the selective increase in catalase did not prevent H 2 O 2 -induced loss in cardiac insulin signaling. Taken together, our results indicate that mitochondrial catalase likely functions to preclude the formation of high levels of H 2 O 2 without perturbing redox-dependent signaling. Copyright © 2016 the American Physiological Society.

  7. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, R.L.; Austen, K.F. (Brigham and Women' s Hospital, Boston, MA (USA)); Fox, C.C.; Lichtenstein, L.M. (Johns Hopkins School of Medicine, Baltimore, MD (USA))

    1988-04-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparin and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

  8. Lymphoid Progenitor Cells from Childhood Acute Lymphoblastic Leukemia Are Functionally Deficient and Express High Levels of the Transcriptional Repressor Gfi-1

    Directory of Open Access Journals (Sweden)

    Jessica Purizaca

    2013-01-01

    Full Text Available Acute lymphoblastic leukemia (ALL is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34+ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

  9. Radiation inactivation studies on the rabbit kidney sodium-dependent glucose transporter.

    Science.gov (United States)

    Takahashi, M; Malathi, P; Preiser, H; Jung, C Y

    1985-09-05

    Rabbit kidney cortical brush-border membrane vesicles were irradiated in the frozen state with increasing doses of high energy electrons from a Van de Graaff generator. Sodium-dependent D-glucose and L-alanine transport showed a simple exponential loss of activity with increasing radiation dosage. Target size calculation based on these data gives estimates of 1.0 X 10(6) daltons for the glucose transporter and 1.2 X 10(6) daltons for the alanine transporter. A highly purified glucose transport protein extracted from rabbit kidney cortex was similarly irradiated both before and after reconstitution into liposomes. The target size of this purified glucose transporter was 343,000 daltons, based on inactivation of transport. The intensity of the major 165,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation was decreased by radiation. The decrease in staining intensity was dose-dependent, yielding a target size of 298,000 daltons, in situ. We propose that the purified glucose transporter reconstituted into liposomes is a tetramer comprised of 85,000-dalton subunits.

  10. Summary of intensive monitoring for radionuclides in fishery products after Fukushima accident and comparison to the results of long term monitoring program in Japan

    Energy Technology Data Exchange (ETDEWEB)

    Fujimoto, Ken; Morita, Takami; Shigenobu, Yuya; Takagi, Kaori; Miki, Shizuho; Kaeriyama, Hideki; Ambe, Daisuke; Ono, Tsuneo [National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4, Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648 (Japan)

    2014-07-01

    Monitoring of artificial radionuclides in fishery products started in late 1950's and has over half a century history in Japan. Fisheries Agency (FA), Fisheries Research Agency (FRA) and prefectural organizations have been conducting the monitoring. The intensive monitoring of radioactive material in fishery products started in the late of March 2011 immediately after the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident for the coastal area faced to the Pacific Ocean in the eastern area of Japan and for the offshore area. The purpose of this monitoring is to avoid the distribution of fishery products containing radioactive cesium in concentration over the Japanese standard limit (Cs-134+Cs-137: 100 Bq/kg). Japanese monitoring data are opened to public on the FA's and FRA's web page. In this study, we resume the results of intensive monitoring started in 2011 and show the temporal change of concentration of radioactive cesium in fishery product. In Fukushima Prefecture, the ratio of samples exceeding the Japanese standard limit shows a steadily decreasing trend from 53% at Mar.- Jun. 2011 to below 3% at Jul. - Sep. 2013. In other prefectures, the ratio was 6.5% at Mar.- Jun. 2011 and fell gradually, and it has been below to 1% at Jul. - Sep. 2013. In presentation, we show the results of monitoring conducted in the coastal area and deeper water around Japan from long gamma ray measurements with ashed samples by high purified germanium gamma spectrometry. (authors)

  11. A stable latent reservoir for HIV-1 in resting CD4+ T lymphocytes in infected children

    Science.gov (United States)

    Persaud, Deborah; Pierson, Theodore; Ruff, Christian; Finzi, Diana; Chadwick, Karen R.; Margolick, Joseph B.; Ruff, Andrea; Hutton, Nancy; Ray, Stuart; Siliciano, Robert F.

    2000-01-01

    HIV-1 persists in a latent state in resting CD4+ T lymphocytes of infected adults despite prolonged highly active antiretroviral therapy (HAART). To determine whether a latent reservoir for HIV-1 exists in infected children, we performed a quantitative viral culture assay on highly purified resting CD4+ T cells from 21 children with perinatally acquired infection. Replication-competent HIV-1 was recovered from all 18 children from whom sufficient cells were obtained. The frequency of latently infected resting CD4+ T cells directly correlated with plasma virus levels, suggesting that in children with ongoing viral replication, most latently infected cells are in the labile preintegration state of latency. However, in each of 7 children who had suppression of viral replication to undetectable levels for 1–3 years on HAART, latent replication-competent HIV-1 persisted with little decay, owing to a stable reservoir of infected cells in the postintegration stage of latency. Drug-resistance mutations generated by previous nonsuppressive regimens persisted in this compartment despite more than 1 year of fully suppressive HAART, rendering untenable the idea of recycling drugs that were part of failed regimens. Thus the latent reservoir for HIV-1 in resting CD4+ T cells will be a major obstacle to HIV-1 eradication in children. PMID:10749578

  12. Nano-Objects and Ions in Liquid Crystals: Ion Trapping Effect and Related Phenomena

    Directory of Open Access Journals (Sweden)

    Yuriy Garbovskiy

    2015-11-01

    Full Text Available The presence of ions in liquid crystals is one of the grand challenges that hinder the application of liquid crystals in various devices, which include advanced 3-D and flexible displays, tunable lenses, etc. Not only do they compromise the overall performance of liquid crystal devices, ions are also responsible for slow response, image sticking, and image flickering, as well as many other negative effects. Even highly purified liquid crystal materials can get contaminated during the manufacturing process. Moreover, liquid crystals can degrade over time and generate ions. All of these factors raise the bar for their quality control, and increase the manufacturing cost of liquid crystal products. A decade of dedicated research has paved the way to the solution of the issues mentioned above through merging liquid crystals and nanotechnology. Nano-objects (guests that are embedded in the liquid crystals (hosts can trap ions, which decreases the ion concentration and electrical conductivity, and improves the electro-optical response of the host. In this paper, we (i review recently published works reporting the effects of nanoscale dopants on the electrical properties of liquid crystals; and (ii identify the most promising inorganic and organic nanomaterials suitable to capture ions in liquid crystals.

  13. The evolutionary consequences of blood-stage vaccination on the rodent malaria Plasmodium chabaudi.

    Directory of Open Access Journals (Sweden)

    Victoria C Barclay

    Full Text Available Malaria vaccine developers are concerned that antigenic escape will erode vaccine efficacy. Evolutionary theorists have raised the possibility that some types of vaccine could also create conditions favoring the evolution of more virulent pathogens. Such evolution would put unvaccinated people at greater risk of severe disease. Here we test the impact of vaccination with a single highly purified antigen on the malaria parasite Plasmodium chabaudi evolving in laboratory mice. The antigen we used, AMA-1, is a component of several candidate malaria vaccines currently in various stages of trials in humans. We first found that a more virulent clone was less readily controlled by AMA-1-induced immunity than its less virulent progenitor. Replicated parasites were then serially passaged through control or AMA-1 vaccinated mice and evaluated after 10 and 21 rounds of selection. We found no evidence of evolution at the ama-1 locus. Instead, virulence evolved; AMA-1-selected parasites induced greater anemia in naïve mice than both control and ancestral parasites. Our data suggest that recombinant blood stage malaria vaccines can drive the evolution of more virulent malaria parasites.

  14. Photosynthetic CO2 fixation in guard cells (GC)

    International Nuclear Information System (INIS)

    Gotow, K.; Taylor, S.; Zeiger, E.

    1987-01-01

    Recent studies indicate that carbon metabolism in GC is modulated by light quality. The fate of 14 CO 2 supplied to highly purified Vicia GC protoplasts irradiated with red light was investigated. The suspension was stirred at 25 0 C and dark-adapted for 5 min. After 5 min. in red light, 4.8 uCi of NaH 14 CO 3 was added (final concentration: 100 uM). Metabolism was quenched after 30 s with boiling ethanol. Anionic compounds were separated by 2D PC and TLC, and quantified. Rates of CO 2 fixation were 5- to 8-fold higher in the light. In the dark, malate and aspartate had 90% of the total label; in the light, 3-PGA, sugar monophosphates (SMP) and sugar diophosphates (SDP) had up to 60% of the label. Phosphates treatment and rechromatography of labelled SDP showed the presence of ribulose, a specific PCRP metabolite. In time-course experiments, labelled 3-PGA was detected within 5 s. With time, the percentage of label in 3-PGA decreased and that in SMP increased. The authors conclude that 3-PGA is a primary carboxylation product of the PCRP in GC and that the activity of the PCRP and PEP-carboxylase is metabolically regulated

  15. Altered cell mechanics from the inside: dispersed single wall carbon nanotubes integrate with and restructure actin.

    Science.gov (United States)

    Holt, Brian D; Shams, Hengameh; Horst, Travis A; Basu, Saurav; Rape, Andrew D; Wang, Yu-Li; Rohde, Gustavo K; Mofrad, Mohammad R K; Islam, Mohammad F; Dahl, Kris Noel

    2012-05-23

    With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs) are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  16. Carbon Catabolite Repression and the Related Genes of ccpA, ptsH and hprK in Thermoanaerobacterium aotearoense.

    Directory of Open Access Journals (Sweden)

    Muzi Zhu

    Full Text Available The strictly anaerobic, Gram-positive bacterium, Thermoanaerobacterium aotearoense SCUT27, is capable of producing ethanol, hydrogen and lactic acid by directly fermenting glucan, xylan and various lignocellulosically derived sugars. By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG. However, the xylose and mannose consumptions were not markedly affected by 2-DG at the concentration of one-tenth of the metabolizable sugar. Accordingly, T. aotearoense SCUT27 could consume xylose and mannose in the presence of glucose. The carbon catabolite repression (CCR related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization. The highly purified Histidine-containing Protein (HPr could be specifically phosphorylated at Serine 46 by HPr kinase/phosphatase (HPrK/P with no need to add fructose-1,6-bisphosphate (FBP or glucose-6-phosphate (Glc-6-P in the reaction mixture. The specific protein-interaction of catabolite control protein A (CcpA and phosphorylated HPr was proved via affinity chromatography in the absence of formaldehyde. The equilibrium binding constant (KD of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR analysis, indicating the high affinity between these two proteins.

  17. Novel purification method of human immunoglobulin by using a thermo-responsive protein A.

    Science.gov (United States)

    Koguma, Ichiro; Yamashita, Shuntaro; Sato, Satoshi; Okuyama, Kazuo; Katakura, Yoshinori

    2013-08-30

    We attempted to evaluate a novel purification method of immunoglobulins (IgGs) by using a mutant type of protein A. Although this mutant protein A binds to IgGs at 5°C, the IgGs are released at 40°C; hence, it was designated as thermo-responsive protein A (TRPA). We aimed to purify IgG1 from the culture supernatant of CHO cells producing AE6F4 human monoclonal IgG1. AE6F4 IgG1 was purified using only a TRPA-filled column and by modifying the temperature, without any exposure to acidic conditions. Furthermore, the purified AE6F4 IgG1 maintained the inherent binding affinity to antigen, while this property was lost in AE6F4 IgG1 purified using a conventional protein A (CPA) column possibly because of product aggregation and fragmentation. These data suggest that IgG is sensitive to acid treatment; however, it can be highly purified with retention of high affinity by using a TRPA column. Further, this purification method can be used on an industrial scale for the purification of antibody drugs. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Facile N-oxygenation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by the flavin-containing monooxygenase. A convenient synthesis of tritiated [methyl-3H]-4-phenyl-2,3-dihydropyridinium species

    International Nuclear Information System (INIS)

    Cashman, J.R.

    1988-01-01

    A rapid, efficient procedure useful for the radiosynthesis of [Me- 3 H]-MPDP+ ([methyl- 3 H]-4-phenyl-2,3-dihydropyridinium species) is described. Hog liver microsomes or the highly purified flavin-containing monooxygenase from hog liver quantitatively biotransforms [Me- 3 H]-MPTP to its corresponding radiolabeled N-oxide. For the small-scale synthesis required for radiolabeling procedures, this enzymatic process is superior to H 2 O 2 -mediated N-oxygenation of MPTP. In the presence of 0.5 mM NADPH, 4.5 mM n-octylamine, and 2 microCi [Me- 3 H]-MPTP, the only product detected in extracts from incubations performed with hog liver microsomes or purified hog liver flavin-containing monooxygenase is [Me- 3 H]-MPTP N-oxide. [Me- 3 H]-MPTP N-oxide is almost completely converted to [Me- 3 H]-MPDP+ by the action of trifluoroacetic anhydride. This procedure has the advantage of using a commercially available tritiated starting material, efficient transformations, and easily accomplished purification to afford a rapid synthesis of [Me- 3 H]-MPDP+

  19. Phosphorylation states of the (Na+ + K+)-transporting ATPase in preparations from lamb kidney and electric-eel (Electophorus electricus) electric organ.

    Science.gov (United States)

    Harris, W E; Stahl, W L

    1984-01-01

    Phosphorylation states of the (Na+ + K+)-transporting ATPase were studied in highly purified preparations isolated from electric-eel electric organ and from lamb kidney. The steady-state level of phosphorylated lamb kidney enzyme, obtained by reaction with [gamma-32P]ATP, was not appreciably reduced in the presence of ADP unless oligomycin was present. The phosphorylated form of the electric-eel electric-organ enzyme was reduced by at least 95% under the same conditions, suggesting that the E1P state in the kidney enzyme is more transitory than that in electric organ. The level of phosphorylation from [32P]Pi was higher in the lamb kidney preparation than in the electric-organ preparation, and the difference in stimulation of phosphorylation by ouabain in the two preparations was striking. Ouabain increased the level of phosphorylation by 35% in the kidney preparation and 734% in the electric-organ preparation. The E2P state seems to be stabilized by ouabain in the latter preparation. These findings, as well as the different reactivities of the thiol groups to blocking reagents in these preparations, suggest that the tertiary structure in the enzyme isolated from these two sources is different. PMID:6324756

  20. Generation of tunable glycosaminoglycan hydrogels to mimic extracellular matrices.

    Science.gov (United States)

    Herrero-Mendez, A; Palomares, T; Castro, B; Herrero, J; Alonso-Varona, A

    2016-12-01

    Biomaterials and, especially, scaffolds may function as temporary extracellular matrix (ECM), mimicking in vivo environmental structures and facilitating cell growth and tissue regeneration. ECM is composed mostly of glycosaminoglycans (GAGs) and proteins, the ratio of GAGs, hyaluronic acid (HA):sulphated GAGs (sGAGs) being characteristic of each type of tissue. Umbilical cord (UC) and particularly Wharton's jelly (WJ) have been proposed as good sources for obtaining GAGs. In this work, we present a novel methodology for the extraction, purification and separation of GAGs from UC obtained from two different species, human and pig. The new methodology is based on enzymatic digestion of WJ, precipitation of GAGs with organic solvents, purification steps and chromatographic separation of GAGs using ion exchange columns. This novel process allows highly purified HA and sGAGs to be obtained from human and pig WJ. The composition of sGAGs and molecular weight of HA were very similar in the two species and GAGs are haemocompatible and non-cytotoxic. Finally, these new biomaterials have significant bioactive properties, increasing proliferation rates of two cell lines, human adipose mesenchymal stem cells (ASCs) and fibroblasts. In summary, the separation of HA and sGAGs, linked to the improvement in the GAG quantification method described in this paper, opens new avenues for the formulation of natural biomaterials with various ratios of GAGs, mimicking tissue matrix for different tissue-engineering applications. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  1. DNA polymerase betas from liver and testes of cherry salmon, Oncorhynchus masou: purification and characterization of DNA polymerase betas with acidic isoelectric points.

    Science.gov (United States)

    Yamaguchi, T; Nishimura, S; Takahashi, K; Yoshikuni, M; Masaki, J; Hirai, T; Saneyoshi, M

    1996-01-01

    DNA polymerase betas from cherry salmon, Oncorhynchus masou, liver and testes were purified to near homogeneity, and no substantial differences between the enzymes were observed. The molecular weight of both enzymes, determined by SDS-polyacrylamide gel electrophoresis, was 39,000. The amino acid sequences of the N-terminus of the liver and testes enzymes were determined and compared with that of the rat enzyme. Of the N-terminal 30 amino acid residues of salmon liver DNA polymerase beta, 21 (70%) were identical to those of the rat enzyme sequence. However, unlike most eukaryotic DNA polymerase betas, the isoelectric points (pIs) of the DNA polymerase betas from salmon liver and testes were both estimated to be 6.2, which is significantly different from the alkaline isoelectric points (pI = 8.5-9.5) established for other highly purified vertebrate DNA polymerase betas. The cherry salmon DNA polymerase betas were still active at below 10 degrees C, compared with the rat enzyme.

  2. Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells.

    Science.gov (United States)

    Armstrong, Lynne; Medford, Andrew R L; Uppington, Kay M; Robertson, John; Witherden, Ian R; Tetley, Teresa D; Millar, Ann B

    2004-08-01

    The recognition of potentially harmful microorganisms involves the specific recognition of pathogen-associated molecular patterns (PAMPs) and the family of Toll-like receptors (TLRs) is known to play a central role in this process. TLR-4 is the major recognition receptor for lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, whereas TLR-2 responds to bacterial products from gram-positive organisms. Although resident alveolar macrophages are the first line of defense against microbial attack, it is now understood that the alveolar epithelium also plays a pivotal role in the innate immunity of the lung. The purpose of the current study was to determine whether human primary type II alveolar epithelial cells (ATII) express functional TLR-2 and TLR-4 and how they may be regulated by inflammatory mediators. We have used reverse transcriptase-polymerase chain reaction and flow cytometry to determine basal and inducible expression on ATII. We have used highly purified preparations of the gram-positive bacterial product lipoteichoic acid (LTA) and LPS to look at the functional consequences of TLR-2 and TLR-4 ligation, respectively, in terms of interleukin-8 release. We have shown that human primary ATII cells express mRNA and protein for both TLR-2 and TLR-4, which can be modulated by incubation with LPS and tumor necrosis factor. Furthermore, we have demonstrated that these receptors are functional. This suggests that ATII have the potential to contribute significantly to the host defense of the human alveolus against bacteria.

  3. Effect of light on respiration and development of photosynthetic cells. Progress report, September 1, 1975--June 1, 1976

    Energy Technology Data Exchange (ETDEWEB)

    Gibbs, M

    1976-06-01

    Adaptation and deadaptation of unicellular algal cells to a hydrogen metabolism may involve not only hydrogenase but also the site of entry of electrons from NADH. NADPH can also serve as an electron donor in a chloroplast preparation fortified with hydrogenase but the rate of photoevolution falls off rapidly perhaps due to competition with NADP for electrons. The evolution of H/sub 2/ with reduced pyridine nucleotide as electron donor is coupled to ATP formation. Evidence is presented that soluble ferredoxin may not be the immediate donor of H/sub 2/. Hydrogenases from Chlamydomonas and Scenedesmus have been highly purified and will be used to test the stoichiometry of NADH disappearance, ATP and H/sub 2/ formation and also whether H/sub 2/-photoevolution in contrast to dark evolution is ferredoxin dependent. Studies with intact Chlamydomonas provide evidence that a reduced carbon compound and not water is the source of H/sub 2/. The presence of acetate eliminates the CO/sub 2/ production. The ratio of CO/sub 2/:H/sub 2/ is 1:2 and evolution is blocked by inhibitors of glycolysis. Acetate has a strong effect on H/sub 2/O. Cells starved over a long period still retain photoreduction but will not evolve H/sub 2/ until glucose is added.

  4. Mechanisms of cholesterol and saturated fatty acid lowering by Quillaja saponaria extract, studied by in vitro digestion model.

    Science.gov (United States)

    Vinarova, Liliya; Vinarov, Zahari; Damyanova, Borislava; Tcholakova, Slavka; Denkov, Nikolai; Stoyanov, Simeon

    2015-04-01

    Quillaja saponin extracts are known to reduce plasma cholesterol levels in humans. Here we study the mechanism of this effect with Quillaja Dry saponin extract (QD). In vitro model of triglyceride lipolysis is used to quantify the effect of QD on the solubilization of cholesterol and of the lipolysis products (fatty acids and monoglycerides) in the dietary mixed micelles (DMM). We found that QD extract decreases significantly both the cholesterol (from 80% to 20%) and saturated fatty acids (SFA, from 70% to 10%) solubilised in DMM. Series of dedicated experiments prove that QD may act by two mechanisms: (1) direct precipitation of cholesterol and (2) displacement of cholesterol from the DMM. Both mechanisms lead to increased cholesterol precipitation and, thus, render cholesterol bio-inaccessible. We prove also that the saponin molecules are not the active component of QD, because highly purified Quillaja extract with very similar saponin composition does not exhibit cholesterol-lowering or SFA-lowering effect. The effect of QD extract on cholesterol solubilisation is most probably caused by the high-molecular weight polyphenol molecules, present in this extract. The reduced SFA solubilisation is caused by Ca(2+) ions of relatively high concentration (1.25 wt%), also present in QD extract, which precipitate the fatty acids into calcium soaps.

  5. The Oligomeric Form of the Escherichia coli Dps Protein Depends on the Availability of Iron Ions

    Directory of Open Access Journals (Sweden)

    Sergey Antipov

    2017-11-01

    Full Text Available The Dps protein of Escherichia coli, which combines ferroxidase activity and the ability to bind DNA, is effectively used by bacteria to protect their genomes from damage. Both activities depend on the integrity of this multi-subunit protein, which has an inner cavity for iron oxides; however, the diversity of its oligomeric forms has only been studied fragmentarily. Here, we show that iron ions stabilize the dodecameric form of Dps. This was found by electrophoretic fractionation and size exclusion chromatography, which revealed several oligomers in highly purified protein samples and demonstrated their conversion to dodecamers in the presence of 1 mM Mohr’s salt. The transmission electron microscopy data contradicted the assumption that the stabilizing effect is given by the optimal core size formed in the inner cavity of Dps. The charge state of iron ions was evaluated using Mössbauer spectroscopy, which showed the presence of Fe3O4, rather than the expected Fe2O3, in the sample. Assuming that Fe2+ can form additional inter-subunit contacts, we modeled the interaction of FeO and Fe2O3 with Dps, but the binding sites with putative functionality were predicted only for Fe2O3. The question of how the dodecameric form can be stabilized by ferric oxides is discussed.

  6. Selective ion-permeation through strained and charged graphene membranes

    Science.gov (United States)

    Li, Kun; Tao, Yi; Li, Zhongwu; Sha, Jingjie; Chen, Yunfei

    2018-01-01

    By means of molecular dynamics simulations and density functional theory calculations, we demonstrate that stretched and charged graphene can act as ion sieve membranes. It is observed that loading 30% strain on graphene can induce pores in the dense electron cloud to allow ions to pass through the aromatic rings. Meanwhile, a charged surface is helpful to peel the hydration layers from the ions and decrease the energy barrier for ion translocation through nanopores. Our results suggest that with a membrane charge density of 6.80 e nm‑2, Li+ can be highly purified from the mixed solution including Li+, K+, Na+ and Cl‑ ions. Further increasing the charge density to 15.78 e nm‑2 can obtain excellent Na+/K+ selectivity. The potential of mean force profiles of ion permeation reveal that the potential for each ion is quite different. By fine tuning membrane charge density, pristine monolayer graphene can act as ion sieves with both high permeability and high selectivity.

  7. Characterization and Small RNA Content of Extracellular Vesicles in Follicular Fluid of Developing Bovine Antral Follicles.

    Science.gov (United States)

    Navakanitworakul, Raphatphorn; Hung, Wei-Ting; Gunewardena, Sumedha; Davis, John S; Chotigeat, Wilaiwan; Christenson, Lane K

    2016-05-09

    Exosomes and microvesicles (i.e., extracellular vesicles: EVs) have been identified within ovarian follicular fluid and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, whether EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth remains unknown. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle.

  8. Biological activity evaluation of cloned and expressed caprine growth hormone from local Pakistani goat breed beetal

    International Nuclear Information System (INIS)

    Butt, H.I.; Shahzad, M.I.; Bashir, Q.

    2011-01-01

    Growth hormone cDNA of local goat breed-beetal was amplified by RT PCR and gene including leader sequence was cloned in pTZR57 cloning vector. The cGH-pTZR57 clone was confirmed by restriction digestion and sequence analyses before finally sub-cloning the gene in pND- a mammalian expression vector. The clones were again confirmed by restriction digestion and PCR analyses. Highly purified, supercoiled cGH-pND construct was used to transfect Vero cell lines for expression studies. The in vitro expression of cGH was checked by dot-ELISA technique. After confirming its in vitro cell line based expression, the construct was injected to 4 weeks old balb/c mice intramuscularly. Two animals were euthanized per week till four weeks to monitor the in vivo biological activity by evaluating the tibia epiphyseal width and body weight gain assays. Significant increase in tibia epiphyseal width and gain in body weight was observed from vaccinated animals. The study supports the concept that DNA based therapeutics are an efficient and cost effective method for gene delivery and in vivo transgene expressions. (author)

  9. Purification of uranium metal

    International Nuclear Information System (INIS)

    Suzuki, Kenji; Shikama, Tatsuo; Ochiai, Akira.

    1993-01-01

    We developed the system for purifying uranium metal and its metallic compounds and for growing highly pure uranium compounds to study their intrinsic physical properties. Uranium metal was zone refined under low contamination conditions as far as possible. The degree of the purity of uranium metal was examined by the conventional electrical resistivity measurement and by the chemical analysis using the inductive coupled plasma emission spectrometry (ICP). The results show that some metallic impurities evaporated by the r.f. heating and other usual metallic impurities moved to the end of a rod with a molten zone. Therefore, we conclude that the zone refining technique is much effective to the removal of metallic impurities and we obtained high purified uranium metal of 99.99% up with regarding to metallic impurities. The maximum residual resistivity ratio, the r.r.r., so far obtained was about 17-20. Using the purified uranium, we are attempting to grow a highly pure uranium-titanium single crystals. (author)

  10. Relation of structure to function for the US reference standard endotoxin after exposure to 60Co radiation

    International Nuclear Information System (INIS)

    Csako, G.; Suba, E.A.; Ahlgren, A.; Tsai, C.M.; Elin, R.J.

    1986-01-01

    The structure and function of the highly purified US reference standard endotoxin (RSE) were studied after exposure to ionizing radiation from a 60 Co source. With increasing doses of radiation, the trilaminar ribbon-like structure of untreated endotoxin exhibited focal swelling, after which only spherical particles were seen by electron microscopy. These morphological changes were paralleled by the respective loss of O-side chain repeating units and pieces of the R-core from the lipopolysaccharide molecules, as demonstrated by electrophoresis. The biologic function of the irradiated endotoxin was assessed with a variety of tests. At higher doses of radiation, a direct relation was observed between the degradation of the molecular and supramolecular structure and the loss of biologic function. At lower doses of radiation, however, there was variability among the functional assays in their rate of change with progressive irradiation of the RSE. The results suggest that the carbohydrate moiety plays an important role both in determining the supramolecular structure and in modulating certain biologic activities of bacterial endotoxins

  11. Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation.

    Science.gov (United States)

    Greco, Fabio; Ciana, Annarita; Pietra, Daniela; Balduini, Cesare; Minetti, Giampaolo; Torti, Mauro

    2006-03-01

    Recent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the beta2 subunit of the Na+/K+ -ATPase, as verified according to the recently proposed "beta-profiling test" [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+ -ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+ -activated RBCs, suggesting a possible role for this GTPase in membrane shedding.

  12. Tissue-specific mitotic bookmarking by hematopoietic transcription factor GATA1.

    Science.gov (United States)

    Kadauke, Stephan; Udugama, Maheshi I; Pawlicki, Jan M; Achtman, Jordan C; Jain, Deepti P; Cheng, Yong; Hardison, Ross C; Blobel, Gerd A

    2012-08-17

    Tissue-specific transcription patterns are preserved throughout cell divisions to maintain lineage fidelity. We investigated whether transcription factor GATA1 plays a role in transmitting hematopoietic gene expression programs through mitosis when transcription is transiently silenced. Live-cell imaging revealed that a fraction of GATA1 is retained focally within mitotic chromatin. ChIP-seq of highly purified mitotic cells uncovered that key hematopoietic regulatory genes are occupied by GATA1 in mitosis. The GATA1 coregulators FOG1 and TAL1 dissociate from mitotic chromatin, suggesting that GATA1 functions as platform for their postmitotic recruitment. Mitotic GATA1 target genes tend to reactivate more rapidly upon entry into G1 than genes from which GATA1 dissociates. Mitosis-specific destruction of GATA1 delays reactivation selectively of genes that retain GATA1 during mitosis. These studies suggest a requirement of mitotic "bookmarking" by GATA1 for the faithful propagation of cell-type-specific transcription programs through cell division. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.L.; Rajagopalan, K.V. (Duke Univ. Medical Center, Durham, NC (USA)); London, R.E. (National Institute of Environmental Health Science, Research Triangle Park, NC (USA))

    1989-09-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and {sup 31}P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.

  14. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Directory of Open Access Journals (Sweden)

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  15. Enhancement of acetate productivity in a thermophilic (55 °C) hollow-fiber membrane biofilm reactor with mixed culture syngas (H2/CO2) fermentation.

    Science.gov (United States)

    Wang, Yun-Qi; Yu, Shi-Jin; Zhang, Fang; Xia, Xiu-Yang; Zeng, Raymond J

    2017-03-01

    Conversion of organic wastes to syngas is an attractive way to utilize wastes. The produced syngas can be further used to produce a variety of chemicals. In this study, a hollow-fiber membrane biofilm reactor with mix cultures was operated at 55 °C to convert syngas (H 2 /CO 2 ) into acetate. A high concentration of acetate (42.4 g/L) was reached in batch experiment while a maximum acetate production rate of 10.5 g/L/day was achieved in the continuous-flow mode at hydraulic retention time (HRT) of 1 day. Acetate was the main product in both batch and continuous-flow experiments. n-Butyrate was the other byproduct in the reactor. Acetate accounted for more than 98.5 and 99.1% of total volatile fatty acids in batch and continuous modes, respectively. Illumina Miseq high-throughput sequencing results showed that microorganisms were highly purified and enriched in the reactor. The main genus was Thermoanaerobacterium (66% of relative abundance), which was usually considered as H 2 producer in the literature, however, likely played a role as a H 2 consumer in this study. This study provides a new method to generate the high producing rate and purity of acetate from syngas.

  16. Prions efficiently cross the intestinal barrier after oral administration: Study of the bioavailability, and cellular and tissue distribution in vivo

    Science.gov (United States)

    Urayama, Akihiko; Concha-Marambio, Luis; Khan, Uffaf; Bravo-Alegria, Javiera; Kharat, Vineetkumar; Soto, Claudio

    2016-01-01

    Natural forms of prion diseases frequently originate by oral (p.o.) infection. However, quantitative information on the gastro-intestinal (GI) absorption of prions (i.e. the bioavailability and subsequent biodistribution) is mostly unknown. The main goal of this study was to evaluate the fate of prions after oral administration, using highly purified radiolabeled PrPSc. The results showed a bi-phasic reduction of PrPSc with time in the GI, except for the ileum and colon which showed sustained increases peaking at 3–6 hr, respectively. Plasma and whole blood 125I-PrPSc reached maximal levels by 30 min and 3 hr, respectively, and blood levels were constantly higher than plasma. Upon crossing the GI-tract 125I-PrPSc became associated to blood cells, suggesting that binding to cells decreased the biological clearance of the agent. Size-exclusion chromatography revealed that oligomeric 125I-PrPSc were transported from the intestinal tract, and protein misfolding cyclic amplification showed that PrPSc in organs and blood retained the typical prion self-replicating ability. Pharmacokinetic analysis found the oral bioavailability of 125I-PrPSc to be 33.6%. Interestingly, 125I-PrPSc reached the brain in a quantity equivalent to the minimum amount needed to initiate prion disease. Our findings provide a comprehensive and quantitative study of the fate of prions upon oral infection. PMID:27573341

  17. Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

    Science.gov (United States)

    Njengele, Zikhona; Kleynhans, Ronel; Sayed, Yasien; Mosebi, Salerwe

    2016-12-01

    Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Structural requirements for the action of parathyroid hormone-related protein (PTHrP) on bone resorption by isolated osteoclasts

    Energy Technology Data Exchange (ETDEWEB)

    Evely, R.S.; Bonomo, A.; Schneider, H.G.; Moseley, J.M.; Gallagher, J.; Martin, T.J. (St. Vincent' s Institute of Medical Research, Melbourne (Australia))

    1991-01-01

    Parathyroid hormone-related protein (PTHrP) plays a major role in the syndrome of humoral hypercalcemia of malignancy (HHM) by its actions on bone and kidney. In this study an isolated osteoclast bone resorption assay was used to investigate the actions of this peptide and the structure-activity relationships for its resorption effect. As with PTH, neither synthetic nor recombinant PTHrP preparations stimulated resorption within highly purified osteoclast populations. Resorption was stimulated only in the presence of contaminating osteoblasts or in cocultures with the osteoblast-like cell line UMR-106. In the presence of osteoblasts PTHrP-(1-34) and PTHrP-(1-84) stimulated bone resorption in a dose-dependent manner with a potency comparable to that of PTH-(1-34) on a molar basis. The biologic activity of the PTHrP was shown to reside in the first 34 amino acids, and within that region the structural requirements for promotion of osteoclastic resorption resembled closely those for promotion of cyclic AMP formation in osteoblast-like cells. Using emulsion autoradiography with iodinated PTHrP-(1-34) and PTHrP-(1-84) on mixed bone cell preparations from neonatal rats, specific binding was demonstrated only to osteoblasts, not to osteoclasts. These results clearly demonstrate that PTHrP is a potent stimulator of bone resorption and that these effects are, like those of PTH, mediated by initial actions upon cells of the osteoblast lineage.

  19. Exit of pediatric pre-B acute lymphoblastic leukaemia cells from the bone marrow to the peripheral blood is not associated with cell maturation or alterations in gene expression

    Directory of Open Access Journals (Sweden)

    Wiebe Thomas

    2008-08-01

    Full Text Available Abstract Background Childhood pre-B acute lymphoblastic leukemia (ALL is a bone marrow (BM derived disease, which often disseminates out of the BM cavity, where malignant cells to a variable degree can be found circulating in the peripheral blood (PB. Normal pre-B cells are absolutely dependent on BM stroma for survival and differentiation. It is not known whether transformed pre-B ALL cells retain any of this dependence, which possibly could impact on drug sensitivity or MRD measurements. Results Pre-B ALL cells, highly purified by a novel method using surface expression of CD19 and immunoglobulin light chains, from BM and PB show a very high degree of similarity in gene expression patterns, with differential expression of vascular endothelial growth factor (VEGF as a notable exception. In addition, the cell sorting procedure revealed that in 2 out of five investigated patients, a significant fraction of the malignant cells had matured beyond the pre-B cell stage. Conclusion The transition of ALL cells from the BM into the circulation does not demand, or result in, major changes of gene expression pattern. This might indicate an independence of BM stroma on the part of transformed pre-B cells, which contrasts with that of their normal counterparts.

  20. Technology development for recovery of individual rare earth elements at high purity from Dong-Pao rare earth concentrated ore of Vietnam

    International Nuclear Information System (INIS)

    Hoang Nhuan; Le Ba Thuan; Luu Xuan Dinh; Tran Hoang Mai; Tran Thi Hong Thai; Yoshiuyki Aiba; Hiroaki Nishimura

    2015-01-01

    In this work, the research results on RE processing process at laboratory scale and pilot scale was reported and discussed. Experimental research on thermal decomposition and sulfate process of bastnaesite ore with sulfuric acid in electric furnace was carried out, the different roasting conditions, mass transfer rate, reactions and RE and/or non-RE behaviors during roasting and leaching were investigated. The roasting temperatures were 450 o C and 550 o C. With higher roasting temperature and longer roasting time, the RE recovery yield reduced. The RE recovery yield reached the highest (over 94%) at roasting temperature of 550 o C for 2 hrs. The different extracting conditions for separation of REEs were investigated in laboratory scale as well as pilot scale. At pilot scale, the separation of REEs was performed on 120-stage extraction system produced by Japan, using PC88A solvent dissolved in IP2028. The volume of each stage was 20 L. The results showed that REEs were separated from RE resource of Vietnam and individual RE elements such as La, Ce, Pr, and Nd were obtained at high purity. The parameters for each extraction stage were reported in this work. The results indicated that in order to obtain highly purified Nd (>99%), it needs to use an extraction system with higher stage number, about 200 stages. The extraction data at pilot scale of this investigation was used as basic data for calculating parameters for extraction system in industrial scale. (author)

  1. Synthesis, growth and characterization of organic nonlinear optical material: N-benzyl-2-methyl-4-nitroaniline (BNA)

    Science.gov (United States)

    Kalaivanan, R.; Srinivasan, K.

    2017-05-01

    Synthesis of the organic nonlinear optical compound N-benzyl-2-methyl-4-nitroaniline (BNA) was carried out in a newer chemical environment using the mixture of benzyl chloride and 2-methl-4-nitroaniline by a preferred laboratory synthesis process. The synthesized BNA compound was separated by column chromatography (CC) with low pressure silica gell using petrollium benzine and purity of the separated resultant product was confirmed by thin layer chromatography (TLC). Further, the material was recrystallized atleast four times in methanol and the highly purified BNA was used for the growth of single crystals from solutions with selected solvents by slow evaporation method at room temperature. Single crystals having natural growth morphology were harvested and their different growth faces were identified by optical goniometry. The grown crystals were subjected to different characterization techniques such as powder x-ray diffraction (PXRD), fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and UV-vis-Near IR spectroscopy. Further, the second harmonic generation (SHG) efficiency of the grown BNA crystal was studied by Kurtz and Perry powder technique using Nd:YAG laser as fundamental source and found to be twice that of inorganic standard KDP.

  2. [Pilot-scale purification of lipopeptide from marine-derived Bacillus marinus].

    Science.gov (United States)

    Gu, Kangbo; Guan, Cheng; Xu, Jiahui; Li, Shulan; Luo, Yuanchan; Shen, Guomin; Zhang, Daojing; Li, Yuanguang

    2016-11-25

    This research was aimed at establishing the pilot-scale purification technology of lipopeptide from marine-derived Bacillus marinus. We studied lipopeptide surfactivity interferences on scale-up unit technologies including acid precipitation, methanol extraction, solvent precipitation, salting out, extraction, silica gel column chromatography and HZ806 macroporous absorption resin column chromatography. Then, the unit technologies were combined in a certain order, to remove the impurities gradually, and to gain purified lipopeptide finally, with high recovery rate throughout the whole process. The novel pilot-scale purification technology could effectively isolate and purify lipopeptide with 87.51% to 100% purity in hectograms from 1 ton of Bacillus marinus B-9987 fermentation broth with more than 81.73% recovery rate. The first practical hectogram production of highly purified lipopeptide derived from Bacillus marinus was achieved. With this new purification method, using complex media became possible in fermentation process to reduce the fermentation cost and scale-up the purification for lipopeptide production. For practicability and economy, foaming problem resulting from massive water evaporation was avoided in this technology.

  3. Role of RNase MRP in viral RNA degradation and RNA recombination.

    Science.gov (United States)

    Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

    2011-01-01

    RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

  4. Development of homologous radioimmunoassays for equine growth hormone and equine prolactin and their application to the detection of circulating levels of hormone in horse plasma

    Energy Technology Data Exchange (ETDEWEB)

    Cahill, C.M.; Hayden, T.J. [University Coll., Dublin (Ireland); Ven der Kolk, H. [Rijksuniversiteit Utrecht (Netherlands); Goode, J.A. [Agricultural Research Council, Cambridge (United Kingdom). Inst. of Animal Physiology

    1994-12-31

    Highly purified and well-characterized preparations of equine prolactin and growth hormone from equine pituitary glands were employed to set up highly sensitive and specific homologous radioimmunoassays (RIA) for the measurement of hormone in horse plasma. The limit of sensitivity of the GH RIA was 1.2 ng/ml with mean intra -and inter- assay coefficients of variation (CV) of 6.6 and 10%, respectively. The sensitivity of the equine prolactin (ePRL) RIA was 0.5 ng/ml with mean intra and inter-assay CV of 9.1 and 15.6%, respectively. Dose-response curves of a crude pituitary gland extract and plasma samples collected from a mare and foal were parallel to the standards and the PRL RIA was clinically validated by administration of thyrotropin-releasing hormone (TRH). Plasma samples taken at 15 min intervals over 24 h from lactating mares gave 24 h mean GH values in the range 5.5 to 7.95 ng/ml. Large intermittent elevations of GH activity were detected. The mean 24 h PRL concentrations were between 3.2-10.4 ng/ml in the lactating animals, with higher concentrations earlier in lactation. Long episodic bursts of PRL were detected. (authors). 48 refs., 9 figs.

  5. Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic evidence supporting the Warburg theory of cancer.

    Science.gov (United States)

    Kiebish, Michael A; Han, Xianlin; Cheng, Hua; Chuang, Jeffrey H; Seyfried, Thomas N

    2008-12-01

    Otto Warburg first proposed that cancer originated from irreversible injury to mitochondrial respiration, but the structural basis for this injury has remained elusive. Cardiolipin (CL) is a complex phospholipid found almost exclusively in the inner mitochondrial membrane and is intimately involved in maintaining mitochondrial functionality and membrane integrity. Abnormalities in CL can impair mitochondrial function and bioenergetics. We used shotgun lipidomics to analyze CL content and composition in highly purified brain mitochondria from the C57BL/6J (B6) and VM/Dk (VM) inbred strains and from subcutaneously grown brain tumors derived from these strains to include an astrocytoma and ependymoblastoma (B6 tumors), a stem cell tumor, and two microgliomas (VM tumors). Major abnormalities in CL content or composition were found in all tumors. The compositional abnormalities involved an abundance of immature molecular species and deficiencies of mature molecular species, suggesting major defects in CL synthesis and remodeling. The tumor CL abnormalities were also associated with significant reductions in both individual and linked electron transport chain activities. A mathematical model was developed to facilitate data interpretation. The implications of our findings to the Warburg cancer theory are discussed.

  6. Biotransformation of catechin and extraction of active polysaccharide from green tea leaves via simultaneous treatment with tannase and pectinase.

    Science.gov (United States)

    Baik, Joo Hyun; Shin, Kwang-Soon; Park, Yooheon; Yu, Kwang-Won; Suh, Hyung Joo; Choi, Hyeon-Son

    2015-08-30

    Green tea is a dietary source of bioactive compounds for human health. Enzymatic treatments induce the bioconversion of bioactive components, which can improve biological activities. In this study, we investigated the effect of simultaneous treatment with tannase and Rapidase on biotransformation of catechins and extraction of polysaccharide from green tea extract (GTE). Tannase and pectinase treatments induced the biotransformation of catechins and altered tea polysaccharide () content. The addition of GTE to the enzyme reaction resulted in a significant increase in degallated catechins, including gallic acid, a product of the tannase reaction (314.5-4076.0 µg mL(-1)) and a reduction in epigallocatechin gallate (EGCG). Biotransformation of catechins improved the radical scavenging activity of GTE. Pectinase treatment led to change of TPS composition in GTE by hydrolyzing polysaccharides. In addition, pectinase-driven hydrolysis in polysaccharides significantly increased TPS-induced Interleukin 6 (IL-6) production in macrophages. In particular, treatment of Rapidase (TPS-Ra) led to the highest IL-6 production among TPS samples, similar to treatment of highly purified pectinase (TPS-GTE), a positive control. Simultaneous processing with tannase and Rapidase can be an efficient method for the extraction of bioactive polysaccharides and biotransformation of catechins with enhanced radical scavenging activity from green tea. © 2014 Society of Chemical Industry.

  7. Laser material purification of neodymium. Hikari reiki seiseiho ni yoru neodymium no kojundoka

    Energy Technology Data Exchange (ETDEWEB)

    Ogawa, Y.; Ozaki, T.; Yoshimatsu, S. (National Research Institute for Metals, Tokyo (Japan)); Chiba, K.; Umeda, H.; Saeki, M. (Nippon Steel Corp., Tokyo (Japan))

    1991-05-20

    Selective photoexcitation and photoionization of neodymium atoms were studied basically by using laser. Also, using their properties, feasibility of laser material purification (LPM) technology in gaseous phase was presented. In the selective 3-step ionization, laser beam with two kinds of proper wave length causes resonance exitation of the target element, which goes up to the excitation level of the first step, and the element is ionized by the second laser beam and the third laser beam. The 3-step ionization scheme by a single wave length laser beam of 577.612nm was used for the ionization of Nd. Nd ionized selectively by laser beam was recovered as thin layer at the negative potential side of plane electrodes placed at both sides of the laser irradiation area. In the layer formed by the TPD technology with this scheme, it is possible to decrease impuritis such as Pr by 1/16 and to form highly purified thin layer. 13 refs., 12 figs., 1 tab.

  8. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR

    Directory of Open Access Journals (Sweden)

    Roman Wölfel

    2012-08-01

    Full Text Available Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  9. Characterization of recombinant glyoxylate reductase from thermophile Thermus thermophilus HB27.

    Science.gov (United States)

    Ogino, Hiroyasu; Nakayama, Hitoshi; China, Hideyasu; Kawata, Takuya; Doukyu, Noriyuki; Yasuda, Masahiro

    2008-01-01

    A glyoxylate reductase gene from the thermophilic bacterium Thermus thermophilus HB27 (TthGR) was cloned and expressed in Escherichia coli cells. The recombinant enzyme was highly purified to homogeneity and characterized. The purified TthGR showed thermostability up to 70 degrees C. In contrast, the maximum reaction condition was relatively mild (45 degrees C and pH 6.7). Although the kcat values against co-enzyme NADH and NADPH were similar, the Km value against co-enzyme NADH was approximately 18 times higher than that against NADPH. TthGR prefers NADPH rather than NADH as an electron donor. These results indicate that a phosphate group of a co-enzyme affects the binding affinity rather than the reaction efficiency, and TthGR demands appropriate amount of phosphate for a high activity. Furthermore, it was found that the half-lives of TthGR in the presence of 25% dimethyl sulfoxide and diethylene glycol were significantly longer than that in the absence of an organic solvent.

  10. Transcriptional Changes during Naturally Acquired Zika Virus Infection Render Dendritic Cells Highly Conducive to Viral Replication.

    Science.gov (United States)

    Sun, Xiaoming; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Einkauf, Kevin; Tse, Samantha; Ard, Kevin; Ciaranello, Andrea; Yawetz, Sigal; Sax, Paul; Rosenberg, Eric S; Lichterfeld, Mathias; Yu, Xu G

    2017-12-19

    Although dendritic cells are among the human cell population best equipped for cell-intrinsic antiviral immune defense, they seem highly susceptible to infection with the Zika virus (ZIKV). Using highly purified myeloid dendritic cells isolated from individuals with naturally acquired acute infection, we here show that ZIKV induces profound perturbations of transcriptional signatures relative to healthy donors. Interestingly, we noted a remarkable downregulation of antiviral interferon-stimulated genes and innate immune sensors, suggesting that ZIKV can actively suppress interferon-dependent immune responses. In contrast, several host factors known to support ZIKV infection were strongly upregulated during natural ZIKV infection; these transcripts included AXL, the main entry receptor for ZIKV; SOCS3, a negative regulator of ISG expression; and IDO-1, a recognized inducer of regulatory T cell responses. Thus, during in vivo infection, ZIKV can transform the transcriptome of dendritic cells in favor of the virus to render these cells highly conducive to ZIKV infection. Published by Elsevier Inc.

  11. Dissociation of Survival, Proliferation, and State Control in Human Hematopoietic Stem Cells.

    Science.gov (United States)

    Knapp, David J H F; Hammond, Colin A; Miller, Paul H; Rabu, Gabrielle M; Beer, Philip A; Ricicova, Marketa; Lecault, Véronique; Da Costa, Daniel; VanInsberghe, Michael; Cheung, Alice M; Pellacani, Davide; Piret, James; Hansen, Carl; Eaves, Connie J

    2017-01-10

    The role of growth factors (GFs) in controlling the biology of human hematopoietic stem cells (HSCs) remains limited by a lack of information concerning the individual and combined effects of GFs directly on the survival, Mitogenesis, and regenerative activity of highly purified human HSCs. We show that the initial input HSC activity of such a purified starting population of human cord blood cells can be fully maintained over a 21-day period in serum-free medium containing five GFs alone. HSC survival was partially supported by any one of these GFs, but none were essential, and different combinations of GFs variably stimulated HSC proliferation. However, serial transplantability was not detectably compromised by many conditions that reduced human HSC proliferation and/or survival. These results demonstrate the dissociated control of these three human HSC bio-responses, and set the stage for future improvements in strategies to modify and expand human HSCs ex vivo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. PDGFRα and CD51 mark human nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic progenitor cell expansion.

    Science.gov (United States)

    Pinho, Sandra; Lacombe, Julie; Hanoun, Maher; Mizoguchi, Toshihide; Bruns, Ingmar; Kunisaki, Yuya; Frenette, Paul S

    2013-07-01

    The intermediate filament protein Nestin labels populations of stem/progenitor cells, including self-renewing mesenchymal stem cells (MSCs), a major constituent of the hematopoietic stem cell (HSC) niche. However, the intracellular location of Nestin prevents its use for prospective live cell isolation. Hence it is important to find surface markers specific for Nestin⁺ cells. In this study, we show that the expression of PDGFRα and CD51 among CD45⁻ Ter119⁻ CD31⁻ mouse bone marrow (BM) stromal cells characterizes a large fraction of Nestin⁺ cells, containing most fibroblastic CFUs, mesenspheres, and self-renewal capacity after transplantation. The PDGFRα⁺ CD51 ⁺subset of Nestin⁺ cells is also enriched in major HSC maintenance genes, supporting the notion that niche activity co-segregates with MSC activity. Furthermore, we show that PDGFRα⁺ CD51⁺ cells in the human fetal BM represent a small subset of CD146⁺ cells expressing Nestin and enriched for MSC and HSC niche activities. Importantly, cultured human PDGFRα⁺ CD51⁺ nonadherent mesenspheres can significantly expand multipotent hematopoietic progenitors able to engraft immunodeficient mice. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified nonadherent cultures of niche cells may represent a useful novel technology to culture human hematopoietic stem and progenitor cells.

  13. Pre-Transplantation Blockade of TNF-α-Mediated Oxygen Species Accumulation Protects Hematopoietic Stem Cells.

    Science.gov (United States)

    Ishida, Takashi; Suzuki, Sachie; Lai, Chen-Yi; Yamazaki, Satoshi; Kakuta, Shigeru; Iwakura, Yoichiro; Nojima, Masanori; Takeuchi, Yasuo; Higashihara, Masaaki; Nakauchi, Hiromitsu; Otsu, Makoto

    2017-04-01

    Hematopoietic stem cell (HSC) transplantation (HSCT) for malignancy requires toxic pre-conditioning to maximize anti-tumor effects and donor-HSC engraftment. While this induces bone marrow (BM)-localized inflammation, how this BM environmental change affects transplanted HSCs in vivo remains largely unknown. We here report that, depending on interval between irradiation and HSCT, residence within lethally irradiated recipient BM compromises donor-HSC reconstitution ability. Both in vivo and in vitro we demonstrate that, among inflammatory cytokines, TNF-α plays a role in HSC damage: TNF-α stimulation leads to accumulation of reactive oxygen species (ROS) in highly purified hematopoietic stem/progenitor cells (HSCs/HSPCs). Transplantation of flow-cytometry-sorted murine HSCs reveals damaging effects of accumulated ROS on HSCs. Short-term incubation either with an specific inhibitor of tumor necrosis factor receptor 1 signaling or an antioxidant N-acetyl-L-cysteine (NAC) prevents TNF-α-mediated ROS accumulation in HSCs. Importantly, pre-transplantation exposure to NAC successfully demonstrats protective effects in inflammatory BM on graft-HSCs, exhibiting better reconstitution capability than that of nonprotected control grafts. We thus suggest that in vivo protection of graft-HSCs from BM inflammation is a feasible and attractive approach, which may lead to improved hematopoietic reconstitution kinetics in transplantation with myeloablative conditioning that inevitably causes inflammation in recipient BM. Stem Cells 2017;35:989-1002. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  14. High-throughput protein crystallography and drug discovery.

    Science.gov (United States)

    Tickle, Ian; Sharff, Andrew; Vinkovic, Mladen; Yon, Jeff; Jhoti, Harren

    2004-10-20

    Single crystal X-ray diffraction is the technique of choice for studying the interactions of small organic molecules with proteins by determining their three-dimensional structures; however the requirement for highly purified protein and lack of process automation have traditionally limited its use in this field. Despite these shortcomings, the use of crystal structures of therapeutically relevant drug targets in pharmaceutical research has increased significantly over the last decade. The application of structure-based drug design has resulted in several marketed drugs and is now an established discipline in most pharmaceutical companies. Furthermore, the recently published full genome sequences of Homo sapiens and a number of micro-organisms have provided a plethora of new potential drug targets that could be utilised in structure-based drug design programs. In order to take maximum advantage of this explosion of information, techniques have been developed to automate and speed up the various procedures required to obtain protein crystals of suitable quality, to collect and process the raw X-ray diffraction data into usable structural information, and to use three-dimensional protein structure as a basis for drug discovery and lead optimisation. This tutorial review covers the various technologies involved in the process pipeline for high-throughput protein crystallography as it is currently being applied to drug discovery. It is aimed at synthetic and computational chemists, as well as structural biologists, in both academia and industry, who are interested in structure-based drug design.

  15. Directed evolution of an endoinulinase from Talaromyces purpureogenus toward efficient production of inulooligosaccharides.

    Science.gov (United States)

    Afriat-Jurnou, Livnat; Cohen, Rami; Paluy, Irina; Ben-Adiva, Ran; Yadid, Itamar

    2018-02-01

    Inulinases are fructofuranosyl hydrolases that target the β-2,1 linkage of inulin and hydrolyze it into fructose, glucose and inulooligosaccharides (IOS), the latter are of growing interest as dietary fibers. Inulinases from various microorganisms have been purified, characterized and produced for industrial applications. However, there remains a need for inulinases with increased catalytic activity and better production yields to improve the hydrolysis process and fulfill the growing industrial demands for specific fibers. In this study, we used directed enzyme evolution to increase the yield and activity of an endoinulinase enzyme originated from the filamentous fungus Talaromyces purpureogenus (Penicillium purpureogenum ATCC4713). Our directed evolution approach yielded variants showing up to fivefold improvements in soluble enzyme production compared to the starting point which enabled high-yield production of highly purified recombinant enzyme. The distribution of the enzymatic reaction products demonstrated that after 24 h of incubation, the main product (57%) had a degree of polymerization of 3 (DP3). To the best of our knowledge, this is the first application of directed enzyme evolution to improve inulooligosaccharide production. The approach enabled the screening of large genetic libraries within short time frames and facilitated screening for improved enzymatic activities and properties, such as substrate specificity, product range, thermostability and pH optimum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

  16. Photosynthesis of hydrogen and methane as key components for clean energy system

    Directory of Open Access Journals (Sweden)

    Seng Sing Tan et al

    2007-01-01

    Full Text Available While researchers are trying to solve the world's energy woes, hydrogen is becoming the key component in sustainable energy systems. Hydrogen could be produced through photocatalytic water-splitting technology. It has also been found that hydrogen and methane could be produced through photocatalytic reduction of carbon dioxide with water. In this exploratory study, instead of coating catalysts on a substrate, pellet form of catalyst, which has better adsorption capacity, was used in the photo-reduction of carbon dioxide with water. In the experiment, some water was first absorbed into titanium dioxide pellets. Highly purified carbon dioxide gas was then discharged into a reactor containing these wet pellets, which were then illuminated continuously using UVC lamps. Gaseous samples accumulated in the reactor were extracted at different intervals to analyze the product yields. The results confirmed that methane and hydrogen were photosynthesized using pellet form of TiO2 catalysts. Hydrogen was formed at a rate as high as 0.16 micromoles per hour (μmol h−1. The maximum formation rate of CH4 was achieved at 0.25 μmol h−1 after 24 h of irradiation. CO was also detected.

  17. A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum

    Directory of Open Access Journals (Sweden)

    Lorena Vázquez-Iglesias

    2017-06-01

    Full Text Available Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

  18. Functional role of zinc in poly(A) synthesis catalyzed by nuclear poly(A) polymerase

    International Nuclear Information System (INIS)

    Rose, K.M.; Allen, M.S.; Crawford, I.L.; Jacob, S.T.; Pennsylvania State Univ., Hershey; Texas Univ., Dallas; Texas Univ., Dallas

    1978-01-01

    The functional role of transition metals in poly(A) synthesis was elucidated by investigating the effect of the metal chelator o-phenanthroline on purified nuclear poly(A) polymerase. This chelator inhibited the enzyme activity in a manner competitive with respect to the polynucleotide primer concentration. o-phenanthroline was a non-competitive inhibitor with regard to ATP concentration and an 'uncompetitive' inhibitor with regard to dithiothreitor levels. The metal content of the purified enzyme preparations from rat liver and Morris hepatoma 3924A was determined using atomic absorption spectrometry. Of the transition metals measured, only zinc was present in detectable quantities, at levels less than 1 mol/mol of enzyme. Hepatoma enzyme contained 2-3 times as much zinc as the corresponding liver enzyme. Hepatoma poly(A) polymerase was also radioactively labelled in vivo by injection of tumor-bearing animals with 65 Zn. Dialysis experiments with highly purified radiolabelled poly(A) polymerase showed that the enzyme-zinc complex was labile and that a reduction in 65 Zn content correlated with a loss in enzyme activity. (orig./AJ) [de

  19. Activated human neonatal CD8+ T cells are subject to immunomodulation by direct TLR2 or TLR5 stimulation.

    LENUS (Irish Health Repository)

    McCarron, Mark

    2012-02-01

    In conditions of optimal priming, the neonate possesses competency to mount quantitatively adult-like responses. Vaccine formulations containing sufficiently potent adjuvants may overcome the neonate\\'s natural tendency for immunosuppression and provoke a similarly robust immune response. TLR expression on T cells represents the possibility of directly enhancing T cell immunity. We examined the ex vivo responsiveness of highly purified human cord blood-derived CD8(+) T cells to direct TLR ligation by a repertoire of TLR agonists. In concert with TCR stimulation, only Pam(3)Cys (palmitoyl-3-Cys-Ser-(Lys)(4)) and flagellin monomers significantly enhanced proliferation, CD25(+) expression, IL-2, IFN-gamma, TNF-alpha, and intracellular granzyme B expression. TLR2 and TLR5 mRNA was detected in the CD8(+) T cells. Blocking studies confirmed that the increase in IFN-gamma production was by the direct triggering of surface TLR2 or TLR5. The simultaneous exposure of CD8(+) T cells to both TLR agonists had an additive effect on IFN-gamma production. These data suggest that a combination of the two TLR ligands would be a potent T cell adjuvant. This may represent a new approach to TLR agonist-based adjuvant design for future human neonatal vaccination strategies requiring a CD8(+) component.

  20. Identification of luteolin as enterovirus 71 and coxsackievirus A16 inhibitors through reporter viruses and cell viability-based screening.

    Science.gov (United States)

    Xu, Lin; Su, Weiheng; Jin, Jun; Chen, Jiawen; Li, Xiaojun; Zhang, Xuyuan; Sun, Meiyan; Sun, Shiyang; Fan, Peihu; An, Dong; Zhang, Huafei; Zhang, Xiguang; Kong, Wei; Ma, Tonghui; Jiang, Chunlai

    2014-07-17

    Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.

  1. Method of separating short half-life radionuclides from a mixture of radionuclides

    Science.gov (United States)

    Bray, L.A.; Ryan, J.L.

    1999-03-23

    The present invention is a method of removing an impurity of plutonium, lead or a combination thereof from a mixture of radionuclides that contains the impurity and at least one parent radionuclide. The method has the steps of (a) insuring that the mixture is a hydrochloric acid mixture; (b) oxidizing the acidic mixture and specifically oxidizing the impurity to its highest oxidation state; and (c) passing the oxidized mixture through a chloride form anion exchange column whereupon the oxidized impurity absorbs to the chloride form anion exchange column and the {sup 229}Th or {sup 227}Ac ``cow`` radionuclide passes through the chloride form anion exchange column. The plutonium is removed for the purpose of obtaining other alpha emitting radionuclides in a highly purified form suitable for medical therapy. In addition to plutonium, lead, iron, cobalt, copper, uranium, and other metallic cations that form chloride anionic complexes that may be present in the mixture are removed from the mixture on the chloride form anion exchange column. 8 figs.

  2. Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.

    Science.gov (United States)

    Flannery, J G; Zolotukhin, S; Vaquero, M I; LaVail, M M; Muzyczka, N; Hauswirth, W W

    1997-06-24

    We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

  3. Radioreceptor assay for epidermal growth factor

    International Nuclear Information System (INIS)

    Ladda, R.L.; Bullock, L.P.; Gianopoulos, T.; McCormick, L.

    1979-01-01

    An established cell line of human lung fibroblasts with a high number of surface receptors for mouse epidermal growth factor (mEGF) was used to deveop a simple and highly sensitive radioreceptor assay for EGF. 125 I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF

  4. Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis. VII. Hemoglobin does not inhibit clearance of Escherichia coli from the peritoneal cavity

    International Nuclear Information System (INIS)

    Dunn, D.L.; Barke, R.A.; Lee, J.T. Jr.; Condie, R.M.; Humphrey, E.W.; Simmons, R.L.

    1983-01-01

    Hemoglobin has been shown to be a potent adjuvant in experimental Escherichia coli peritonitis, although a satisfactory mechanistic rationale is still obscure. Hemoglobin has been thought to impair intraperitoneal neutrophil function, delay clearance of bacteria from the peritoneal cavity by the normal absorptive mechanisms, or directly enhance bacterial growth. Using highly purified stroma-free hemoglobin (SFHgb), we have largely discounted any direct effect of hemoglobin on peritoneal white blood cell function. In the present study, we confirmed that uncontrolled proliferation of bacteria takes place in the presence of hemoglobin in the peritoneal cavity. Nonviable 5-iododeoxyuridine 125 I-labelled bacteria were then used to directly study peritoneal clearance kinetics, eliminating the problem of bacterial growth. SFHgb had no influence on the removal of intraperitoneal bacteria. The rate of bloodstream appearance of radiolabel was similar with or without intraperitoneal SFHgb. Thus, SFHgb does not prevent clearance of bacteria from the peritoneal cavity by interfering with normal host clearance mechanisms. SFHgb may act as a bacterial growth adjuvant, either by serving as a bacterial nutrient or by suitably modifying the environment so that extensive bacterial proliferation can occur. The latter hypothesis appears to be an area in which investigation concerning the adjuvant effect of hemoglobin may prove most fruitful

  5. Monoclonal antibodies that bind the renal Na/sup +//glucose symport system. 1. Identification

    Energy Technology Data Exchange (ETDEWEB)

    Wu, J.S.R.; Lever, J.E.

    1987-09-08

    Phlorizin is a specific, high-affinity ligand that binds the active site of the Na/sup +//glucose symporter by a Na/sup +/-dependent mechanism but is not itself transported across the membrane. The authors have isolated a panel of monoclonal antibodies that influence high-affinity, Na/sup +/-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK/sub 1/, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG/sub 2b/, reproducibly stimulated Na/sup +/-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na/sup +/-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na/sup +//glucose symporter.

  6. Nanotechnology: MEMS and NEMS and their applications to smart systems and devices

    Science.gov (United States)

    Varadan, Vijay K.

    2003-10-01

    civil strutures and food and medical industries. This unique combination of technologies also results in novel conformal sensors that can be remotely sensed by an antenna system with the advantage of no power requirements at the sensor site. This paper provides a brief review of MEMS and NEMS based smart systems for various applications mentioned above. Carbon Nano Tubes (CNT) with their unique structure, have already proven to be valuable in their application as tips for scanning probe microscopy, field emission devices, nanoelectronics, H2-storage, electromagnetic absorbers, ESD, EMI films and coatings and structural composites. For many of these applications, highly purified and functionalized CNT which are compatible with many host polymers are needed. A novel microwave CVD processing technique to meet these requirements has been developed at Penn State Center for the Engineering of Electronic and Acoustic Materials and Devices (CEEAMD). This method enables the production of highly purified carbon nano tubes with variable size (from 5 - 40 nm) at low cost (per gram) and high yield. Whereas, carbon nano tubes synthesized using the laser ablation or arc discharge evaporation method always include impurity due to catalyst or catalyst support. The Penn State research is based on the use of zeolites over other metal/metal oxides in the microwave field for a high production and uniformity of the product. An extended coventional purification method has been employed to purify our products in order to remove left over impurity. A novel composite structure can be tailored by functionalizing carbon nano tubes and chemically bonding them with the polymer matrix e.g. block or graft copolymer, or even cross-linked copolymer, to impart exceptional structural, electronic and surface properties. Bio- and Mechanical-MEMS devices derived from this hybrid composites will be presented.

  7. MEMS- and NEMS-based smart devices and systems

    Science.gov (United States)

    Varadan, Vijay K.

    2001-11-01

    structures and food and medical industries. This unique combination of technologies also results in novel conformal sensors that can be remotely sensed by an antenna system with the advantage of no power requirements at the sensor site. This paper provides a brief review of MEMS and NEMS based smart systems for various applications mentioned above. Carbon Nano Tubes (CNT) with their unique structure, have already proven to be valuable in their application as tips for scanning probe microscopy, field emission devices, nanoelectronics, H2-storage, electromagnetic absorbers, ESD, EMI films and coatings and structural composites. For many of these applications, highly purified and functionalized CNT which are compatible with many host polymers are needed. A novel microwave CVD processing technique to meet these requirements has been developed at Penn State Center for the engineering of Electronic and Acoustic Materials and Devices (CEEAMD). This method enables the production of highly purified carbon nano tubes with variable size (from 5-40 nm) at low cost (per gram) and high yield. Whereas, carbon nano tubes synthesized using the laser ablation or arc discharge evaporation method always include impurity due to catalyst or catalyst support. The Penn State research is based on the use of zeolites over other metal/metal oxides in the microwave field for a high production and uniformity of the product. An extended conventional purification method has been employed to purify our products in order to remove left over impurity. A novel composite structure can be tailored by functionalizing carbon nano tubes and chemically bonding them with the polymer matrix e.g. block or graft copolymer, or even cross- linked copolymer, to impart exceptional structural, electronic and surface properties. Bio- and Mechanical-MEMS devices derived from this hybrid composites will be presented.

  8. The effect of different molecular weight hyaluronan on macrophage physiology.

    Science.gov (United States)

    Krejcova, Daniela; Pekarova, Michaela; Safrankova, Barbora; Kubala, Lukas

    2009-01-01

    Hyaluronan, a linear glycosaminoglycan, is an abundant component of extracellular matrix. In its native form, the high-molar-mass hyaluronan polymers have an array of structural and regulatory, mainly anti-inflammatory and anti-angiogenic, functions. In contradiction, the biological effects of fragmented low molecular weight hyaluronan are suggested to be pro-angiogenic and pro-inflammatory. The effects of highly purified pharmacological grade hyaluronan of defined molecular weights 11, 52, 87, 250 and 970 kilodaltons were tested on mouse macrophage cell lines RAW 264.7 and MHS. The surface expression of CD44 and Toll-like receptor 2, surface receptors for hyaluronan, was determined by flow cytometry. Activation of macrophages was determined based on nitric oxide and tumour necrosis factor alpha production, inducible nitric oxide synthase expression, and the activation of the nuclear factor kappa B transcriptional factor. Both macrophage cell lines expressed CD44 and Toll-like receptor 2, which were significantly increased by the pre-treatment of macrophages with bacterial lipopolysaccharide. Hyaluronan of any molecular weight did not activate production of nitric oxide or tumour necrosis factor alpha in any mouse macrophage cell lines. Correspondingly, hyaluronan of any tested molecular weight did not stimulate nuclear factor kappa B activation. Similarly, hyaluronan of any molecular weight neither exerted stimulatory nor inhibitory effects on macrophages pre-treated by lipopolysaccharide. Interestingly, the data does not support the current view of low molecular weight hyaluronan as a pro-inflammatory mediator for macrophages. Further studies are necessary to clarify the effects of different molecular weight hyaluronan on phagocytes.

  9. Characterization of osteopontin-uranyl interaction: role of multiple phosphorylations

    International Nuclear Information System (INIS)

    Qi, Lei

    2014-01-01

    While some metals are essential for Life, other ones are only toxicants for living organisms, tolerated below well-definite concentrations. This is the case for uranium, a natural element which has no known biological function. It is a low α emitter and its chemical toxicity rather than its radiological toxicity is a subject of concern. Once in the body, this metal reaches the blood and accumulates in the bones under the action of unknown mechanisms. Uranium mainly exists in form of uranyl ion (UO 2 2+ ) in aqueous media and particularly reacts with carboxylates, phenolates and phosphates of the proteins. Previous studies have highlighted that UO 2 2+ modulates the SPP1 expression, a gene which codes for osteopontin (OPN). This highly phosphorylated glycoprotein plays an important role in bone homeostasis. This role and its biochemical properties led us to hypothesize that OPN might be a potential target of UO 2 2+ and involved in its accumulation in bones. A simple and original purification process was optimized to produce very highly purified OPN starting from human and bovine milk. Various biophysical approaches were set up and confirmed that both bovine and human OPN display very high affinity for UO 2 2+ . Moreover, the formation of stable UO 2 -protein complexes originating from structural changes was evidenced. The major role of phosphorylations, both on the OPN's affinity for UO 2 2+ and the stability of the UO 2 -protein complexes, was confirmed. These results demonstrate that OPN presents all the characteristics to be a major UO 2 2+ binding-protein in vitro, and they open new insights in the understanding of the UO 2 2+ mineralization process mechanisms. (author) [fr

  10. Comprehensive proteomic analysis of developing protein bodies in maize (Zea mays) endosperm provides novel insights into its biogenesis.

    Science.gov (United States)

    Wang, Guifeng; Wang, Gang; Wang, Jiajia; Du, Yulong; Yao, Dongsheng; Shuai, Bilian; Han, Liang; Tang, Yuanping; Song, Rentao

    2016-12-01

    Prolamins, the major cereal seed storage proteins, are sequestered and accumulated in the lumen of the endoplasmic reticulum (ER), and are directly assembled into protein bodies (PBs). The content and composition of prolamins are the key determinants for protein quality and texture-related traits of the grain. Concomitantly, the PB-inducing fusion system provides an efficient target to produce therapeutic and industrial products in plants. However, the proteome of the native PB and the detailed mechanisms underlying its formation still need to be determined. We developed a method to isolate highly purified and intact PBs from developing maize endosperm and conducted proteomic analysis of intact PBs of zein, a class of prolamine protein found in maize. We thus identified 1756 proteins, which fall into five major categories: metabolic pathways, response to stimulus, transport, development, and growth, as well as regulation. By comparing the proteomes of crude and enriched extractions of PBs, we found substantial evidence for the following conclusions: (i) ribosomes, ER membranes, and the cytoskeleton are tightly associated with zein PBs, which form the peripheral border; (ii) zein RNAs are probably transported and localized to the PB-ER subdomain; and (iii) ER chaperones are essential for zein folding, quality control, and assembly into PBs. We futher confirmed that OPAQUE1 (O1) cannot directly interact with FLOURY1 (FL1) in yeast, suggesting that the interaction between myosins XI and DUF593-containing proteins is isoform-specific. This study provides a proteomic roadmap for dissecting zein PB biogenesis and reveals an unexpected diversity and complexity of proteins in PBs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  11. Characterization of bacterium isolated from the sediment at coastal area of Omura Bay in Japan and several biological activities of pigment produced by this isolate.

    Science.gov (United States)

    Nakashima, Takuji; Kurachi, Maki; Kato, Yoko; Yamaguchi, Kenichi; Oda, Tatsuya

    2005-01-01

    Recently we discovered a bacterial strain (MS-02-063) that produces large amounts of red pigment from coastal area of Nagasaki Prefecture, Japan. Comparative 16S rDNA gene sequencing analysis revealed that strain MS-02-063 was phylogenetically closely related to gamma-proteobacterium Hahella sp. MBIC 3957 that produces prodigiosin. However, some physiological and biochemical differences between strain MS-02-063 and Hahella sp. MBIC 3957 were observed. The red pigment (RP-063) produced by this isolate was highly purified from the culture supernatant. It was speculated that RP-063 might be prodigiosin-like pigment in physical properties and biological activities such as antibacterial and cytotoxic activity. Antibacterial activity of RP-063 was examined by an agar dilution method. The results indicated that RP-063 showed antibacterial activity for specific for pathogenic gram-positive bacteria such as Staphylococcus aureus. The potency of antibacterial activity against S. aureus was nearly equal to those of tetracycline. Moreover, RP-063 showed inhibition of the superoxide generation by 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated mouse macrophage RAW 264.7 cell line. Prodigiosin members have a wide variety of biological properties, including anticancer and antimalarial, etc. Especially, potent immunosuppressive properties have been reported for prodigiosin members with the mechanism of action different from that of the other well known immunosuppressors in atopic dermatitis therapy such as cyclosporin A, FK506 and rapamycin. It is suggested that RP-063 may be able to arrest the inflammation caused by superantigens secreted from S. aureus, which colonized skin on atopic dermatitis as well as suppression of activated lymphocyte proliferation and superoxide generation from leucocytes.

  12. Effect of ultraviolet and x-ray radiation on optical properties of epoxy polymers dyed with organic phosphors

    Science.gov (United States)

    Laurinas, V. CH; Kasymov, S. S.; Yurov, V. M.; Eremin, E. N.; Vedyashkin, M. V.

    2017-01-01

    Highly purified industrial bisphenol and cycloaliphatic epoxy oligomers of ED-24 and UP-612 brands were used to produce optically transparent products. UV radiation of a low-pressure mercury lamp with 80% of the light energy at 254 nm was used to study photodegradation. X-ray apparatus with 0.7BSV- Ag tube was used as an ionizing radiation source to investigate the effect of X-rays on the spectra of organic dyes in epoxy polymer. The threshold value of the energy generated by ruby laser which indicated the degradation in the test samples recorded by light scattering method was determined to study radiation resistance of epoxy polymers. Basically, all the dyes exhibited high resistance to UV light. The observation of the absorption spectra showed that on average, a third of the dye molecules in the matrix experienced photobleaching within 200 hour exposure. The exception was coumarin 1, which was completely decolourized after 40 hours of exposure. X-ray irradiation of the samples for two hours results in the change in the optical density equivalent to that caused by 40 hour exposure to UV irradiation. However, in the first case, the matrix optical density is proportional to the irradiation time, and in the second case, it remains stable upon further UV irradiation. The comparison of photoaging processes in dyed and undyed epoxy polymers showed that the investigated organic dyes do not have a sensitizing effect on the matrix. The stability of the optical properties of the epoxy matrices exposed to the effects of different factors was found to depend on the nature of epoxy polymer and the technique of its production. The results of these effects are significantly different in the character of the change in the optical density and mechanisms of chemical transformations in polymer.

  13. A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.

    Science.gov (United States)

    Sonderegger, Christoph; Galgóczy, László; Garrigues, Sandra; Fizil, Ádám; Borics, Attila; Manzanares, Paloma; Hegedüs, Nikoletta; Huber, Anna; Marcos, Jose F; Batta, Gyula; Marx, Florentine

    2016-11-11

    Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15 N-/ 13 C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays. This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of

  14. Induction of mast cell accumulation, histamine release and skin edema by N49 phospholipase A2

    Directory of Open Access Journals (Sweden)

    Mo Ya-Zhen

    2009-04-01

    Full Text Available Abstract Background It has been recognized that phospholipase A2 (PLA2 is a crucial component of snake venom, which contributes greatly to snake venom induced inflammation in man. However, the mechanisms through which N49 PLA2 provoke inflammation remain unclear. Recently, a N49 PLA2, TM-N49 from Protobothrops mucrosquamatus crude venom was characterized in our laboratory. Since the purification procedure developed is able to supply us with relatively large quantity of highly purified TM-N49, we investigated the ability of TM-N49 in induction of inflammation. Results The results showed that TM-N49 provoked a dose dependent increase in microvascular leakage in the skin of rats. The potency of TM-N49 in induction of skin edema appeared similar potency of bradykinin and histamine. Pretreatment of rats with compound 48/80 diminished TM-N49 induced skin reaction and reduced mast cell numbers in rats. Ginkgolide B and cyproheptadine, but not terfenadine and quinacrine, inhibited TM-N49 elicited microvascular leakage when they were co-injected with the stimulus to rat skin. Moreover, TM-N49 was found to induce histamine release from human colon, lung and tonsil mast cells, and both metabolic inhibitors and pertussis toxin were capable of inhibiting TM-N49 elicited histamine release. TM-N49 induced mast cell accumulation in the peritoneum of mice, which was inhibited by co-injection of ginkgolide B, cyproheptadine and terfenadine. Intravenous injection of monoclonal antibodies against CD18, ICAM-1 and CD11a also blocked TM-N49 induced mast cell accumulation. Conclusion TM-N49 is a potent stimulus for skin edema, mast cell activation and accumulation.

  15. Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Meier, P.J.; Knickelbein, R.; Moseley, R.H.; Dobbins, J.W.; Boyer, J.L.

    1985-04-01

    To determine whether anion exchangers might play a role in hepatic bile formation, the authors looked for the presence of Cl/sup -/:OH/sup -/ and Cl/sup -/:HCO3/sup -/ exchange in highly purified canalicular (c) and basolateral (bl) rat liver plasma membrane (LPM) vesicles. In cLPM vesicles, a pH gradient stimulated /sup 36/Cl- uptake twofold above values obtained during pH-equilibrated conditions. When 50 mM HCO3/sup -/ was also present inside the vesicles, the same pH gradient resulted in Cl/sup -/ uptake to levels fourfold above pH- and HCO3--equilibrated controls and two- to threefold above Cl- equilibrium. Initial rates of both pH and HCO3/sup -/ gradient-stimulated Cl/sup -/ uptake were completely inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). A valinomycin-induced K/sup +/ diffusion potential (inside positive) also stimulated Cl/sup -/ uptake in cLPM, but this conductive Cl- pathway was insensitive to DIDS. The DIDS-sensitive, pH and HCO3- gradient-stimulated Cl/sup -/ uptake demonstrated: saturation with Cl/sup -/; partial inhibition by bumetanide (26%), furosemide (33%), probenecid (37%), and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (49%); cis-inhibition by chloride and nitrate but not by sulfate and various organic anions, and independence from the membrane potential. These data demonstrate the presence of an electroneutral Cl/sup -/:OH/sup -/ and Cl/sup -/:HCO3/sup -/ exchanger in rat liver canalicular membranes that favors Cl/sup -/:HCO3/sup -/ exchange. In contrast, no evidence was found for the presence of a Cl/sup -/:HCO3/sup -/ (OH/sup -/) exchange system in blLPM vesicles.

  16. [Value of Ginkgo biloba in treatment of Alzheimer dementia].

    Science.gov (United States)

    Loew, D

    2002-01-01

    During the last decade, there has been an explosive growth of research concerning the extract of Ginkgo biloba termed Egb 761. In experimental studies, animal studies and clinical studies Ginkgo biloba has shown a similar pharmacological potency and clinical efficacy like synthetic defined drugs in the therapy of reduced cerebral performance. Ginkgo biloba special extract Egb 761 is a standardized and highly purified extract of Ginkgo leaves. Among the active constituents are the ginkgo-flavone glycosides and the terpene-lactones (ginkgolides, bilobalide). The multifactorial principle of action of Ginkgo biloba is characterized by rheological and blood-flow-promoting properties, protective effects against ischaemia and hypoxia, effects on nerve cell energy metabolism, antioedematous and myelin-protective effects, radical-scavenger activity, effects on various cerebral transmitter and receptor systems. These action principles constitute the rationale for clinical trials in vascular dementia and primary degenerative dementia of the Alzheimer type, and in mixed forms of both. The cerebral bioavailability of Ginkgo biloba extract has been demonstrated by electroencephalography. In clinical trials of different working-groups, effects of Ginkgo biloba on the cognitive performance, global function, and activities of the daily living have been found. Metaanalysis in the indication--demential disorders--comparing Ginkgo biloba versus acetylcholinesterase inhibitors have shown a similar clinical efficacy of both therapy regimens with an additional drug safety benefit for Ginkgo. Due to the clinical efficacy the WHO accepted Ginkgo biloba as an antidementiv drug and add it in January 2000 into the recent ATC-Classification Index. In future antidementive therapy drugs with an different mode of action should be given in combination. Furthermore clinical trials with fixed combinations of acetylcholinesterase inhibitors with Ginkgo biloba extracts in moderate or severe dementia

  17. n-3 fatty acids effectively improve the reference memory-related learning ability associated with increased brain docosahexaenoic acid-derived docosanoids in aged rats.

    Science.gov (United States)

    Hashimoto, Michio; Katakura, Masanori; Tanabe, Yoko; Al Mamun, Abdullah; Inoue, Takayuki; Hossain, Shahdat; Arita, Makoto; Shido, Osamu

    2015-02-01

    We investigated whether a highly purified eicosapentaenoic acid (EPA) and a concentrated n-3 fatty acid formulation (prescription TAK-085) containing EPA and docosahexaenoic acid (DHA) ethyl ester could improve the learning ability of aged rats and whether this specific outcome had any relation with the brain levels of EPA-derived eicosanoids and DHA-derived docosanoids. The rats were tested for reference memory errors (RMEs) and working memory errors (WMEs) in an eight-arm radial maze. Fatty acid compositions were analyzed by GC, whereas brain eicosanoid/docosanoids were measured by LC-ESI-MS-MS-based analysis. The levels of lipid peroxides (LPOs) were measured by thiobarbituric acid reactive substances. The administration of TAK-085 at 300 mg·kg⁻¹day⁻¹ for 17 weeks reduced the number of RMEs in aged rats compared with that in the control rats. Both TAK-085 and EPA administration increased plasma EPA and DHA levels in aged rats, with concurrent increases in DHA and decreases in arachidonic acid in the corticohippocampal brain tissues. TAK-085 administration significantly increased the formation of EPA-derived 5-HETE and DHA-derived 7-, 10-, and 17-HDoHE, PD1, RvD1, and RvD2. ARA-derived PGE2, PGD2, and PGF2α significantly decreased in TAK-085-treated rats. DHA-derived mediators demonstrated a significantly negative correlation with the number of RMEs, whereas EPA-derived mediators did not exhibit any relationship. Furthermore, compared with the control rats, the levels of LPO in the plasma, cerebral cortex, and hippocampus were significantly reduced in TAK-085-treated rats. The findings of the present study suggest that long-term EPA+DHA administration may be a possible preventative strategy against age-related cognitive decline. Copyright © 2014. Published by Elsevier B.V.

  18. Guideline for dialysate quality of Spanish Society of Nephrology (second edition, 2015).

    Science.gov (United States)

    Pérez-García, Rafael; García Maset, Rafael; Gonzalez Parra, Emilio; Solozábal Campos, Carlos; Ramírez Chamond, Rafael; Martín-Rabadán, Pablo; Sobrino Pérez, Pedro Enrique; Gallego Pereira, Ovidio; Dominguez, Jon; de la Cueva Matute, Enrique; Ferllen, Ricardo

    2016-01-01

    A Best Practice Guideline about Dialysis fluid purity was developed under the leadership of the Spanish Society of Nephrology in 2004. The second edition revised Guideline considered new evidences and International Standard. The Guideline has established recommendations for standards for preparing dialysate: water, concentrates and hemodialysis proportioning systems. This Guideline is based on the ISO13959, European Pharmacopoeia, the Real Farmacopea Española, the AAMI Standards and Recommended Practices, European Best Practice Guidelines for Haemodialysis, literature reviews, according to their level of evidence, and the opinion of the expert Spanish group. Two levels of quality of water were defined: purified water and high purified water (ultra pure) and for dialysate: ultra pure dialysate. Regular use of ultra pure dialysate is recommended for all type of hemodialysis to prevent and delay the occurrence of complications: inflammation, malnutrition, anaemia and amiloidosis. Water, concentrates and dialysate quality requirements are defined as maximum allowable contaminant levels: chemicals (4.1.2), conductivity, microbial and endotoxins (4.1.1): Monitoring frequency, maintenance and corrective actions were specified. Methods of sampling and analysis were described in appendix (anexos). For microbiological monitoring, R2A medium is recommended, incubated during 7-14 days at a temperature of 17-23°C. The dialysate quality assurance process involves all dialysis staff members and requires strict protocols. The physician in charge of hemodialysis has the ultimate responsibility for dialysate quality. All suggestions and questions about this Guideline are wellcome to www.senefro.org. Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

  19. Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs.

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    Marie-Clotilde Bernard

    Full Text Available HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29 has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529 derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a

  20. PRODUCTION OF POLYCLONAL ANTIBODY TO THE COAT PROTEIN OF CITRUS TRISTEZA VIRUS IN CHICKEN EGGS

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    Nurhadi Nurhadi

    2016-10-01

    Full Text Available Citrus tristeza virus (CTV is one of the most destructive diseases in many citrus growing areas of Indonesia. Effective strategies for controlling CTV depend on diagnostic procedure namely enzyme-linked immunosorbent assay (ELISA. Study aimed to purify the CTV antigen and produced its polyclonal antibody. Virion of the severe CTV isolate designated UPM/ T-002 was concentrated by polyethylene glycol (PEG precipitation combined with low speed centrifugation. Semipurified antigen was further purified by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE. The specific coat protein (CP band of CTV with molecular weight of 25 kD was excised and eluted using elution buffer containing 0.25 M Tris-HCl pH 6.8 + 0.1% SDS, then used as antigen for injection into 6-month-old female of White Leghorn chicken. Results, showed than the specific polyclonal antibody raised against the 25-kDa CP had a titer of approximately 104, gave low background reaction with healthy plant sap and reacted specifically with CTV isolates. The reaction was equally strong for a severe, a moderate, a mild, and a symptomless isolate, suggesting a broad reaction range of this antibody toward different CTV isolates. Optimal virus titer can be obtained since virus loss during purification could be minimized and the highly purified antigen as an immunogen could be obtained by cutting out the CP band from SDS-PAGE gels. Large amount of highly titer of CTV antibody can be produced in chicken egg. The simplicity of the procedure makes it economically acceptable and technically adoptable because the antibody can be produced in basic laboratory.

  1. Characterization of chemokine receptor utilization of viruses in the latent reservoir for human immunodeficiency virus type 1.

    Science.gov (United States)

    Pierson, T; Hoffman, T L; Blankson, J; Finzi, D; Chadwick, K; Margolick, J B; Buck, C; Siliciano, J D; Doms, R W; Siliciano, R F

    2000-09-01

    Latently infected resting CD4(+) T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1 env genes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4(+) T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4(+) T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established.

  2. Are the sea foods on our table safe after the Fukushima Nuclear Power Plant Accident?

    International Nuclear Information System (INIS)

    Enriquez, E.B.; Palad, L.J.H.; Encabo, R.R.; Cruz, P.T.F.; Garcia, T.Y.

    2015-01-01

    The Fukushima Nuclear Power Plant Accident that occurred in March 2011 raised immediate public concern on the safety of marine organisms caught in the Philippine waters. This is because of Japan’s proximity to the Philippine archipelago and the threat of contamination reaching our shores became a major issue. Immediately after the accident, the Philippine Nuclear Research Institute (PNRI) established an extensive marine monitoring program to assess any possible effect of one of the worst nuclear accidents that occurred in recent time. This study is under the framework of the project entitled “Radiological Impact Assessment of the Fukushima Nuclear Accident in the Philippine Marine Environment”. Species of fish, mollusks and crustaceans were analyzed for anthropogenic radionuclides cesium-134 (Cs-134) and Cesium-137 (Cs-137) Using a high purify Germanium (HPGe) detector from ORTEC Inc. Major fishing grounds and coastal areas in the west and eastern seaboards as well as in the north and south were selected as sources of biota samples. Commonly eaten and popular species of fish such as tuna, mackerel, sardines, etc were purchased from local markets in the area; processed and analyzed by gamma spectrometry. The results showed that the average Cs-137 activity concentration in fish samples (n=103) was found to be 0.74 ± 0.28 Becquerel/kilogram wet. The Cs-137 concentrations in mollusks (n=12) and crustaceans (n=4) were all below the Lowest Limit of Detection (LLD). Cs-134 was not detected in any of the samples analyzed. The low concentration of the radionuclides studied showed that, thus far, the Fukushima NPP accident has no Impact to the Philippines marine environment. (author)

  3. Self-assembling protein nanoparticles in the design of vaccines

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    Jacinto López-Sagaseta

    2016-01-01

    Full Text Available For over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estimated to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. Compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, modern vaccines containing isolated, highly purified antigenic protein subunits are safer but tend to induce lower levels of protective immunity. One strategy to overcome the latter is to design antigen nanoparticles: assemblies of polypeptides that present multiple copies of subunit antigens in well-ordered arrays with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity and can provide improved antigen stability and immunogenicity. Several exciting advances have emerged lately, including preclinical evidence that this strategy may be applicable for the development of innovative new vaccines, for example, protecting against influenza, human immunodeficiency virus, and respiratory syncytial virus. Here, we provide a concise review of a critical selection of data that demonstrate the potential of this field. In addition, we highlight how the use of self-assembling protein nanoparticles can be effectively combined with the emerging discipline of structural vaccinology for maximum impact in the rational design of vaccine antigens.

  4. Decreased capacity for sodium export out of Arabidopsis chloroplasts impairs salt tolerance, photosynthesis and plant performance.

    Science.gov (United States)

    Müller, Maria; Kunz, Hans-Henning; Schroeder, Julian I; Kemp, Grant; Young, Howard S; Neuhaus, H Ekkehard

    2014-05-01

    Salt stress is a widespread phenomenon, limiting plant performance in large areas around the world. Although various types of plant sodium/proton antiporters have been characterized, the physiological function of NHD1 from Arabidopsis thaliana has not been elucidated in detail so far. Here we report that the NHD1-GFP fusion protein localizes to the chloroplast envelope. Heterologous expression of AtNHD1 was sufficient to complement a salt-sensitive Escherichia coli mutant lacking its endogenous sodium/proton exchangers. Transport competence of NHD1 was confirmed using recombinant, highly purified carrier protein reconstituted into proteoliposomes, proving Na(+) /H(+) antiport. In planta NHD1 expression was found to be highest in mature and senescent leaves but was not induced by sodium chloride application. When compared to wild-type controls, nhd1 T-DNA insertion mutants showed decreased biomasses and lower chlorophyll levels after sodium feeding. Interestingly, if grown on sand and supplemented with high sodium chloride, nhd1 mutants exhibited leaf tissue Na(+) levels similar to those of wild-type plants, but the Na(+) content of chloroplasts increased significantly. These high sodium levels in mutant chloroplasts resulted in markedly impaired photosynthetic performance as revealed by a lower quantum yield of photosystem II and increased non-photochemical quenching. Moreover, high Na(+) levels might hamper activity of the plastidic bile acid/sodium symporter family protein 2 (BASS2). The resulting pyruvate deficiency might cause the observed decreased phenylalanine levels in the nhd1 mutants due to lack of precursors. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  5. Peripheral blood B lymphocytes derived from patients with idiopathic pulmonary arterial hypertension express a different RNA pattern compared with healthy controls: a cross sectional study

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    Huber Lars C

    2008-02-01

    Full Text Available Abstract Background Idiopathic pulmonary arterial hypertension (IPAH is a progressive and still incurable disease. Research of IPAH-pathogenesis is complicated by the lack of a direct access to the involved tissue, the human pulmonary vasculature. Various auto-antibodies have been described in the blood of patients with IPAH. The purpose of the present work was therefore to comparatively analyze peripheral blood B lymphocyte RNA expression characteristics in IPAH and healthy controls. Methods Patients were diagnosed having IPAH according to WHO (mean pulmonary arterial pressure ≥ 25 mmHg, pulmonary capillary occlusion pressure ≤ 15 mmHg, absence of another explaining disease. Peripheral blood B-lymphocytes of patients and controls were immediately separated by density gradient centrifugation and magnetic beads for CD19. RNA was thereafter extracted and analyzed by the use of a high sensitivity gene chip (Affymetrix HG-U133-Plus2 able to analyze 47000 transcripts and variants of human genes. The array data were analyzed by two different softwares, and up-and down-regulations were defined as at least 1.3 fold with standard deviations smaller than fold-changes. Results Highly purified B-cells of 5 patients with IPAH (mean pulmonary artery pressure 51 ± 13 mmHg and 5 controls were analyzed. Using the two different analyzing methods we found 225 respectively 128 transcripts which were up-regulated (1.3–30.7 fold in IPAH compared with healthy controls. Combining both methods, there were 33 overlapping up-regulated transcripts and no down-regulated B-cell transcripts. Conclusion Patients with IPAH have a distinct RNA expression profile of their peripheral blood B-lymphocytes compared to healthy controls with some clearly up-regulated genes. Our finding suggests that in IPAH patients B cells are activated.

  6. Role of enzymatic activity in muscle damage and cytotoxicity induced by Bothrops asper Asp49 phospholipase A2 myotoxins: are there additional effector mechanisms involved?

    Science.gov (United States)

    Mora-Obando, Diana; Díaz, Cecilia; Angulo, Yamileth; Gutiérrez, José María; Lomonte, Bruno

    2014-01-01

    Viperid venoms often contain mixtures of Asp49 and Lys49 PLA2 myotoxin isoforms, relevant to development of myonecrosis. Given their difference in catalytic activity, mechanistic studies on each type require highly purified samples. Studies on Asp49 PLA2s have shown that enzyme inactivation using p-bromophenacyl bromide (p-BPB) drastically affects toxicity. However, based on the variable levels of residual toxicity observed in some studies, it has been suggested that effector mechanisms independent of catalysis may additionally be involved in the toxicity of these enzymes, possibly resembling those of the enzymatically inactive Lys49 myotoxins. A possibility that Lys49 isoforms could be present in Asp49 PLA2 preparations exists and, if undetected in previous studies, could explain the variable residual toxicity. This question is here addressed by using an enzyme preparation ascertained to be free of Lys49 myotoxins. In agreement with previous reports, inactivation of the catalytic activity of an Asp49 myotoxin preparation led to major inhibition of toxic effects in vitro and in vivo. The very low residual levels of myotoxicity (7%) and cytotoxicity (4%) observed can be attributed to the low, although detectable, enzyme remaining active after p-BPB treatment (2.7%), and would be difficult to reconcile with the proposed existence of additional catalytic-independent toxic mechanisms. These findings favor the concept that the effector mechanism of toxicity of Asp49 PLA2 myotoxins from viperids fundamentally relies on their ability to hydrolyze phospholipids, arguing against the proposal that membrane disruption may also be caused by additional mechanisms that are independent of catalysis.

  7. Role of enzymatic activity in muscle damage and cytotoxicity induced by Bothrops asper Asp49 phospholipase A2 myotoxins: are there additional effector mechanisms involved?

    Directory of Open Access Journals (Sweden)

    Diana Mora-Obando

    2014-09-01

    Full Text Available Viperid venoms often contain mixtures of Asp49 and Lys49 PLA2 myotoxin isoforms, relevant to development of myonecrosis. Given their difference in catalytic activity, mechanistic studies on each type require highly purified samples. Studies on Asp49 PLA2s have shown that enzyme inactivation using p-bromophenacyl bromide (p-BPB drastically affects toxicity. However, based on the variable levels of residual toxicity observed in some studies, it has been suggested that effector mechanisms independent of catalysis may additionally be involved in the toxicity of these enzymes, possibly resembling those of the enzymatically inactive Lys49 myotoxins. A possibility that Lys49 isoforms could be present in Asp49 PLA2 preparations exists and, if undetected in previous studies, could explain the variable residual toxicity. This question is here addressed by using an enzyme preparation ascertained to be free of Lys49 myotoxins. In agreement with previous reports, inactivation of the catalytic activity of an Asp49 myotoxin preparation led to major inhibition of toxic effects in vitro and in vivo. The very low residual levels of myotoxicity (7% and cytotoxicity (4% observed can be attributed to the low, although detectable, enzyme remaining active after p-BPB treatment (2.7%, and would be difficult to reconcile with the proposed existence of additional catalytic-independent toxic mechanisms. These findings favor the concept that the effector mechanism of toxicity of Asp49 PLA2 myotoxins from viperids fundamentally relies on their ability to hydrolyze phospholipids, arguing against the proposal that membrane disruption may also be caused by additional mechanisms that are independent of catalysis.

  8. Safety and tolerability of intra-articular hyaluronic acid (Sinovial®/GELSYN-3tm) injections in the treatment of knee osteoarthritis.

    Science.gov (United States)

    Abate, M; Salini, V

    2017-01-01

    Osteoarthritis (OA) is a progressively degenerative joint disease, with a very high prevalence rate that is expected to increase worldwide with the ageing of the population. Considering that OA requires long-term treatment, therapies with minimal side effects and which can be repeated as needed are warranted. Hyaluronic acid (HA), a natural glycosaminoglycan with viscoelastic properties, is a major component of synovial fluid and the extracellular matrix of the joint cartilage, and plays key roles in maintaining synovial fluid viscosity and the bio-mechanical integrity of healthy cartilage. Intra-articular administration of exogenous HA has therefore been used to successfully improve the viscoelastic properties of the joint to improve lubrication, modulate inflammation and modify the catabolic micro-environment. Sinovial®/GELSYN-3TM is a sterile, non-pyrogenic formulation of highly purified, chemically unmodified HA of bio-fermentative origin, which has been introduced in several different concentrations in clinical use within the European market. This expert opinion reports on the published data regarding the efficacy and tolerability of first and multiple injection series of Sinovial®-based product formulations. The data regarding the tolerability of Sinovial® in patients with knee osteoarthritis were analyzed, showing that this formulation, beside favourable therapeutic effects, has a very good tolerability profile, with only mild, transient, and easily managed, local injection-site reactions and absence of systemic reactions. In particular, repetitive cycles of HA have been shown to yield positive results in terms of both efficacy and safety and therefore should be offered to patients who had undergone a successful first course of therapy when their symptoms reoccur.

  9. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin.

    Science.gov (United States)

    Gabe, Claire M; Brookes, Steven J; Kirkham, Jennifer

    2017-01-01

    acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

  10. Experimental Granulomatous Pulmonary Nocardiosis in BALB/C Mice

    Science.gov (United States)

    Mifuji Lira, Roque M.; Limón Flores, Alberto Yairh; Salinas Carmona, Mario César

    2016-01-01

    Pulmonary nocardiosis is a granulomatous disease with high mortality that affects both immunosuppressed and immunocompetent patients. The mechanisms leading to the establishment and progression of the infection are currently unknown. An animal model to study these mechanisms is sorely needed. We report the first in vivo model of granulomatous pulmonary nocardiosis that closely resembles human pathology. BALB/c mice infected intranasally with two different doses of GFP-expressing Nocardia brasiliensis ATCC700358 (NbGFP), develop weight loss and pulmonary granulomas. Mice infected with 109 CFUs progressed towards death within a week while mice infected with 108 CFUs died after five to six months. Histological examination of the lungs revealed that both the higher and lower doses of NbGFP induced granulomas with NbGFP clearly identifiable at the center of the lesions. Mice exposed to 108 CFUs and subsequently to 109 CFUs were not protected against disease severity but had less granulomas suggesting some degree of protection. Attempts to identify a cellular target for the infection were unsuccessful but we found that bacterial microcolonies in the suspension used to infect mice were responsible for the establishment of the disease. Small microcolonies of NbGFP, incompatible with nocardial doubling times starting from unicellular organisms, were identified in the lung as early as six hours after infection. Mice infected with highly purified unicellular preparations of NbGFP did not develop granulomas despite showing weight loss. Finally, intranasal delivery of nocardial microcolonies was enough for mice to develop granulomas with minimal weight loss. Taken together these results show that Nocardia brasiliensis microcolonies are both necessary and sufficient for the development of granulomatous pulmonary nocardiosis in mice. PMID:27303806

  11. Experimental Granulomatous Pulmonary Nocardiosis in BALB/C Mice.

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    Roque M Mifuji Lira

    Full Text Available Pulmonary nocardiosis is a granulomatous disease with high mortality that affects both immunosuppressed and immunocompetent patients. The mechanisms leading to the establishment and progression of the infection are currently unknown. An animal model to study these mechanisms is sorely needed. We report the first in vivo model of granulomatous pulmonary nocardiosis that closely resembles human pathology. BALB/c mice infected intranasally with two different doses of GFP-expressing Nocardia brasiliensis ATCC700358 (NbGFP, develop weight loss and pulmonary granulomas. Mice infected with 109 CFUs progressed towards death within a week while mice infected with 108 CFUs died after five to six months. Histological examination of the lungs revealed that both the higher and lower doses of NbGFP induced granulomas with NbGFP clearly identifiable at the center of the lesions. Mice exposed to 108 CFUs and subsequently to 109 CFUs were not protected against disease severity but had less granulomas suggesting some degree of protection. Attempts to identify a cellular target for the infection were unsuccessful but we found that bacterial microcolonies in the suspension used to infect mice were responsible for the establishment of the disease. Small microcolonies of NbGFP, incompatible with nocardial doubling times starting from unicellular organisms, were identified in the lung as early as six hours after infection. Mice infected with highly purified unicellular preparations of NbGFP did not develop granulomas despite showing weight loss. Finally, intranasal delivery of nocardial microcolonies was enough for mice to develop granulomas with minimal weight loss. Taken together these results show that Nocardia brasiliensis microcolonies are both necessary and sufficient for the development of granulomatous pulmonary nocardiosis in mice.

  12. Proteogenomics analysis reveals specific genomic orientations of distal regulatory regions composed by non-canonical histone variants.

    Science.gov (United States)

    Won, Kyoung-Jae; Choi, Inchan; LeRoy, Gary; Zee, Barry M; Sidoli, Simone; Gonzales-Cope, Michelle; Garcia, Benjamin A

    2015-01-01

    Histone variants play further important roles in DNA packaging and controlling gene expression. However, our understanding about their composition and their functions is limited. Integrating proteomic and genomic approaches, we performed a comprehensive analysis of the epigenetic landscapes containing the four histone variants H3.1, H3.3, H2A.Z, and macroH2A. These histones were FLAG-tagged in HeLa cells and purified using chromatin immunoprecipitation (ChIP). By adopting ChIP followed by mass spectrometry (ChIP-MS), we quantified histone post-translational modifications (PTMs) and histone variant nucleosomal ratios in highly purified mononucleosomes. Subsequent ChIP followed by next-generation sequencing (ChIP-seq) was used to map the genome-wide localization of the analyzed histone variants and define their chromatin domains. Finally, we included in our study large datasets contained in the ENCODE database. We newly identified a group of regulatory regions enriched in H3.1 and the histone variant associated with repressive marks macroH2A. Systematic analysis identified both symmetric and asymmetric patterns of histone variant occupancies at intergenic regulatory regions. Strikingly, these directional patterns were associated with RNA polymerase II (PolII). These asymmetric patterns correlated with the enhancer activities measured using global run-on sequencing (GRO-seq) data. Our studies show that H2A.Z and H3.3 delineate the orientation of transcription at enhancers as observed at promoters. We also showed that enhancers with skewed histone variant patterns well facilitate enhancer activity. Collectively, our study indicates that histone variants are deposited at regulatory regions to assist gene regulation.

  13. Identification of light-independent inhibition of human immunodeficiency virus-1 infection through bioguided fractionation of Hypericum perforatum

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    Widrlechner Mark P

    2009-07-01

    Full Text Available Abstract Background Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated. Results Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1 by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material. Conclusion Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.

  14. Tyrosine nitration affects thymidylate synthase properties.

    Science.gov (United States)

    Dąbrowska-Maś, Elżbieta; Frączyk, Tomasz; Ruman, Tomasz; Radziszewska, Karolina; Wilk, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Jurkiewicz, Agata; Gołos, Barbara; Wińska, Patrycja; Wałajtys-Rode, Elżbieta; Leś, Andrzej; Nizioł, Joanna; Jarmuła, Adam; Stefanowicz, Piotr; Szewczuk, Zbigniew; Rode, Wojciech

    2012-01-14

    Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

  15. A Novel ω-3 Acid Ethyl Ester Formulation Incorporating Advanced Lipid TechnologiesTM(ALT®) Improves Docosahexaenoic Acid and Eicosapentaenoic Acid Bioavailability Compared with Lovaza®.

    Science.gov (United States)

    Lopez-Toledano, Miguel A; Thorsteinsson, Thorsteinn; Daak, Ahmed; Maki, Kevin C; Johns, Colleen; Rabinowicz, Adrian L; Sancilio, Frederick D

    2017-03-01

    The US Food and Drug Administration has approved several highly purified ω-3 fatty acid prescription drugs for the treatment of severe hypertriglyceridemia. These differ in the amounts and forms of docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). This study compared the bioavailability of SC401 (1530 mg EPA-ethyl esters [EEs] and DHA-EEs plus Advanced Lipid Technologies ⁎ [ALT † ], a proprietary lipid-delivery platform to improve absorption), with. Lovaza ‡ (3600 mg ω-3, primarily EPA-EEs and DHA-EEs) under low-fat feeding conditions. This was a Phase I, randomized, open-label, single-dose, 2-way crossover study in healthy participants housed from day -3 to day 2 in each treatment period. Blood samples for pharmacokinetic measurements were collected before and after dosing, and safety profile and tolerability were assessed. In unadjusted analyses, SC401 had 5% lower C max and approximately the same AUC 0-last of EPA + DHA total lipids compared with Lovaza. When adjusted for baseline, SC401 had ~6% higher C max and 18% higher AUC 0-last for EPA + DHA total lipids, and dose- and baseline-adjusted analyses found that SC401 had ~149% higher C max and 178% higher AUC 0-last than Lovaza for EPA + DHA total lipids. The T max was also substantially longer with Lovaza (~10 hours) than with SC401 (~6 hours). These results indicate that SC401, an ω-3 acid EE formulation containing ALT † achieved high bioavailability of EPA and DHA, at a lower dose (1530 mg) than Lovaza (3600 mg), under low-fat feeding conditions. Copyright © 2017 Elsevier HS Journals, Inc. All rights reserved.

  16. Using water wisely: New, affordable, and essential water conservation practices for facility and home hemodialysis.

    Science.gov (United States)

    Agar, John W M; Simmonds, Rosemary E; Knight, Richard; Somerville, Christine A

    2009-01-01

    Despite a global focus on resource conservation, most hemodialysis (HD) services still wastefully or ignorantly discard reverse osmosis (R/O) "reject water" (RW) to the sewer. However, an R/O system is producing the highly purified water necessary for dialysis, it rejects any remaining dissolved salts from water already prefiltered through charcoal and sand filters in a high-volume effluent known as RW. Although the RW generated by most R/O systems lies well within globally accepted potable water criteria, it is legally "unacceptable" for drinking. Consequently, despite being extremely high-grade gray water, under current dialysis practices, it is thoughtlessly "lost-to-drain." Most current HD service designs neither specify nor routinely include RW-saving methodology, despite its simplicity and affordability. Since 2006, we have operated several locally designed, simple, cheap, and effective RW collection and distribution systems in our in-center, satellite, and home HD services. All our RW water is now recycled for gray-water use in our hospital, in the community, and at home, a practice that is widely appreciated by our local health service and our community and is an acknowledged lead example of scarce resource conservation. Reject water has sustained local sporting facilities and gardens previously threatened by indefinite closure under our regional endemic local drought conditions. As global water resources come under increasing pressure, we believe that a far more responsible attitude to RW recycling and conservation should be mandated for all new and existing HD services, regardless of country or region.

  17. Circulating clonotypic B cells in multiple myeloma and monoclonal gammopathy of undetermined significance.

    Science.gov (United States)

    Thiago, Leandro S; Perez-Andres, Martin; Balanzategui, Ana; Sarasquete, Maria E; Paiva, Bruno; Jara-Acevedo, Maria; Barcena, Paloma; Sanchez, Maria Luz; Almeida, Julia; González, Marcos; San Miguel, Jesus F; Garcia-Sanz, Ramón; Orfao, Alberto

    2014-01-01

    The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM(+) CD27(-) naïve B-lymphocytes, plus surface-membrane IgG(+), IgA(+) and IgM(+) memory CD27(+) B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies <0.03 cells/μL (range: 0.0003-0.08 cells/μL); the only exception were the myeloma plasma cells detected and purified from the peripheral blood of four of the seven myeloma patients. These results indicate that circulating B cells from patients with multiple myeloma and monoclonal gammopathies of undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.

  18. Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations

    Directory of Open Access Journals (Sweden)

    Tamborrini Marco

    2011-12-01

    Full Text Available Abstract Background In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP. Methods The highly purified recombinant protein GMZ2 was coupled to phosphatidylethanolamine and the conjugates incorporated into the membrane of IRIVs. The immunogenicity of this adjuvant-free virosomal formulation was compared to GMZ2 formulated with the adjuvants Montanide ISA 720 and Alum in three mouse strains with different genetic backgrounds. Results Intramuscular injections of all three candidate vaccine formulations induced GMZ2-specific antibody responses in all mice tested. In general, the humoral immune response in outbred NMRI mice was stronger than that in inbred BALB/c and C57BL/6 mice. ELISA with the recombinant antigens demonstrated immunodominance of the GLURP component over the MSP3 component. However, compared to the Al(OH3-adjuvanted formulation the two other formulations elicited in NMRI mice a larger proportion of anti-MSP3 antibodies. Analyses of the induced GMZ2-specific IgG subclass profiles showed for all three formulations a predominance of the IgG1 isotype. Immune sera against all three formulations exhibited cross-reactivity with in vitro cultivated blood-stage parasites. Immunofluorescence and immunoblot competition experiments showed that both components of the hybrid protein induced IgG cross-reactive with the corresponding native proteins. Conclusion A virosomal formulation of the chimeric protein GMZ2 induced P. falciparum blood stage parasite cross-reactive IgG responses specific for both MSP3 and GLURP. GMZ2 thus represents a candidate component suitable for inclusion into a multi-valent virosomal

  19. Analysis of recombinant and native human lutropin/luteotrophin and human chorionic gonadotropin by reversed-phase high performance liquid chromatography; Analise de luteotrofina humana e de gonadotrofina corionica humana, recombinante e natural, por cromatografia liquida de alta eficiencia em fase reversa

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, Beatriz Elane de

    2009-07-01

    Specific RP-HPLC conditions for the analysis of recombinant and native hLH and hCG preparations and of their subunits were set up. Heterodimeric hLH and hCG and their alpha and beta subunits all migrated with significantly different retention times (t{sub R}) in the following order of increasing hydrophobicity: alpha-hCG < alpha-hLH < hCG < hLH < -beta-hCG < beta-hLH. With basis on these conditions, a total of eleven preparations were studied: the International Standard of recombinant hLH-WHO 96/602, a commercial recombinant and two highly purified pituitary hLH, a recombinant and two urinary hCG preparations and four heterogeneous urinary products containing hLH + hFSH. All hLH preparations showed very similar retention times for the main peak (t{sub R} = 38.35 +- 0.42 min; RSD = 1.1 %; n = 4 preparations), while the hCG main peak ran about 4 % faster when compared to this average value. Human LH, hFSH and hCG peaks could also be identified in the heterogeneous urinary preparations. Quantitative analysis could be validated for the seven homogeneous preparations and accuracy, precision and sensitivity were calculated on the basis of a highly linear dose-response curve (r=0.99998; p<0.0001; n=20). Quantification of the different gonadotropins in the heterogeneous urinary preparations was also carried out, though with clear accuracy limitations. (author)

  20. New radioimmunoassay for follicle-stimulating hormone in macaques: ovulatory menstrual cycles. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Hodgen, G.D.; Wilks, J.W.; Vaitukaitis, J.L.; Chen, H.C.; Papkoff, H.; Ross, G.

    1976-07-01

    A sensitive and specific radioimmunoassay system for macaque follicle-stimulating hormone (mFSH) was developed utilizing an antiserum (H-31) prepared in a rabbit against purified ovine FSH as the immunogen. Sera from castrated female, adult male, and juvenile rhesus monkeys, as well as urinary extracts from castrated rhesus and bonnet monkeys, were used to demonstrate parallelism with a standard of partially purified monkey pituitary gonadotropins (LER-M-907-D). An extract of baboon pituitary tissue also showed parallelism with the reference standard. A highly purified pituitary extract (WP-X-105-28), containing approximately 75 percent macaque luteinizing hormone (mLH) and 1 percent mFSH, was used to demonstrate the specificity of this mFSH assay system. Sera and urinary extracts obtained from hypophysectomized monkeys did not show cross-reactivity in the assay. Macaque chorionic gonadotropin (mCG) did not produce an inhibition curve in the assay, as determined from serum samples and urinary extracts collected from pregnant monkeys at the time of peak mCG secretion. Serum concentrations of mFSH were suppressed in ovariectomized monkeys by the administration of ethinyl estradiol for 3 days, but returned to near pretreatment values by 96 h after the last estradiol administration. The determination of serum mFSH concentrations in daily blood samples obtained from 20 rhesus monkeys throughout ovulatory menstrual cycles revealed a pattern similar to that previously reported for the rhesus monkey and the woman. The peak value of serum mFSH during the menstrual cycle coincided with the midcycle surge of mLH in each case. The gonadotropin peaks were preceded by increasing serum concentrations of estradiol and followed by rises in the serum concentrations of progesterone.

  1. Sulodexide prevents activation of the PLA2/COX-2/VEGF inflammatory pathway in human retinal endothelial cells by blocking the effect of AGE/RAGE.

    Science.gov (United States)

    Giurdanella, Giovanni; Lazzara, Francesca; Caporarello, Nunzia; Lupo, Gabriella; Anfuso, Carmelina Daniela; Eandi, Chiara M; Leggio, Gian Marco; Drago, Filippo; Bucolo, Claudio; Salomone, Salvatore

    2017-10-15

    Diabetic retinopathy is characterized by the breakdown of endothelial blood-retinal barrier. We tested the hypothesis that sulodexide (SDX), a highly purified glycosaminoglycan composed of 80% iduronylglycosaminoglycan sulfate and 20% dermatan sulfate, protects human retinal endothelial cells (HREC) from high glucose (HG)-induced damage, through the suppression of inflammatory ERK/cPLA2/COX-2/PGE 2 pathway, by blocking the effect of advanced glycation end-products (AGEs). HREC were treated with HG (25mM) or AGEs (glycated-BSA, 2mg/ml) for 48h, with or without SDX (60μg/ml) or aflibercept (AFL, 40μg/ml), a VEGF-trap. SDX protected HREC from HG-induced damage (MTT and LDH release) and preserved their blood-retinal barrier-like properties (Trans Endothelial Electrical Resistance and junction proteins, claudin-5, VE-cadherin and occludin, immunofluorescence and immunoblot) as well as their angiogenic potential (Tube Formation Assay). Both HG and AGEs increased phosphoERK and phospho-cPLA 2 , an effect counteracted by SDX and, less efficiently, by AFL. Both HG and exogenous VEGF (80ng/ml) increased PGE 2 release, an effect partially reverted by SDX for HG and by AFL for VEGF. Analysis of NFκB activity revealed that HG increased the abundance of p65 in the nuclear fraction (nuclear translocation), an effect entirely reverted by SDX, but only partially by AFL. SDX, AFL and SDX+AFL protected HREC even when added 24h after HG. These data show that SDX protects HREC from HG damage and suggest that it counteracts the activation of ERK/cPLA2/COX-2/PGE 2 pathway by reducing AGE-related signaling and downstream NFκB activity. This mechanism, partially distinct from VEGF blockade, may contribute to the therapeutic effect of SDX. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. The fate of purified radio-labelled alkaline phosphatase from the liver in the organism

    International Nuclear Information System (INIS)

    Herbert, V.

    1981-01-01

    Alkaline phosphatase (AP) from dog liver was enriched by a factor of 5.444 in various steps. Rabbit antiserum to the purified AP was produced; 125-I was used then to radiolabel the highly purified AP. Four dogs were cholecystectomized and subsequently received an extracorporal drainage of the bile ducts. Decrease rate of total radio-activity and of PBI in the serum was determined in one dog; likewise in three other dogs before and one week after occlusion of their main bile ducts. In addition, radioactivity above the organs was measured in some animals at short intervals. In the dogs with main bile duct drainage, bile was collected continuously for up to 70 h, samples were taken, and residual bile plus native dog bile were re-infused into the distal choledochus catheter. Total radioactivity, PBI and immunoprecipitability with antibodies were determined in the bile and serum samples. AP, GOT, CPT and bilirubin were determined in some serum samples. In addition, total radioactivity excreted by urine was established. Results show injected 125-I-AP to be rapidly stored in the liver and not to be excreted via bile to a decisive extent. The fact that 125-I-AP is not excreted via bile is further indicated by the identical decrease rate of injected 125-I-AP in the serum in dogs with and without main bile duct occlusion. Injected 125-I-AP appears to be metabolized very rapidly in the liver as is indicated by the rapid decrease of immuno precipitability of 125-I-AP in the serum. (orig./MG) [de

  3. Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions

    Energy Technology Data Exchange (ETDEWEB)

    Antanasijevic, Aleksandar; Kingsley, Carolyn [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States); Basu, Arnab; Bowlin, Terry L. [Microbiotix Inc. (United States); Rong, Lijun [University of Illinois at Chicago, Department of Microbiology and Immunology (United States); Caffrey, Michael, E-mail: caffrey@uic.edu [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States)

    2016-03-15

    The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.

  4. Synthesis and multiferroic properties of M-type SrFe{sub 12}O{sub 19} hexaferrite ceramics

    Energy Technology Data Exchange (ETDEWEB)

    Kostishyn, V.G.; Panina, L.V., E-mail: lpanina@plymouth.ac.uk; Kozhitov, L.V.; Timofeev, A.V.; Kovalev, A.N.

    2015-10-05

    Highlights: • SrFe{sub 12}O{sub 19}-hexaferrites show strong room-temperature multiferroic properties. • Modified ceramic technique helps to increase resistivity of hexaferrites. • Large spontaneous polarisation is observed in single-phase SrFe{sub 12}O{sub 19.} • External electric field deceases the remanent magnetisation of SrFe{sub 12}O{sub 19} by 10%. - Abstract: The coexistence of strong ferromagnetism and large ferroelectricity has been observed in pure strontium hexaferrite SrFe{sub 12}O{sub 19} fabricated by a modified ceramic technological method from highly purified initial materials with addition of boron oxide in mass concentration of less than 1.5%. The structure of the samples confirmed by X-ray diffraction patterns is consistent with a single hexagonal phase. The samples were polycrystalline with the grain size of 300–400 nm and the intergrain space was field with B{sub 2}O{sub 3}. The use of boron oxide resulted in enhanced resistivity enabling proper electric polarisation measurements. The magnetic properties are characterised by a standard ferrimagnetic behaviour with the Neel temperature of about 450 °C. Large electric polarisation was observed with the remnant value of 22 μC/cm{sup 2} and the maximal value of 45 μC/cm{sup 2} under the electric field of 300 kV/m. A strong coupling between magnetic and electric ordering was confirmed by measuring the magnetoelectric parameter and the electric field-controlled magnetic hysteresis. The change in magnetization by applying an electric field was in the range of 9–10%, which is even greater than that observed in some substituted hexaferrites with a magnetically induced electric polarisation. The combination of room-temperature multiferroic properties and electrically tuneable magnetization is promising for applications in magnetoelectric devices.

  5. Facile synthesis of ZnFe{sub 2}O{sub 4}-graphene aerogels composites as high-performance anode materials for lithium ion batteries

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yu [Beijing Key Laboratory of Electrochemical Process and Technology for Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Jin, Yuhong, E-mail: jinyh@bjut.edu.cn [Beijing Guyue New Materials Research Institute, Beijing University of Technology, Beijing 100124 (China); Zhang, Rupeng [Beijing Key Laboratory of Electrochemical Process and Technology for Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Jia, Mengqiu, E-mail: jiamq@mail.buct.edu.cn [Beijing Key Laboratory of Electrochemical Process and Technology for Materials, Beijing University of Chemical Technology, Beijing 100029 (China)

    2017-08-15

    Highlights: • 3D ZnFe{sub 2}O{sub 4}-graphene aerogel composites are obtained by a facile method. • The specific capacity of as-prepared 3D ZnFe{sub 2}O{sub 4}-graphene aerogel composites are 1049 mAh g{sup −1} at 100 mA g{sup −1} after 100 cycles. • Excellent rate capabilities are observed for 3D ZnFe{sub 2}O{sub 4}-graphene aerogel. • 3D ZnFe{sub 2}O{sub 4}-graphene aerogel shows enhanced cyclic stability. - Abstract: ZnFe{sub 2}O{sub 4}-graphene aerogels (ZnFe{sub 2}O{sub 4}/GAs) composites are prepared by two-step method (hydrothermal-calcination). Highly-purified ZnFe{sub 2}O{sub 4} nanoparticles are dispersed uniformly on three-dimensional (3D) GAs substrate. The mass loading of ZnFe{sub 2}O{sub 4} in ZnFe{sub 2}O{sub 4}/GAs composites is 89.3%. Compared with pure ZnFe{sub 2}O{sub 4} sample, the ZnFe{sub 2}O{sub 4}/GAs composites exhibit much higher irreversible capacity of 1449.4 mAh g{sup −1} and enhanced cycling stability (1049 mAh g{sup −1} at 100 mA g{sup −1} after 100 cycles). The improved electrochemical performance of the ZnFe{sub 2}O{sub 4}/GAs composites could be attributed from the synergetic effect between 3D conductive GAs and nanostructured ZnFe{sub 2}O{sub 4}.

  6. Control of mucosal virus infection by influenza nucleoprotein-specific CD8+ cytotoxic T lymphocytes

    Directory of Open Access Journals (Sweden)

    Couch Robert B

    2007-06-01

    Full Text Available Abstract Background MHC class I-restricted CD8+ cytotoxic T lymphocytes (CTL are thought to play a major role in clearing virus and promoting recovery from influenza infection and disease. This has been demonstrated for clearance of influenza virus from the lungs of infected mice. However, human influenza infection is primarily a respiratory mucosal infection involving the nasopharynx and tracheobronchial tree. The role of CD8+ CTL directed toward the influenza nucleoprotein (NP in defense against influenza virus infection at the respiratory mucosa was evaluated in two separate adoptive transfer experiments. Methods Influenza nucleoprotein (NP-specific CD8+ CTL were generated from splenocytes obtained from Balb/c mice previously primed with influenza A/Taiwan/1/86 (H1N1 infection or with influenza A/PR/8/34 (H1N1-derived NP plasmid DNA vaccine followed by infection with A/Hong Kong/68 (H3N2 virus. After in vitro expansion by exposure to an influenza NP-vaccinia recombinant, highly purified CD8+ T cells exhibited significant lysis in vitro of P815 target cells infected with A/Hong Kong/68 (H3N2 virus while the CD8- fraction (CD4+ T cells, B cells and macrophages had no CTL activity. Purified CD8+ and CD8- T cells (1 × 107 were injected intravenously or interperitoneally into naive mice four hours prior to intranasal challenge with A/HK/68 (H3N2 virus. Results The adoptively transferred NP-vaccinia-induced CD8+ T cells caused significant reduction of virus titers in both the lungs and nasal passages when compared to CD8- cells. Neither CD8+ nor CD8- T cells from cultures stimulated with HIV gp120-vaccinia recombinant reduced virus titers. Conclusion The present data demonstrate that influenza NP-specific CD8+ CTL can play a direct role in clearance of influenza virus from the upper respiratory mucosal surfaces.

  7. El diabético insulinodependiente: Estrategia terapéutica actual

    Directory of Open Access Journals (Sweden)

    Francisco Carvajal Martínez

    1998-10-01

    Full Text Available Se analiza la estrategia terapéutica actual en el diabético insulinodependiente. Se insiste en la necesidad de lograr la normoglicemia como criterio de control metabólico expresado en valores normales de hemoglobina glicolisada o Hb Alc trimestral. Se debe enfatizar en la educación diabetológica reforzándola en cada consulta y reevaluando cada cierto tiempo. El cumplimiento de la dieta es fundamental. Debemos utilizar insulina altamente purificada con esquemas insulínicos principalmente basados en mezclas de insulinas o regímenes de múltiples dosis de insulina. Es necesario lograr el monitoreo glicémico por el paciente. La práctica sistemática del ejercicio físico diario o tres veces a la semana es importante. Es preciso tratar de localizar y estudiar los posibles grupos de riesgo a desarrollar esta enfermedadThe present therapeutic estrategy for the insulin-dependent diabetic is analyzed. It is stressed the need of attaining normoglycaemia as a criterion of metabolic control expressed in normal values of glycosylated hemoglobin or HB Alc quarterly. Emphasis shoud be made on the education received by the diabetic patients in connection with the disease, reinforcing it at every office visit, and reevaluating it periodically. Being on a diet is very important, too. We should use highly purified insulin with insulin schemes based mainly on mixtures of insulins or regimes of multiple doses of insulin. It is necessary that the patient attains the glycaemic control. We should try to locate and study the possible risk groups that may develop this disease

  8. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

    Directory of Open Access Journals (Sweden)

    Claire M. Gabe

    2017-06-01

    suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

  9. Prion removal capacity of plasma protein manufacturing processes: a data collection from PPTA member companies.

    Science.gov (United States)

    Cai, Kang; Gröner, Albrecht; Dichtelmüller, Herbert O; Fabbrizzi, Fabrizio; Flechsig, Eckhard; Gajardo, Rodrigo; von Hoegen, Ilka; Jorquera, Juan I; Kempf, Christoph; Kreil, Thomas R; Lee, Douglas C; Moscardini, Mila; Pölsler, Gerhard; Roth, Nathan J

    2013-09-01

    The variant Creutzfeldt-Jakob disease incidence peaked a decade ago and has since declined. Based on epidemiologic evidence, the causative agent, pathogenic prion, has not constituted a tangible contamination threat to large-scale manufacturing of human plasma-derived proteins. Nonetheless, manufacturers have studied the prion removal capabilities of various manufacturing steps to better understand product safety. Collectively analyzing the results could reveal experimental reproducibility and detect trends and mechanisms driving prion removal. Plasma Protein Therapeutics Association member companies collected more than 200 prion removal studies on plasma protein manufacturing steps, including precipitation, adsorption, chromatography, and filtration, as well as combined steps. The studies used a range of model spiking agents and bench-scale process replicas. The results were grouped based on key manufacturing variables to identify factors impacting removal. The log reduction values of a group are presented for comparison. Overall prion removal capacities evaluated by independent groups were in good agreement. The removal capacity evaluated using biochemical assays was consistent with prion infectivity removal measured by animal bioassays. Similar reduction values were observed for a given step using various spiking agents, except highly purified prion protein in some circumstances. Comparison between combined and single-step studies revealed complementary or overlapping removal mechanisms. Steps with high removal capacities represent the conditions where the physiochemical differences between prions and therapeutic proteins are most significant. The results support the intrinsic ability of certain plasma protein manufacturing steps to remove prions in case of an unlikely contamination, providing a safeguard to products. © 2012 American Association of Blood Banks.

  10. Catalytic antibodies in clinical and experimental pathology: human and mouse models.

    Science.gov (United States)

    Ponomarenko, Natalya A; Durova, Oxana M; Vorobiev, Ivan I; Aleksandrova, Elena S; Telegin, Georgy B; Chamborant, Olga G; Sidorik, Lyudmila L; Suchkov, Sergei V; Alekberova, Zemfira S; Gnuchev, Nikolay V; Gabibov, Alexander G

    2002-11-01

    Most of the data accumulated through studies on natural catalytic autoantibodies indicate that production scales up markedly in pathological abnormalities. We have previously described an increased level of DNA-hydrolyzing autoantibodies in the sera of patients with various autoimmune disorders [systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma], HIV infection and lymphoproliferative diseases accompanied by autoimmune manifestations. In the present study, we show that an increased level of catalytic activity of autoantibodies can be observed in the sera of autoimmune mice, thus providing a fundamental insight into the medical relevance of abzymes. Polyclonal autoantibodies purified from sera of NZB/W, MRL-lpr/lpr and SJL/J mice show proteolytic and DNA-hydrolyzing activities, as opposed to those harvested from non-autoimmune BALB/c mice. The expressiveness of the catalytic activity was strongly dependent on the age of the animal. The highest levels of catalytic activity were found in the sera of mice aged between 8 and 12 months; the lowest level was typical of younger animals whose age ranged from 6 to 8 weeks. Specific inhibition assays of the catalytic activities were performed to throw light on the nature of the abzyme activity. Within a cohort of aging animals, a strong correlation between marked autoimmune abnormalities and levels of catalytic activities has been established. Nonimmunized SJL/J mice revealed specific immune responses to myelin basic protein (MBP), skeletal muscle myosin (skMyo) and cardiac myosin (Myo), and highly purified antibodies from their serum show specific proteolytic attack against the target antigens. This finding prompted us to undertake a more detailed study of specific antibody-mediated proteolysis in diseased humans. A targeted catalytic response was originally demonstrated against MBP and Myo in multiple sclerosis and myocarditis patients, respectively.

  11. Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species.

    Science.gov (United States)

    Carmona-Salazar, Laura; El Hafidi, Mohammed; Gutiérrez-Nájera, Nora; Noyola-Martínez, Liliana; González-Solís, Ariadna; Gavilanes-Ruíz, Marina

    2015-01-01

    It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical-chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Plasma membrane isolation using immobilized concanavalin A magnetic beads.

    Science.gov (United States)

    Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

    2012-01-01

    Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

  13. Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes

    International Nuclear Information System (INIS)

    Chen, J.W.; Cunningham, M.D.; Galton, N.; Michaelis, E.K.

    1988-01-01

    Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an ∼ 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger M/sub r/ protein in synaptic membranes. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 + M, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L[ 3 H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes

  14. [Osteoplastic materials in the treatment of parodontosis].

    Science.gov (United States)

    Okropiridze, T V; Menabde, G T; Gogilashvili, K T; Margvelashvili, V V

    2008-01-01

    Efficiency of bioactive osteoplastic composition HA-phage at a surgical stage of combined treatment of chronic generalized periodontitis was examined. The results obtained in the control and experimental groups proved that HA-phage allows reducing the time of treatment and to achieve remission of the disease. Out of 92 patients with chronic generalized periodontitis 52 undertook microplasmic scalpel-irradiator in complex treatment. The treatment course comprises 3-5 procedures. The quantity of manipulations is determined by the pathological process. Before and after the treatment the radiographic investigations were carried out in persons of both groups. 18 months later after the operation a bone regeneration was shown in 87,8% cases in the investigated group and 60,1%--in the control group patients. The therapy has shown positive results. So, the new method of treatment of chronic generalized periodontitis may prove to be more practical in clinical dentistry. Currently, it is possible to isolate highly purified BMSSCs from bone marrow using STRO-1. The management of periodontal defects has been an ongoing challenge in clinical periodontics. This is mainly a result of the fact that the tissues which comprise the periodontium, the periodontal ligament, and the cementum and alveolar bone represent three unique tissues in their own right. Thus, reconstruction of the periodontium is not just a simple matter of regenerating one tissue but involves at least three quite diverse and unique tissues. Taking into account the results of recent studies we hypothesize that mesenchymal stem cells transplantation should be a perspective approach in the treatment of periodontal defects. For this purpose we plan to perform an experimental investigations which will include an induction of periodontitis, purification of the bone marrow stem cells and their transplantation.

  15. Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Elke Gabriel

    Full Text Available Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in

  16. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

    Directory of Open Access Journals (Sweden)

    Geyer Matthias

    2007-10-01

    Full Text Available Abstract Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.

  17. Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells.

    Directory of Open Access Journals (Sweden)

    Yan Cheng

    Full Text Available Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.

  18. Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells.

    Directory of Open Access Journals (Sweden)

    Robin R Craven

    Full Text Available Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal alpha-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus alpha-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant alpha-hemolysin, we now demonstrate that alpha-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant alpha-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to alpha-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by alpha-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs to induce secretion of pro-inflammatory cytokines IL-1beta and IL-18. Additionally, alpha-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1. These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.

  19. Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

    Science.gov (United States)

    Craven, Robin R.; Gao, Xi; Allen, Irving C.; Gris, Denis; Wardenburg, Juliane Bubeck; McElvania-TeKippe, Erin; Ting, Jenny P.; Duncan, Joseph A.

    2009-01-01

    Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation. PMID:19826485

  20. Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells.

    Science.gov (United States)

    Craven, Robin R; Gao, Xi; Allen, Irving C; Gris, Denis; Bubeck Wardenburg, Juliane; McElvania-Tekippe, Erin; Ting, Jenny P; Duncan, Joseph A

    2009-10-14

    Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal alpha-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus alpha-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant alpha-hemolysin, we now demonstrate that alpha-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant alpha-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to alpha-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by alpha-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1beta and IL-18. Additionally, alpha-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.

  1. Expansion of mycobacterium-reactive gamma delta T cells by a subset of memory helper T cells.

    Science.gov (United States)

    Vila, L M; Haftel, H M; Park, H S; Lin, M S; Romzek, N C; Hanash, S M; Holoshitz, J

    1995-04-01

    Human gamma delta T cells expressing the V gamma 9/V delta 2 T-cell receptor have been previously found to proliferate in response to certain microorganisms and to expand throughout life, presumably because of extrathymic activation by foreign antigens. In vitro expansion of V gamma 9/V delta 2 cells by mycobacteria has been previously shown to be dependent on accessory cells. In order to gain an insight into the mechanisms involved in the expansion of these cells, we have undertaken to identify the peripheral blood subset of cells on which proliferation of V gamma 9/V delta 2 cells in response to mycobacteria is dependent. Contrary to their role in antigen presentation to alpha beta T cells, professional antigen-presenting cells, such as monocytes, B cells, and dendritic cells, were unable to provide the cellular support for the expansion of V gamma 9/V delta 2 cells. Selective depletion of T-cell subsets, as well as the use of highly purified T-cell populations, indicated that the only subset of peripheral blood cells that could expand V gamma 9/V delta 2 cells were CD4+ CD45RO+ CD7- alpha beta T cells. These cells underwent distinct intracellular signaling events after stimulation with the mycobacterial antigen. Expansion of V gamma 9/V delta 2 cells by alpha beta T cells was dependent on cell-cell contact. This is the first evidence that a small subset of the memory helper T-cell population is exclusively responsible for the peripheral expansion of V gamma 9/V delta 2 cells. These data illustrate a unique aspect of antigen recognition by gamma delta T cells and provide new means to study their immune defense role.

  2. Sequential Versus Continual Purified Urinary FSH/hCG in Men With Idiopathic Hypogonadotropic Hypogonadism.

    Science.gov (United States)

    Zhang, Manna; Tong, Guoyu; Liu, Yanling; Mu, Yiming; Weng, Jianping; Xue, Yaoming; Luo, Zuojie; Xue, Yuanming; Shi, Lixin; Wu, Xueyan; Sun, Shouyue; Zhu, Yanhua; Cao, Ying; Zhang, Jie; Huang, Hong; Niu, Ben; Li, Hong; Guo, Qinghua; Gao, Yan; Li, Zhibin; Ning, Guang; Zhu, Dalong; Li, Xiaoying

    2015-06-01

    Gonadotropin therapy using a human chorionic gonadotropin (hCG) and FSH preparation is an effective regimen in inducing masculinization and spermatogenesis in men with idiopathic hypogonadotropic hypogonadism (IHH). However, the high cost of medication and frequent injections affect compliance. The aim of this study was to determine the efficacy of sequential use of highly purified urinary FSH (uFSH)/hCG in men with IHH. A randomized, open-label, prospective, controlled noninferiority trial with an 18-month follow-up was conducted in 9 tertiary hospitals. A total of 67 Chinese men with IHH were randomly allocated into group A receiving continual uFSH (75 U, 3 times a week) and hCG (2000 U, twice a week) injection and group B receiving sequential uFSH (75 U, 3 times a week every other 3 months) and hCG (2000 U, twice a week) injection. The primary outcome was the proportion of subjects with a sperm concentration of ≥ 1.0 × 10(6)/mL during the 18 months. The efficacy between groups A and B was compared for noninferiority. Of the patients, 17/33 (51.5%) receiving continual uFSH/hCG and 19/34 (55.9%) receiving sequential uFSH/hCG achieved sperm concentrations of ≥ 1.0 × 10(6)/mL. The efficacy in the sequential uFSH/hCG group was not inferior to that in the continual uFSH/hCG group (noninferiority, P = .008) by intention-to-treat analysis. The efficacy of the sequential uFSH/hCG regimen is not inferior to that of the continual uFSH/hCG regimen in inducing spermatogenesis and masculinization of patients with IHH.

  3. Seawater-driven forward osmosis for enriching nitrogen and phosphorous in treated municipal wastewater: effect of membrane properties and feed solution chemistry.

    Science.gov (United States)

    Xue, Wenchao; Tobino, Tomohiro; Nakajima, Fumiyuki; Yamamoto, Kazuo

    2015-02-01

    Seawater-driven forward osmosis (FO) is considered to be a novel strategy to concentrate nutrients in treated municipal wastewater for further recovery as well as simultaneous discharge of highly purified wastewater into the sea with low cost. As a preliminary test, the performance of FO membranes in concentrating nutrients was investigated by both batch experiments and model simulation approaches. With synthetic seawater as the draw solution, the dissolved organic carbon, phosphate, and ammonia in the effluent from a membrane bioreactor (MBR) treating municipal wastewater were 2.3-fold, 2.3-fold, and 2.1-fold, respectively, concentrated by the FO process with approximately 57% of water reduction. Most of the dissolved components, including trace metals in the MBR effluent, were highly retained (>80%) in the feed side, indicating high water quality of permeate to be discharged. The effect of membrane properties on the nutrient enrichment performance was investigated by comparing three types of FO membranes. Interestingly, a polyamide membrane possessing a high negative charge demonstrated a poor capability of retaining ammonia, which was hypothesized because of an ion exchange-like mechanism across the membrane prompted by the high ionic concentration of the draw solution. A feed solution pH of 7 was demonstrated to be an optimum condition for improving the overall retention of nutrients, especially for ammonia because of the pH-dependent speciation of ammonia/ammonium forms. The modeling results showed that higher than 10-fold concentrations of ammonia and phosphate are achievable by seawater-driven FO with a draw solution to feed solution volume ratio of 2:1. The enriched municipal wastewater contains nitrogen and phosphorous concentrations comparable with typical animal wastewater and anaerobic digestion effluent, which are used for direct nutrient recovery. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Anaerobic degradation of benzene by enriched consortia with humic acids as terminal electron acceptors

    Energy Technology Data Exchange (ETDEWEB)

    Cervantes, Francisco J., E-mail: fjcervantes@ipicyt.edu.mx [Division de Ciencias Ambientales, Instituto Potosino de Investigacion Cientifica y Tecnologica (IPICyT), Camino a la Presa San Jose 2055, Col. Lomas 4a. Seccion, San Luis Potosi, SLP, 78216 Mexico (Mexico); Mancilla, Ana Rosa; Toro, E. Emilia Rios-del [Division de Ciencias Ambientales, Instituto Potosino de Investigacion Cientifica y Tecnologica (IPICyT), Camino a la Presa San Jose 2055, Col. Lomas 4a. Seccion, San Luis Potosi, SLP, 78216 Mexico (Mexico); Alpuche-Solis, Angel G.; Montoya-Lorenzana, Lilia [Division de Biologia Molecular, Instituto Potosino de Investigacion Cientifica y Tecnologica (IPICyT), Camino a la Presa San Jose 2055, Col. Lomas 4a. Seccion, San Luis Potosi, SLP, 78216 Mexico (Mexico)

    2011-11-15

    Highlights: {yields} Enriched consortia were able to couple the anaerobic degradation of benzene to the reduction of humic acids. {yields} Electron-equivalents derived from anaerobic benzene oxidation were highly recovered as reduced humic acids. {yields} Several species from classes {beta}-, {delta}- and {gamma}-Proteobacteria were enriched during the anaerobic degradation of benzene. - Abstract: The anaerobic degradation of benzene coupled to the reduction of humic acids (HA) was demonstrated in two enriched consortia. Both inocula were able to oxidize benzene under strict anaerobic conditions when the humic model compound, anthraquinone-2,6-disulfonate (AQDS), was supplied as terminal electron acceptor. An enrichment culture originated from a contaminated soil was also able to oxidize benzene linked to the reduction of highly purified soil humic acids (HPSHA). In HPSHA-amended cultures, 9.3 {mu}M of benzene were degraded, which corresponds to 279 {+-} 27 micro-electron equivalents ({mu}Eq) L{sup -1}, linked to the reduction of 619 {+-} 81 {mu}Eq L{sup -1} of HPSHA. Neither anaerobic benzene oxidation nor reduction of HPSHA occurred in sterilized controls. Anaerobic benzene oxidation did not occur in soil incubations lacking HPSHA. Furthermore, negligible reduction of HPSHA occurred in the absence of benzene. The enrichment culture derived from this soil was dominated by two {gamma}-Proteobacteria phylotypes. A benzene-degrading AQDS-reducing enrichment originated from a sediment sample showed the prevalence of different species from classes {beta}-, {delta}- and {gamma}-Proteobacteria. The present study provides clear quantitative demonstration of anaerobic degradation of benzene coupled to the reduction of HA.

  5. Preparation of mono-radioiodinated tracers for study of the in vivo metabolism of atrial natriuretic peptide in humans

    International Nuclear Information System (INIS)

    Clerico, A.; Marastoni, M.; Del Chicca, M.G.; Giannessi, D.; Del Ry, S.; Andreassi, M.G.; Sabatino, L.; Iascone, M.R.; Biagini, A.; Donato, L.

    1995-01-01

    The authors evaluate the optimum chemical conditions for labelling atrial natriuretic peptide (ANP) and its metabolites and for preparing highly purified radiotracers which can be used for in vivo kinetic studies of ANP in humans. Synthetic α h 1-28 ANP and some hormone metabolites were iodinated with Na 125 I or Na 131 I by means of the lactoperoxidase (ANP) or the chloramine-T (ANP metabolites) technique. The biological activity of labelled ANP was tested by means of a binding study using mouse cardiac membranes. A high-performance liquid chromatography (HPLC) procedure was used to purify the labelled hormone and the principal labelled metabolites in venous plasma samples collected up to 50 min after the injection of 125 I-labelled ANP from nine healthy men. The main ANP kinetic parameters were derived from the disappearance curves of the [ 125 I]ANP, which were satisfactorily fitted by a bi-exponential function in all subjects. The main advantages of this tracer technique are high accuracy, allowing the identification of the metabolites produced in vivo under steady-state conditions after injection of the precursor (labelled hormone) high sensitivity, allowing the detection of minimal quantities of metabolites high specificity, allowing the detection of possible in vitro artefactual generation of cleavage products of ANP using an internal labelled standard. Utilizing this tracer method, it was possible to estimate the principal parameters of ANP kinetics and also to plot the appearance curves of the labelled metabolites produced in vivo after the injection of the labelled precursor. (orig.). With 5 figs

  6. Hepatic taurine transport: a Na+-dependent carrier on the basolateral plasma membrane

    International Nuclear Information System (INIS)

    Bucuvalas, J.C.; Goodrich, A.L.; Suchy, F.J.

    1987-01-01

    Highly purified rat basolateral liver plasma membrane vesicles were used examine the mechanism and the driving forces for hepatic uptake of the β-amino acid, taurine. An inwardly directed 100 mM NaCl gradient stimulated the initial rate of taurine uptake and energized a transient twofold accumulation of taurine above equilibrium (overshoot). In contrast, uptake was slower and no overshoot was detected in the presence of a KCl gradient. A negative intravesicular electrical potential generated by the presence of permeant anions or an outwardly directed K + gradient with valinomycin increased Na + -stimulated taurine uptake. External Cl - stimulated Na + -dependent taurine uptake independent of effects on the transmembrane electrical potential difference. Na + -dependent taurine uptake showed a sigmoidal dependence on extravesicular Na + concentration, suggesting multiple Na + ions are involved in the translocation of each taurine molecule. Na + -dependent taurine uptake demonstrated Michaelis-Menten kinetics with a maximum velocity of 0.537 nmol x mg protein -1 x min -1 and an apparent K/sub m/ of 174 μM. [ 3 H]taurine uptake was inhibited by the presence of excess unlabeled taurine, β-alanine, or hypotaurine but not by L-glutamine or L-alanine. In summary, using basolateral liver plasma membrane vesicles, the authors have shown that hepatic uptake of taurine occurs by a carrier-mediated, secondary active transport process specific for β-amino acids. Uptake is electrogenic, stimulated by external Cl - , and requires multiple Na + ions for the translocation of each taurine molecule

  7. [The role of balanced low-protein diet in inhibition of predialysis chronic kidney disease progression in patients with systemic diseases].

    Science.gov (United States)

    Milovanov, Iu S; Lysenko, L V; Milovanova, L Iu; Dobrosmyslov, I A

    2009-01-01

    To evaluate the effects of low-protein diet (LPD) balanced by addition of highly energetic mix and essential keto/amino acids on inhibition of renal failure in patients with systemic diseases with predialysis stages of chronic disease of the kidney (CDK). Forty six patients with stage III--IV of CDK in systemic diseases (33 SLE patients and 13 with systemic vasculitis) were randomized into three groups. Group 1 consisted of 18 patients with CDK (10 with stage III and 8 with stage IV). They received LPD (0.6 g/kg/day) with addition of essential keto/amino acids for 24-48 months. Group 2 of 18 CDK patients with the same stages received the same diet but greater amount of vegetable protein (highly purified soya protein) to 0.3 g/kg/day in highly energetic nutrient mixture. Group 3--10 CDK patients (7 with stage III and 3 with stage IV) received free diet. Group 1 and 2 patients received LPD irrespective of the nutrient status assessed basing on anthropometric and other data. Protein consumption and caloric value were estimated by 3-day food diary. Before diet therapy, out of 46 examinees nutrient status was abnormal in 45.7% patients. Both variants of LPD were well tolerated and nutrient status was corrected while the rate of nutritive disorders in group 3 increased 1.5-fold (from 40 to 60%) with progression of renal failure. Intake of LPD diet for at least a year reduced glomerular filtration rate inhibition, especially in addition of highly energetic mixture. Early (predialysis) restriction of diet protein (0.6 g/kg/day) with addition of highly energetic mixture and essential keto/amino acids improves a nutritive status of CDK patients and inhibits GFR decline.

  8. Establishing a comprehensive risk assessment document for Fernald

    International Nuclear Information System (INIS)

    Nelson, K.A.; Janke, R.C.

    1995-01-01

    While in active operation from 1952 until 1989, the Feed Material Production Center (FMPC),currently referred to as the Fernald Environmental Management Project (FEMP), produced highly purified uranium metal for ultimate use in the manufacture of nuclear weapons. In 1986 the U.S. Environmental Protection Agency (EPA) and the DOE entered into a Federal Facility Compliance Agreement covering environmental impacts associated with the FMPC. In response to the FFCA, a sitewide Remedial Investigation/Feasibility Study (RI/FS) was initiated. Production was permanently suspended at the facility in 1989, and the focus has since shifted to environmental restoration and waste management activities. The original RI/FS work plan identified 39 units, but these were later categorized and grouped into 5 larger, more manageable units as follows: (1) operable unit 1 - waste pit area; (2) operable unit 2 - other solid waste units; (3) operable unit 3 - former production area; (4) operable unit 4- silos I through 4; and (5) operable unit 5 - environmental media. On June 29, 1990, a consent agreement (under Sec. 120 and 106[a] of CERCLA) between the DOE and EPA became effective. The purpose of this agreement was to achieve consistency between the operable units and ensure commitments to the RI/FS program without altering the underlying objectives. The consent agreement was amended the following year to revise the schedules for completing the remediation of the five operable units and to direct operable unit integration to ensure compliance with the residual risk requirements of the National Hazardous Substances and Oil Contingency Plan. This Amended Consent Agreement (ACA) was signed on September 20, 1991, and became effective on December 19, 1991. In accordance with provisions of the ACA, a methodology has been prepared for performing risk assessments and establishing risk-based remedial action goals at the FEMP

  9. Identification and Characterization of a Bovine Sperm Acrosomal Matrix Protein and its Mechanism of Interaction with Acrosomal Hydrolases

    Science.gov (United States)

    Nagdas, Subir K; Smith, Linda; Mcnamara, Allen; Hernandez–Encarnacion, Luisa; Medina-Ortiz, Ilza

    2015-01-01

    Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads is comprised of 54, 50, 45, and 38–19kDa polypeptides. The objective of this study is to identify and to characterize the 45kDa (OMC45) polypeptide and to define its role in binding acrosomal hydrolases and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI–TOF–TOF yielded 8 peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X–100–permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti–OMC45 and anti–TEKT3. The OMC45 polypeptide was solubilized by RIPA (radio-immunoprecipitation assay) buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti–OMC45 immunoprecipitation pellet. An identical blot stained with anti–TEKT3 exhibited the presence of TEKT3 polypeptide in the anti–OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide; that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N–linked and O–linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N–acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid

  10. Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity

    Energy Technology Data Exchange (ETDEWEB)

    Nakanishi-Matsui, Mayumi, E-mail: nakanim@iwate-med.ac.jp [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Futai Special Laboratory, Yahaba, Iwate 028-3694 (Japan); Yano, Shio; Matsumoto, Naomi; Futai, Masamitsu [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Futai Special Laboratory, Yahaba, Iwate 028-3694 (Japan)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. Black-Right-Pointing-Pointer The multinuclear cells are formed through cell-cell fusion in the presence of Ca{sup 2+}. Black-Right-Pointing-Pointer The multinuclear cells do not express osteoclast-specific enzymes. Black-Right-Pointing-Pointer They internalized more and larger beads than mononuclear cells and osteoclasts. -- Abstract: Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca{sup 2+}. The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor {kappa}B ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6-15 {mu}m) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies.

  11. Cellular content of biomolecules in sub-seafloor microbial communities

    Science.gov (United States)

    Braun, Stefan; Morono, Yuki; Becker, Kevin W.; Hinrichs, Kai-Uwe; Kjeldsen, Kasper U.; Jørgensen, Bo B.; Lomstein, Bente Aa.

    2016-09-01

    Microbial biomolecules, typically from the cell envelope, can provide crucial information about distribution, activity, and adaptations of sub-seafloor microbial communities. However, when cells die these molecules can be preserved in the sediment on timescales that are likely longer than the lifetime of their microbial sources. Here we provide for the first time measurements of the cellular content of biomolecules in sedimentary microbial cells. We separated intact cells from sediment matrices in samples from surficial, deeply buried, organic-rich, and organic-lean marine sediments by density centrifugation. Amino acids, amino sugars, muramic acid, and intact polar lipids were analyzed in both whole sediment and cell extract, and cell separation was optimized and evaluated in terms of purity, separation efficiency, taxonomic resemblance, and compatibility to high-performance liquid chromatography and mass spectrometry for biomolecule analyses. Because cell extracts from density centrifugation still contained considerable amounts of detrital particles and non-cellular biomolecules, we further purified cells from two samples by fluorescence-activated cell sorting (FACS). Cells from these highly purified cell extracts had an average content of amino acids and lipids of 23-28 fg cell-1 and 2.3 fg cell-1, respectively, with an estimated carbon content of 19-24 fg cell-1. In the sediment, the amount of biomolecules associated with vegetative cells was up to 70-fold lower than the total biomolecule content. We find that the cellular content of biomolecules in the marine subsurface is up to four times lower than previous estimates. Our approach will facilitate and improve the use of biomolecules as proxies for microbial abundance in environmental samples and ultimately provide better global estimates of microbial biomass.

  12. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  13. Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex.

    Science.gov (United States)

    Azarkan, Mohamed; Matagne, André; Wattiez, Ruddy; Bolle, Laetitia; Vandenameele, Julie; Baeyens-Volant, Danielle

    2011-10-01

    The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Environmental factors influencing gene transfer agent (GTA mediated transduction in the subtropical ocean.

    Directory of Open Access Journals (Sweden)

    Lauren D McDaniel

    Full Text Available Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT. However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI and ambient bacterial abundance. These results indicate that GTA

  15. Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs.

    Science.gov (United States)

    Bernard, Marie-Clotilde; Barban, Véronique; Pradezynski, Fabrine; de Montfort, Aymeric; Ryall, Robert; Caillet, Catherine; Londono-Hayes, Patricia

    2015-01-01

    HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.

  16. Anthelmintic Effect of Biocompatible Zinc Oxide Nanoparticles (ZnO NPs on Gigantocotyle explanatum, a Neglected Parasite of Indian Water Buffalo.

    Directory of Open Access Journals (Sweden)

    Yasir Akhtar Khan

    Full Text Available Helminth parasites of veterinary importance cause huge revenue losses to agrarian economy worldwide. With the emergence of drug resistance against the current formulations, there is a need to focus on the alternative approaches in order to control this menace. In the present study, biocompatible zinc oxide nanoparticles (ZnO NPs were used to see their in vitro effect on the biliary amphistomes, Gigantocotyle explanatum, infecting Bubalus bubalis because these nanoparticles are involved in generation of free radicals that induce oxidative stress, resulting in disruption of cellular machinery. The ZnO NPs were synthesized by using egg albumin as a biotemplate and subsequently characterized by Scanning Electron Microscopy (SEM, Transmission Electron Microscopy (TEM, X-ray Diffraction and Spectrophotometrical, which showed that ZnO NPs were highly purified wurtzite type polycrystals, with a mean size of 16.7 nm. When the parasites were treated with lower concentrations (0.004% and 0.008% of the ZnO NPs, the worms mounted a protective response by stimulating the antioxidant system but the treatment of G. explanatum with 0.012% ZnO NPs produced significant inhibition of the antioxidant enzymes like superoxide dismutase (SOD (p< 0.05 and glutathione S- transferase (GST (p<0.01, while the level of malondialdehyde (MDA, a lipid peroxidation marker, was significantly (p< 0.01 elevated. SEM and histopathology revealed pronounced tegumental damage showing the disruption of surface papillae and the annulations, particularly in the posterior region near acetabulum. The under expression of a number of polypeptides, loss of worm motility in a time dependent manner, further reflect strong anthelmintic potential of ZnO NPs. It can be concluded that the anthelmintic effect might be due to the production of reactive oxygen species that target a variety of macromolecules such as nucleic acid, protein and lipids which are involved in different cellular processes.

  17. Anthelmintic Effect of Biocompatible Zinc Oxide Nanoparticles (ZnO NPs) on Gigantocotyle explanatum, a Neglected Parasite of Indian Water Buffalo

    Science.gov (United States)

    Khan, Yasir Akhtar; Singh, Braj Raj; Ullah, Rizwan; Shoeb, Mohd; Naqvi, Alim H.; Abidi, Syed M. A.

    2015-01-01

    Helminth parasites of veterinary importance cause huge revenue losses to agrarian economy worldwide. With the emergence of drug resistance against the current formulations, there is a need to focus on the alternative approaches in order to control this menace. In the present study, biocompatible zinc oxide nanoparticles (ZnO NPs) were used to see their in vitro effect on the biliary amphistomes, Gigantocotyle explanatum, infecting Bubalus bubalis because these nanoparticles are involved in generation of free radicals that induce oxidative stress, resulting in disruption of cellular machinery. The ZnO NPs were synthesized by using egg albumin as a biotemplate and subsequently characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction and Spectrophotometrical, which showed that ZnO NPs were highly purified wurtzite type polycrystals, with a mean size of 16.7 nm. When the parasites were treated with lower concentrations (0.004% and 0.008%) of the ZnO NPs, the worms mounted a protective response by stimulating the antioxidant system but the treatment of G. explanatum with 0.012% ZnO NPs produced significant inhibition of the antioxidant enzymes like superoxide dismutase (SOD) (p< 0.05) and glutathione S- transferase (GST) (p<0.01), while the level of malondialdehyde (MDA), a lipid peroxidation marker, was significantly (p< 0.01) elevated. SEM and histopathology revealed pronounced tegumental damage showing the disruption of surface papillae and the annulations, particularly in the posterior region near acetabulum. The under expression of a number of polypeptides, loss of worm motility in a time dependent manner, further reflect strong anthelmintic potential of ZnO NPs. It can be concluded that the anthelmintic effect might be due to the production of reactive oxygen species that target a variety of macromolecules such as nucleic acid, protein and lipids which are involved in different cellular processes. PMID:26177503

  18. Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.

    Science.gov (United States)

    Shetty, Jagathpala; Sinville, Rondedrick; Shumilin, Igor A; Minor, Wladek; Zhang, Jianhai; Hawkinson, Jon E; Georg, Gunda I; Flickinger, Charles J; Herr, John C

    2016-05-01

    The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

    Energy Technology Data Exchange (ETDEWEB)

    Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D. [Centenary Institute of Cancer Medicine and Cell Biology, Sydney (Australia)] [and others

    1995-05-01

    Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.

  20. Prevention of biofilm formation in dialysis water treatment systems.

    Science.gov (United States)

    Smeets, Ed; Kooman, Jeroen; van der Sande, Frank; Stobberingh, Ellen; Frederik, Peter; Claessens, Piet; Grave, Willem; Schot, Arend; Leunissen, Karel

    2003-04-01

    Biofilm formations in dialysis systems may be relevant because they continuously release bacterial compounds and are resistant against disinfection. The aim of the study was to compare the development of biofilm between a water treatment system based on a single reverse osmosis unit producing purified dialysate water [bacterial count, 350 colony-forming unit (CFU)/L] (center A) and a water treatment system based on double reverse osmosis and electric deionization, which is continuously disinfected with ultraviolet light and treated with ozone once a week (bacterial count, 1 CFU/L) (center B). During a period of 12 weeks, biofilm formation was studied in the tubing segment between the water piping and the dialysis module, using four dialysis monitors in each center. On a weekly basis, tubing samples of 5 cm length (N = 96) were taken under aseptic conditions and investigated for microbiologic contamination [cystine lactose electrolyte-deficient (CLED) Agar], endotoxin levels [limulus amoeben lysate (LAL) gel test, cutoff value, 0.0125 EU/mL], and biofilm formation [electron scanning microscopy (SEM)]. In center A, tube cultures were positive (>100 CFU/mL) in 16% of samples at 22 degrees C and 37 degrees C, compared to 3% of samples of center B (P tubing samples of center A and negative in all of the samples of center B (P < 0.05). Biofilm was present in 91.7% of the samples of center A (Fig. 1), and only present in one sample (taken after 9 weeks) of center B (P < 0.05) (Fig. 2). In center A, biofilm formation was already observed after 1 week. In contrast to a standard water treatment system producing purified water, the use of a system producing highly purified water, which is also treated with regular disinfection procedures, leads to a significant reduction in biofilm formation, bacterial growth, and endotoxin levels in a highly vulnerable part of a water treatment system.

  1. Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

    Directory of Open Access Journals (Sweden)

    H. Carvalho

    2008-04-01

    Full Text Available Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1 plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

  2. Anaerobic degradation of benzene by enriched consortia with humic acids as terminal electron acceptors

    International Nuclear Information System (INIS)

    Cervantes, Francisco J.; Mancilla, Ana Rosa; Toro, E. Emilia Rios-del; Alpuche-Solis, Angel G.; Montoya-Lorenzana, Lilia

    2011-01-01

    Highlights: → Enriched consortia were able to couple the anaerobic degradation of benzene to the reduction of humic acids. → Electron-equivalents derived from anaerobic benzene oxidation were highly recovered as reduced humic acids. → Several species from classes β-, δ- and γ-Proteobacteria were enriched during the anaerobic degradation of benzene. - Abstract: The anaerobic degradation of benzene coupled to the reduction of humic acids (HA) was demonstrated in two enriched consortia. Both inocula were able to oxidize benzene under strict anaerobic conditions when the humic model compound, anthraquinone-2,6-disulfonate (AQDS), was supplied as terminal electron acceptor. An enrichment culture originated from a contaminated soil was also able to oxidize benzene linked to the reduction of highly purified soil humic acids (HPSHA). In HPSHA-amended cultures, 9.3 μM of benzene were degraded, which corresponds to 279 ± 27 micro-electron equivalents (μEq) L -1 , linked to the reduction of 619 ± 81 μEq L -1 of HPSHA. Neither anaerobic benzene oxidation nor reduction of HPSHA occurred in sterilized controls. Anaerobic benzene oxidation did not occur in soil incubations lacking HPSHA. Furthermore, negligible reduction of HPSHA occurred in the absence of benzene. The enrichment culture derived from this soil was dominated by two γ-Proteobacteria phylotypes. A benzene-degrading AQDS-reducing enrichment originated from a sediment sample showed the prevalence of different species from classes β-, δ- and γ-Proteobacteria. The present study provides clear quantitative demonstration of anaerobic degradation of benzene coupled to the reduction of HA.

  3. Passive immunotherapy for influenza A H5N1 virus infection with equine hyperimmune globulin F(ab'2 in mice

    Directory of Open Access Journals (Sweden)

    Li Yanbin

    2006-03-01

    Full Text Available Abstract Background Avian influenza virus H5N1 has demonstrated considerable pandemic potential. Currently, no effective vaccines for H5N1 infection are available, so passive immunotherapy may be an alternative strategy. To investigate the possible therapeutic effect of antibody against highly pathogenic H5N1 virus on a mammal host, we prepared specific equine anti-H5N1 IgGs from horses vaccinated with inactivated H5N1 virus, and then obtained the F(ab'2 fragments by pepsin digestion of IgGs. Methods The horses were vaccinated with inactivated H5N1 vaccine to prepare anti-H5N1 IgGs. The F(ab'2 fragments were purified from anti-H5N1 hyperimmune sera by a protocol for 'enhanced pepsin digestion'. The protective effect of the F(ab'2 fragments against H5N1 virus infection was determined in cultured MDCK cells by cytopathic effect (CPE assay and in a BALB/c mouse model by survival rate assay. Results By the protocol for 'enhanced pepsin digestion', total 16 g F(ab'2 fragments were finally obtained from one liter equine antisera with the purity of over 90%. The H5N1-specific F(ab'2 fragments had a HI titer of 1:1024, and the neutralization titre of F(ab'2 reached 1: 2048. The in vivo assay showed that 100 μg of the F(ab'2 fragments could protect BALB/c mice infected with a lethal dose of influenza H5N1 virus. Conclusion The availability of highly purified H5N1-specific F(ab'2 fragments may be promising for treatment of influenza H5N1 infection. Our work has provided experimental support for the application of the therapeutic equine immunoglobulin in future large primate or human trials.

  4. N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

    International Nuclear Information System (INIS)

    Tang, Nan-Hong; Chen, Yan-Lin; Wang, Xiao-Qian; Li, Xiu-Jin; Wu, Yong; Zou, Qi-Lian; Chen, Yuan-Zhong

    2010-01-01

    Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved. The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively. Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity. Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents

  5. New trends in ganglioside chemistry

    International Nuclear Information System (INIS)

    Sonnino, S.; Ghidoni, R.; Gazzotti, G.; Acquotti, D.; Tettamanti, G.

    1988-01-01

    New methods have been developed for the preparation of highly purified gangliosides, homogeneous in the saccharide, long chain base, and fatty acid moieties and gangliosides carrying different kinds of labelled probes. Gangliosides, homogeneous in the oligosaccharide portion, were prepared by preparative normal phase HPLC on a Lichrosorb-NH-2 column, using a gradient of acetonitrile-phosphate buffer, pH 5.6, as solvent system. Each class of ganglioside (from monosialo- to tetrasialogangliosides) was then submitted to reversed phase HPLC on a preparative RP-8 column, using acetonitrile-5 mM phosphate buffer, pH 7, as solvent system, to obtain gangliosides homogeneous in the long chain base moiety. Gangliosides containing C18 and C20 sphinganine were prepared by catalytic hydrogenation of the corresponding unsaturated gangliosides. GM1 with homogeneous acyl chain was prepared by alkaline hydrolysis in the presence of tetramethylammonium hydroxide, followed by re-N-acylation, carried out in the presence of dimethylaminopropyl, ethylcarbodiimide and natural fatty acids, or of mixed anhydride of ethylchloroformate and 14C-stearic acid, and re-N-acetylation performed with acetic anhydride or labelled acetic anhydride. The GM1 derivative, de-acetylated at the level of sialic acid, also produced by alkaline treatment of GM1, was submitted to re-N-acetylation with 14C-acetic anhydride to produce specifically 14C-labelled GM1. Re-N-acylation was carried out a) in the presence of dimethylaminopropyl, ethylcarbodiimide and natural fatty acids, b) with mixed anhydride of ethylchloroformate and 14C-stearic acid. After re-N-acylations, re-N-acetylation was performed with acetic anhydride or labelled acetic anhydride. 53 references

  6. Monoclonal antibody heterogeneity analysis and deamidation monitoring with high-performance cation-exchange chromatofocusing using simple, two component buffer systems.

    Science.gov (United States)

    Kang, Xuezhen; Kutzko, Joseph P; Hayes, Michael L; Frey, Douglas D

    2013-03-29

    The use of either a polyampholyte buffer or a simple buffer system for the high-performance cation-exchange chromatofocusing of monoclonal antibodies is demonstrated for the case where the pH gradient is produced entirely inside the column and with no external mixing of buffers. The simple buffer system used was composed of two buffering species, one which becomes adsorbed onto the column packing and one which does not adsorb, together with an adsorbed ion that does not participate in acid-base equilibrium. The method which employs the simple buffer system is capable of producing a gradual pH gradient in the neutral to acidic pH range that can be adjusted by proper selection of the starting and ending pH values for the gradient as well as the buffering species concentration, pKa, and molecular size. By using this approach, variants of representative monoclonal antibodies with isoelectric points of 7.0 or less were separated with high resolution so that the approach can serve as a complementary alternative to isoelectric focusing for characterizing a monoclonal antibody based on differences in the isoelectric points of the variants present. Because the simple buffer system used eliminates the use of polyampholytes, the method is suitable for antibody heterogeneity analysis coupled with mass spectrometry. The method can also be used at the preparative scale to collect highly purified isoelectric variants of an antibody for further study. To illustrate this, a single isoelectric point variant of a monoclonal antibody was collected and used for a stability study under forced deamidation conditions. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Eicosapentaenoic acid ameliorates non-alcoholic steatohepatitis in a novel mouse model using melanocortin 4 receptor-deficient mice.

    Science.gov (United States)

    Konuma, Kuniha; Itoh, Michiko; Suganami, Takayoshi; Kanai, Sayaka; Nakagawa, Nobutaka; Sakai, Takeru; Kawano, Hiroyuki; Hara, Mitsuko; Kojima, Soichi; Izumi, Yuichi; Ogawa, Yoshihiro

    2015-01-01

    Many attempts have been made to find novel therapeutic strategies for non-alcoholic steatohepatitis (NASH), while their clinical efficacy is unclear. We have recently reported a novel rodent model of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice, which exhibit the sequence of events that comprise hepatic steatosis, liver fibrosis, and hepatocellular carcinoma with obesity-related phenotypes. In the liver of MC4R-KO mice, there is a unique histological feature termed hepatic crown-like structures (hCLS), where macrophages interact with dead hepatocytes and fibrogenic cells, thereby accelerating inflammation and fibrosis. In this study, we employed MC4R-KO mice to examine the effect of highly purified eicosapentaenoic acid (EPA), a clinically available n-3 polyunsaturated fatty acid, on the development of NASH. EPA treatment markedly prevented the development of hepatocyte injury, hCLS formation and liver fibrosis along with lipid accumulation. EPA treatment was also effective even after MC4R-KO mice developed NASH. Intriguingly, improvement of liver fibrosis was accompanied by the reduction of hCLS formation and plasma kallikrein-mediated transforming growth factor-β activation. Moreover, EPA treatment increased the otherwise reduced serum concentrations of adiponectin, an adipocytokine with anti-inflammatory and anti-fibrotic properties. Collectively, EPA treatment effectively prevents the development and progression of NASH in MC4R-KO mice along with amelioration of hepatic steatosis. This study unravels a novel anti-fibrotic mechanism of EPA, thereby suggesting a clinical implication for the treatment of NASH.

  8. Eicosapentaenoic acid ameliorates non-alcoholic steatohepatitis in a novel mouse model using melanocortin 4 receptor-deficient mice.

    Directory of Open Access Journals (Sweden)

    Kuniha Konuma

    Full Text Available Many attempts have been made to find novel therapeutic strategies for non-alcoholic steatohepatitis (NASH, while their clinical efficacy is unclear. We have recently reported a novel rodent model of NASH using melanocortin 4 receptor-deficient (MC4R-KO mice, which exhibit the sequence of events that comprise hepatic steatosis, liver fibrosis, and hepatocellular carcinoma with obesity-related phenotypes. In the liver of MC4R-KO mice, there is a unique histological feature termed hepatic crown-like structures (hCLS, where macrophages interact with dead hepatocytes and fibrogenic cells, thereby accelerating inflammation and fibrosis. In this study, we employed MC4R-KO mice to examine the effect of highly purified eicosapentaenoic acid (EPA, a clinically available n-3 polyunsaturated fatty acid, on the development of NASH. EPA treatment markedly prevented the development of hepatocyte injury, hCLS formation and liver fibrosis along with lipid accumulation. EPA treatment was also effective even after MC4R-KO mice developed NASH. Intriguingly, improvement of liver fibrosis was accompanied by the reduction of hCLS formation and plasma kallikrein-mediated transforming growth factor-β activation. Moreover, EPA treatment increased the otherwise reduced serum concentrations of adiponectin, an adipocytokine with anti-inflammatory and anti-fibrotic properties. Collectively, EPA treatment effectively prevents the development and progression of NASH in MC4R-KO mice along with amelioration of hepatic steatosis. This study unravels a novel anti-fibrotic mechanism of EPA, thereby suggesting a clinical implication for the treatment of NASH.

  9. Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1

    Science.gov (United States)

    Pierson, Theodore; Hoffman, Trevor L.; Blankson, Joel; Finzi, Diana; Chadwick, Karen; Margolick, Joseph B.; Buck, Christopher; Siliciano, Janet D.; Doms, Robert W.; Siliciano, Robert F.

    2000-01-01

    Latently infected resting CD4+ T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1 env genes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4+ T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4+ T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established. PMID:10933689

  10. Metabolic labeling and membrane fractionation for comparative proteomic analysis of Arabidopsis thaliana suspension cell cultures.

    Science.gov (United States)

    Szymanski, Witold G; Kierszniowska, Sylwia; Schulze, Waltraud X

    2013-09-28

    Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% (1). Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient (2). Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures. We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K(15)NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest (3). By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or

  11. Activity in vivo of anti-Trypanosoma cruzi compounds selected from a high throughput screening.

    Science.gov (United States)

    Andriani, Grasiella; Chessler, Anne-Danielle C; Courtemanche, Gilles; Burleigh, Barbara A; Rodriguez, Ana

    2011-08-01

    Novel technologies that include recombinant pathogens and rapid detection methods are contributing to the development of drugs for neglected diseases. Recently, the results from the first high throughput screening (HTS) to test compounds for activity against Trypanosoma cruzi trypomastigote infection of host cells were reported. We have selected 23 compounds from the hits of this HTS, which were reported to have high anti-trypanosomal activity and low toxicity to host cells. These compounds were highly purified and their structures confirmed by HPLC/mass spectrometry. The compounds were tested in vitro, where about half of them confirmed the anti-T. cruzi activity reported in the HTS, with IC50 values lower than 5 µM. We have also adapted a rapid assay to test anti-T. cruzi compounds in vivo using mice infected with transgenic T. cruzi expressing luciferase as a model for acute infection. The compounds that were active in vitro were also tested in vivo using this assay, where we found two related compounds with a similar structure and low in vitro IC50 values (0.11 and 0.07 µM) that reduce T. cruzi infection in the mouse model more than 90% after five days of treatment. Our findings evidence the benefits of novel technologies, such as HTS, for the drug discovery pathway of neglected diseases, but also caution about the need to confirm the results in vitro. We also show how rapid methods of in vivo screening based in luciferase-expressing parasites can be very useful to prioritize compounds early in the chain of development.

  12. Activity in vivo of anti-Trypanosoma cruzi compounds selected from a high throughput screening.

    Directory of Open Access Journals (Sweden)

    Grasiella Andriani

    2011-08-01

    Full Text Available Novel technologies that include recombinant pathogens and rapid detection methods are contributing to the development of drugs for neglected diseases. Recently, the results from the first high throughput screening (HTS to test compounds for activity against Trypanosoma cruzi trypomastigote infection of host cells were reported. We have selected 23 compounds from the hits of this HTS, which were reported to have high anti-trypanosomal activity and low toxicity to host cells. These compounds were highly purified and their structures confirmed by HPLC/mass spectrometry. The compounds were tested in vitro, where about half of them confirmed the anti-T. cruzi activity reported in the HTS, with IC50 values lower than 5 µM. We have also adapted a rapid assay to test anti-T. cruzi compounds in vivo using mice infected with transgenic T. cruzi expressing luciferase as a model for acute infection. The compounds that were active in vitro were also tested in vivo using this assay, where we found two related compounds with a similar structure and low in vitro IC50 values (0.11 and 0.07 µM that reduce T. cruzi infection in the mouse model more than 90% after five days of treatment. Our findings evidence the benefits of novel technologies, such as HTS, for the drug discovery pathway of neglected diseases, but also caution about the need to confirm the results in vitro. We also show how rapid methods of in vivo screening based in luciferase-expressing parasites can be very useful to prioritize compounds early in the chain of development.

  13. Characterization of purified human liver acid beta-D-galactosidases A2 and A3.

    Science.gov (United States)

    Frost, R G; Holmes, E W; Norden, A G; O'Brien, J S

    1978-01-01

    1. Human liver acid beta-galactosidase A2 and A3 were isolated by chromatography on concanavalin A-Sepharose 4B, Sepharose 6B, and Sepharose 4B-6-aminohexyl 1-thio-beta-D-galactopyranoside. beta-Galactosidase A2 and A3 were purified to final specific activities of 45.5 and 20.6 mumol/min per mg respectively with 4-methylumbelliferyl beta-D-galactopyranoside as substrate. 2. Form A2 had a mol.wt. of 150000 +/- 15000 (gel filtration) and appeared as a single band of protein (mol.wt 65000 +/- 1000) on electrophoresis in the presence of sodium dodecyl sulphate. 3. Form A3 had a mol.wt. (gel filtration) of 660000 +/- 66000. On electrophoresis in the presence of sodium dodecyl sulphate, form A3 appeared as a major band of protein (72% of total) of mol.wt. 65000 +/- 1000 and minor protein bands of mol.wt. 44000 +/- 1000 and 26,000 +/- 1000 and 22000 +/- 1000. 4. Gel-filtration chromatography of purified beta-galactosidase A3 generated approximately equal amounts of forms A3 and A2. beta-Galactosidase A1 was not detected by gel-filtration chromatography of partially or highly purified preparations of forms A2 and A3. 5. Both forms A2 and A3 had identical isoelectric points of 4.42 +/- 0.02. The data suggest that forms A2 and A3 are dimeric and multimeric forms of beta-galactosidase A1. 6. Amino acid analysis of beta-galactosidase A2 gave a ratio of acidic to basic amino acids of 2.6:1. 7. beta-Galactosidase A2 contained 7.5% carbohydrate by weight and sialic acid, D-galactose, D-glucosamine and D-mannose were present in the molar proportions 1.1:1.0:1.7:2.7. Images Fig. 1. PMID:104712

  14. The Radioactive Ion Beams in Brazil (RIBRAS) facility. Description, program, main results, future plans

    Science.gov (United States)

    Lépine-Szily, A.; Lichtenthäler, R.; Guimarães, V.

    2014-08-01

    RIBRAS (Radioactive Ion Beams in Brazil) is a facility installed at the Institute of Physics of the University of São Paulo (IFUSP), Brazil. The RIBRAS system consists of two superconducting solenoids and uses the "in-flight method" to produce radioactive ion beams using the primary beam provided by the 8UD Pelletron Tandem of IFUSP. The ion beams produced so far by RIBRAS are 6He, 8Li, 7Be, 10Be, 8B, 12B with intensities that can vary from 104 to 106 pps. Initially the experimental program covered the study of elastic and inelastic scattering with the objective to study the interaction potential and the reaction mechanisms between weakly bound (RIB) and halo (6He and 8B projectiles on light, medium and heavy mass targets. With highly purified beams, the study of resonant elastic scattering and resonant transfer reactions, using inverse kinematics and thick targets, has also been included in our experimental program. Also, transfer reactions of astrophysical interest and fusion reactions induced by halo nuclei are part of the near-future research program. Our recent results on elastic scattering, alpha-particle production and total reaction cross sections, as well as the resonant elastic and transfer reactions, are presented. Our plans for the near future are related to the installation of a new beam line and a cave for gamma-ray detection. We intend to place in operation a large area neutron detector available in our laboratory. The long-range plans could be the move of the RIBRAS system to the more energetic beam line of the LINAC post-accelerator (10MeV/nucleon primary beams) still in construction in our laboratory.

  15. The Radioactive Ion Beams in Brazil (RIBRAS) facility. Description, program, main results, future plans

    Energy Technology Data Exchange (ETDEWEB)

    Lepine-Szily, A.; Lichtenthaeler, R.; Guimaraes, V. [Instituto de Fisica, Universidade de Sao Paulo (Brazil)

    2014-08-15

    RIBRAS (Radioactive Ion Beams in Brazil) is a facility installed at the Institute of Physics of the University of Sao Paulo (IFUSP), Brazil. The RIBRAS system consists of two superconducting solenoids and uses the ''in-flight method'' to produce radioactive ion beams using the primary beam provided by the 8UD Pelletron Tandem of IFUSP. The ion beams produced so far by RIBRAS are {sup 6}He, {sup 8}Li, {sup 7}Be, {sup 10}Be, {sup 8}B, {sup 12}B with intensities that can vary from 10{sup 4} to 10{sup 6} pps. Initially the experimental program covered the study of elastic and inelastic scattering with the objective to study the interaction potential and the reaction mechanisms between weakly bound (RIB) and halo ({sup 6}He and {sup 8}B) projectiles on light, medium and heavy mass targets. With highly purified beams, the study of resonant elastic scattering and resonant transfer reactions, using inverse kinematics and thick targets, has also been included in our experimental program. Also, transfer reactions of astrophysical interest and fusion reactions induced by halo nuclei are part of the near-future research program. Our recent results on elastic scattering, alpha-particle production and total reaction cross sections, as well as the resonant elastic and transfer reactions, are presented. Our plans for the near future are related to the installation of a new beam line and a cave for gamma-ray detection. We intend to place in operation a large area neutron detector available in our laboratory. The long-range plans could be the move of the RIBRAS system to the more energetic beam line of the LINAC post-accelerator (10MeV/nucleon primary beams) still in construction in our laboratory. (orig.)

  16. High heat load test of molybdenum

    Energy Technology Data Exchange (ETDEWEB)

    Tanabe, T. (Faculty of Engineering, Osaka Univ., Suita (Japan)); Fujine, M.; Noguchi, H. (Daido Steel Co. Ltd., Nagoya (Japan)); Yagi, Y.; Hirano, Y.; Shimizu, H. (Electrotechnical Lab., Umezono, Tsukuba (Japan)); Akiba, M.; Araki, M. (Japan Atomic Energy Research Inst., Naka, Ibaraki (Japan)); Kubota, Y.; Miyahara, A. (National Inst. for Fusion Science, Nagoya (Japan))

    1993-03-01

    Three different types of molybdenum, powder metallurgical polycrystalline (PM-Mo), and as-forged polycrystalline and single crystalline of highly purified electron-beam-melted Mo (AFEB-Mo and SCEB-Mo), have been subjected to high heat load test with neutral beam injection (NBI) stands at Japan Atomic Energy Research Institute (JAERI) and National Institute for Fusion Science (NIFS). These materials have also been tested as a movable limiter in a reversed field pinch machine (RFP:TPE-1RM15) in Electrotechnical Laboratory (ETL). The results are summarized as follows. The SCEB-Mo shows the least damage with slight local melting after a very high heat load of 260 MW/m[sup 2] for 250 ms with NBI, while for the PM-Mo the whole irradiated area melt with many craters due to impurity gas evaporation under less heat load (200 ms). All movable limiter heads of the RFP are severely damaged with partial melting. The appearance of the SCEB-Mo limiter after melting is not good and shows the crystalline cleavage. However, SEM observation of the microstructure opposes the surface appearance. In the SCEB-Mo, appreciable recrystallization is not observed and hence no crack is seen to go into the bulk except the crystalline cleavage. In the PM-Mo, on the other hand, the resolidification to columnar grains as well as the recrystallization is apparent, and the cracks not only go along the columnar grains but also separate the recrystallized region from the matrix. In the AFEB-Mo, a slight grain growth occurs and several cracks enter deep along the grain boundaries. Thus the SCEB-Mo is a very nice plasma-facing material if used under the critical heat load for melting. (orig.).

  17. High-level expression of Staphylococcal Protein A in Pichia pastoris and purification and characterization of the recombinant protein.

    Science.gov (United States)

    Hao, Jing; Xu, Li; He, Hongde; Du, Xiaojun; Jia, Lingyun

    2013-08-01

    Staphylococcal Protein A (SPA), a cell wall protein of Staphylococcus aureus, is in high demand because of its ability to bind immunoglobulins. Much of the SPA that we use today is recombinant SPA (rSPA), which is produced in Escherichia coli. As rSPA is obtained by expressing SPA as an intracellular protein, its purification is tedious and time consuming. In order to obtain a large amount of highly purified rSPA with relative ease, we expressed SPA as a secretory form in the yeast Pichia pastoris. To increase the expression level of SPA and repress its proteolysis during fermentation, the cell density (OD600), temperature and pH at which SPA expression was induced as well as the induction time were optimized. The final yield of SPA obtained was about 8.8 g per liter of culture, which under the optimized fermentation condition, accounted for 80% of the total protein in the culture supernatant. The expressed SPA was purified from the culture supernatant by DEAE ion-exchange chromatography (IEC) after the supernatant was subjected to a desalting step. The purified SPA was resolved as a single band by SDS-PAGE and as a single peak by HPLC. Its identity was confirmed by MALDI-TOF MS and western-blot. Moreover, the protein also exhibited excellent affinity for IgG when tested with human IgG. The production and purification of SPA described in this study offers a new method for obtaining high level of SPA in relatively pure form that is suitable for practical application. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Uranyl Nitrate Flow Loop

    International Nuclear Information System (INIS)

    Ladd-Lively, Jennifer L

    2008-01-01

    The objectives of the work discussed in this report were to: (1) develop a flow loop that would simulate the purified uranium-bearing aqueous stream exiting the solvent extraction process in a natural uranium conversion plant (NUCP); (2) develop a test plan that would simulate normal operation and disturbances that could be anticipated in an NUCP; (3) use the flow loop to test commercially available flowmeters for use as safeguards monitors; and (4) recommend a flowmeter for production-scale testing at an NUCP. There has been interest in safeguarding conversion plants because the intermediate products [uranium dioxide (UO 2 ), uranium tetrafluoride (UF 4 ), and uranium hexafluoride (UF 6 )] are all suitable uranium feedstocks for producing special nuclear materials. Furthermore, if safeguards are not applied virtually any nuclear weapons program can obtain these feedstocks without detection by the International Atomic Energy Agency (IAEA). Historically, IAEA had not implemented safeguards until the purified UF 6 product was declared as feedstock for enrichment plants. H. A. Elayat et al. provide a basic definition of a safeguards system: 'The function of a safeguards system on a chemical conversion plant is in general terms to verify that no useful nuclear material is being diverted to use in a nuclear weapons program'. The IAEA now considers all highly purified uranium compounds as candidates for safeguarding. DOE is currently interested in 'developing instruments, tools, strategies, and methods that could be of use to the IAEA in the application of safeguards' for materials found in the front end of the nuclear fuel cycle-prior to the production of the uranium hexafluoride or oxides that have been the traditional starting point for IAEA safeguards. Several national laboratories, including Oak Ridge, Los Alamos, Lawrence Livermore, and Brookhaven, have been involved in developing tools or techniques for safeguarding conversion plants. This study was sponsored by

  19. Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

    Science.gov (United States)

    Ying, Sheng; Hill, Allyson T; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

    2017-06-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds ( Ricinus communis ) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca 2+ -dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca 2+ -dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: ( i ) a pair of Ca 2+ binding sites with identical dissociation constants of 5.03 μM, ( ii ) a Ca 2+ -dependent electrophoretic mobility shift, and ( iii ) a marked Ca 2+ -independent hydrophobicity. Pull-down experiments established the Ca 2+ -dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca 2+ -dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis ( Arabidopsis thaliana ) CPK4 and soybean ( Glycine max ) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca 2+ signaling and the posttranslational control of respiratory CO 2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. © 2017 American Society of Plant

  20. Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds1[OPEN

    Science.gov (United States)

    Hill, Allyson T.; Anderson, Erin M.; She, Yi-Min

    2017-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC’s BTPC subunit’s at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 μM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. PMID:28363991

  1. Small-scale extraction and radioiodination of human hormones for the substitution of imported radioimmunoassay reagents

    International Nuclear Information System (INIS)

    Gimbo, E.K.; Carvalho Pinto Ribela, M.T. de; Borghi, V.C.; Schwarz, I.; Dias, L.E.M.; Araujo, E.A. de; Bartolini, P.

    1988-08-01

    One of the main objectives of our laboratory is the national production of radioimmunoassay (RIA) reagents so to substitute the use of expensive imported commercial kits or in-bulk reagents such as the following: a) highly purified unlabelled hormones for radioiodination, useful also for preparing secondary standards and specific antisera; b) 125 I-labelled hormones; c) specific high titre antisera. The extraction and purification of human growth hormone (hGH) and human luteinizing hormone (hLH) has been already carried out. We have recently obtained quite purified, active and stable preparation starting from a small number of human pituitaries, the whole process being carried out in just one or two weeks for hGH an hLH respectively. For what concernes 125 I-labelling, we basically utilize two techniques: the Classical Chloramine T method described by Hunter and Roth's stoichiometric method, both followed by a quantitative and qualitative analysis of the reaction mixture, set up in our laboratory. The results of such comparison will be discussed. 125-I-hGH, 125-I-hLH, 125-I-hTSH and 125-I-h Calcitonin (unlabelled synthetic product donated by CIBA, Switzerland) have been prepared and tested in internal and external quality control, in comparison with good quality imported products. Satisfactory results have been obtained through the evaluation of these parameters: maximum binding to specific antiserum (Bo), nonspecific binding (NSB), mean effective dose (ED 50), sensitivity, precision and accuracy. Concerning antisera production, specific and high titre rabbit anti-hGH and anti-hLH antibodies have been obtained, while guinea-pig anti-h Calcitonin antiserum is being produced. Together with 125-I-Calcitonin it will be used in the setting up of the RIA of this hormone whic allows the early detection of Medullary Thyroid Carcinoma. (author) [pt

  2. Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

    Science.gov (United States)

    Henneke, Ghislaine; Gueguen, Yannick; Flament, Didier; Azam, Philippe; Querellou, Joël; Dietrich, Jacques; Hübscher, Ulrich; Raffin, Jean-Paul

    2002-11-08

    The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

  3. Non-chromatographic preparation of a bacterially produced single-shot modular virus-like particle capsomere vaccine for avian influenza.

    Science.gov (United States)

    Wibowo, Nani; Wu, Yang; Fan, Yuanyuan; Meers, Joanne; Lua, Linda H L; Middelberg, Anton P J

    2015-11-04

    Highly pathogenic avian influenza (HPAI) causes significant economic loss, reduced food security and poses an ongoing pandemic threat. Poultry vaccination significantly decreases these problems and recognizes that the health of humans, animals and ecosystems are connected. Low-cost manufacture of poultry vaccine matched quickly to the ever-changing circulating strain is needed for effective vaccination. Here, we re-engineered the process to manufacture bacterially synthesized modular capsomere comprising influenza M2e, previously shown to confer complete protection in challenged mice, for application in poultry. Modular capsomere was prepared using a simplified non-chromatographic salting-out precipitation method and its immunogenicity tested in vivo in poultry. Modular capsomere crudely purified by precipitation (pCapM2e) contained more contaminants than equivalent product purified by chromatography (cCapM2e). Unadjuvanted pCapM2e containing 80 EU of endotoxin per dose was inferior to highly purified and adjuvanted cCapM2e (2 EU per dose). However, addition of adjuvant to pCapM2e resulting in high immunogenicity after only a single dose of vaccination, yet without any local adverse reaction. This finding suggests a strong synergy between adjuvant, antigen and contaminants, and the possible existence of a "Goldilocks" level of contaminants, where high immunogenicity and low reactogenicity can be obtained in a single-shot vaccination. The simplified process offers potential cost and speed advantages to address the needs in influenza poultry vaccination in low-cost veterinary markets. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Black widow spider toxins: the present and the future.

    Science.gov (United States)

    Grishin, E V

    1998-11-01

    The venom of the black widow spider Latrodectus mactans tredisimguttatus was found to contain a family of high molecular weight toxic proteins inducing a sharp increase in transmitter secretion from the affected nerve endings, which are highly specific for vertebrates, or for insects, or for crustaceans. Along with the known alpha-latrotoxin, five latroinsectotoxins affecting the neurotransmitter release from presynaptic endings of insects and one latrocrustatoxin active only for crustaceans were isolated and studied in detail. Alpha-latrotoxin provokes a massive transmitter release from different nerve endings of vertebrates, whereas other toxins increase the secretion process either in insects or crustaceans. The cDNAs encoding the putative alpha-latrotoxin and two latroinsectotoxins (alpha-latroinsectotoxin and delta-latroinsectotoxin) precursors were cloned and sequenced. These toxins are polypeptides of about 1000 amino acids and share a high level of amino acid identity. Analysis of amino acid sequences of the three toxins reveals the central regions being almost entirely composed of series of ankyrin-like repeats. Taking into account the size and multifunctional properties of latrotoxin its molecule can be divided into several functional domains. Immunochemical experiments indicated the presence in the alpha-latrotoxin molecule of distinguishable functional domains responsible for ionophoric and secretogenic actions. The highly purified preparation of alpha-latrotoxin was shown to contain an additional component, a low molecular weight protein structurally related to crustacean hyperglycemic hormones. Several attempts were made to characterize and isolate alpha-latrotoxin receptor components. The existence of Ca-dependent and Ca-independent binding proteins was found in the presynaptic membrane preparations.

  5. T-helper cell-mediated proliferation and cytokine responses against recombinant Merkel cell polyomavirus-like particles.

    Directory of Open Access Journals (Sweden)

    Arun Kumar

    Full Text Available The newly discovered Merkel Cell Polyomavirus (MCPyV resides in approximately 80% of Merkel cell carcinomas (MCC. Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL, suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs, using human bocavirus (HBoV VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-γ, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-γ. The MCPyV antigen tended to induce stronger IFN-γ responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-γ responses. IFN-γ being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance.

  6. Progranulin promotes tumour necrosis factor-induced proliferation of suppressive mouse CD4⁺ Foxp3⁺ regulatory T cells.

    Science.gov (United States)

    Hu, Ya; Xiao, Haitao; Shi, Tingchen; Oppenheim, Joost J; Chen, Xin

    2014-06-01

    Progranulin (PGRN) is a pleiotropic growth factor with immunosuppressive properties. Recently, it was reported that PGRN was an antagonist of tumour necrosis factor (TNF) receptors, preferentially for TNFR2. However, we and others showed that TNF-TNFR2 interaction was critical for the activation and expansion of functional CD4(+)  Foxp3(+) regulatory T (Treg) cells. We therefore examined the effect of PGRN on the proliferation of naturally occurring murine suppressive Treg cells induced by TNF. Consistent with our previous reports, TNF overcame the hyporesponsiveness of highly purified Treg cells to T-cell receptor stimulation. Furthermore, in the presence of interleukin-2, TNF preferentially stimulated proliferation of Treg cells contained in unfractionated CD4 cells. These effects of TNF on suppressive Treg cells were markedly increased by exogenous PGRN. TNF and TNFR2 interactions are required for this effect of PGRN, because the PGRN by itself did not stimulate Treg cell proliferation. The effect of PGRN on Treg cells was abrogated by antibody against TNFR2, and Treg cells deficient in TNFR2 also failed to respond to PGRN. Furthermore, PGRN also enhanced the proliferative responses of effector T cells to TNF, but to a lesser extent than that of Treg cells, presumably caused by the different levels of TNFR2 expression on these two subsets of CD4 cells. Hence, our data clearly show that PGRN promotes, rather than inhibits, the functional consequence of TNF-TNFR2 interaction on Treg cells. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

  7. Purification and characterization of nuclear type II [(3)H]estradiol binding sites from the rat uterus: covalent labeling with [(3)H]luteolin.

    Science.gov (United States)

    Markaverich, B M; Shoulars, K; Brown, M A

    2001-09-01

    Type II [3H]estradiol binding sites play an important role in normal and malignant cell growth and proliferation and the delineation of the precise function of the type II site in cell growth has been hampered by the inability to purify, sequence and or clone this protein. The present manuscript describes methodology for the solubilization, purification and tentative identification of type II sites from the estrogen-treated rat uterus. This protein(s) chromatographs as a single major peak on DNA-cellulose, Affigel Blue dye affinity resin and during high performance liquid chromatography (HPLC) on hydroxyapatite. The purified fractions from these columns elicited classical [3H]estradiol binding characteristics (sigmoidal saturation curve, hyperbolic Scatchard plot, and Hill coefficient of approximately 4) for type II sites that are typically observed in crude or highly purified nuclear fractions or extracts. The type II binding activity also eluted as a single major component from a ligand affinity resin (GT-18-Sepharose) prepared by coupling 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone to epoxy-activated Sepharose (Pharmacia). The molecular weight of the type II site ([3H]estradiol binding activity) under non-denaturing or denaturing conditions was estimated to be approximately 10-15 kDa by gel filtration HPLC. Similarly, nuclear type II sites covalently labeled with the bioflavonoid, [3H]luteolin, migrated in the 10 kDa range on SDS PAGE. Thus, under these various experimental conditions, nuclear type II sites detected by [3H]estradiol or [3H]luteolin labeling techniques displayed little heterogeneity and appear much smaller than ERalpha or ERbeta or other steroid hormone receptors.

  8. Protease domain of human ADAM33 produced by Drosophila S2 cells.

    Science.gov (United States)

    Prosise, Winifred W; Yarosh-Tomaine, Taisa; Lozewski, Zia; Ingram, Richard N; Zou, Jun; Liu, Jian-Jun; Zhu, Feng; Taremi, S Shane; Le, Hung V; Wang, Wenyan

    2004-12-01

    Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies ( approximately 10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].

  9. Activation of the Innate Immune System by Treponema denticola Periplasmic Flagella through Toll-Like Receptor 2.

    Science.gov (United States)

    Ruby, John; Martin, Michael; Passineau, Michael J; Godovikova, Valentina; Fenno, J Christopher; Wu, Hui

    2018-01-01

    Treponema denticola is an indigenous oral spirochete that inhabits the gingival sulcus or periodontal pocket. Increased numbers of oral treponemes within this environment are associated with localized periodontal inflammation, and they are also part of an anaerobic polymicrobial consortium responsible for endodontic infections. Previous studies have indicated that T. denticola stimulates the innate immune system through Toll-like receptor 2 (TLR2); however, the pathogen-associated molecular patterns (PAMPs) responsible for T. denticola activation of the innate immune system are currently not well defined. In this study, we investigated the role played by T. denticola periplasmic flagella (PF), unique motility organelles of spirochetes, in stimulating an innate immune response. Wild-type T. denticola stimulated the production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, and IL-12 by monocytes from human peripheral blood mononuclear cells, while its isogenic nonmotile mutant lacking PF resulted in significantly diminished cytokine stimulation. In addition, highly purified PF were able to dose dependently stimulate cytokine TNF-α, IL-1β, IL-6, IL-10, and IL-12 production in human monocytes. Wild-type T. denticola and the purified PF triggered activation of NF-κB through TLR2, as determined using a variety of TLR-transfected human embryonic 293 cell lines, while the PF-deficient mutants lacked the ability to stimulate, and the complemented PF-positive T. denticola strain restored the activation. These findings suggest that T. denticola stimulates the innate immune system in a TLR2-dependent fashion and that PF are a key bacterial component involved in this process. Copyright © 2017 American Society for Microbiology.

  10. Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system

    International Nuclear Information System (INIS)

    Kuroki, Masahide; Arakawa, Fumiko; Matsunaga, Akira; Okamoto, Naomi; Takakura, Kyoko; Matsuoka, Yuji; Higuchi, Hiroshi.

    1987-01-01

    A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radio-immunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels. (author)

  11. Evidence of glucuronidation of the glycation product LW-1: tentative structure and implications for the long-term complications of diabetes.

    Science.gov (United States)

    Sell, David R; Nemet, Ina; Liang, Zhili; Monnier, Vincent M

    2018-04-01

    LW-1 is a collagen-linked blue fluorophore whose skin levels increase with age, diabetes and end-stage renal disease (ESRD), and correlate with the long-term progression of microvascular disease and indices of subclinical cardiovascular disease in type 1 diabetes. The chemical structure of LW-1 is still elusive, but earlier NMR analyses showed it has a lysine residue in an aromatic ring coupled to a sugar molecule reminiscent of advanced glycation end-products (AGEs). We hypothesized and demonstrate here that the unknown sugar is a N-linked glucuronic acid. LW-1 was extracted and highly purified from ~99 g insoluble skin collagen obtained at autopsy from patients with diabetes/ESRD using multiple rounds of proteolytic digestion and purification by liquid chromatography (LC). Advanced NMR techniques ( 1 H-NMR, 13 C-NMR, 1 H- 13 C HSQC, 1 H- 1 H TOCSY, 1 H- 13 C HMBC) together with LC-mass spectrometry (MS) revealed a loss of 176 amu (atomic mass unit) unequivocally point to the presence of a glucuronic acid moiety in LW-1. To confirm this data, LW-1 was incubated with β-glycosidases (glucosidase, galactosidase, glucuronidase) and products were analyzed by LC-MS. Only glucuronidase could cleave the sugar from the parent molecule. These results establish LW-1 as a glucuronide, now named glucuronidine, and for the first time raise the possible existence of a "glucuronidation pathway of diabetic complications". Future research is needed to rigorously probe this concept and elucidate the molecular origin and biological source of a circulating glucuronidine aglycone.

  12. Human astrocytes: secretome profiles of cytokines and chemokines.

    Directory of Open Access Journals (Sweden)

    Sung S Choi

    Full Text Available Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1, IL-6, IL-8 (CXCL8, MCP-1 (CCL2, MIF and Serpin E1. Following stimulation with IL-1β and TNF-α, activated astrocytes newly produced IL-1β, IL-1ra, TNF-α, IP-10 (CXCL10, MIP-1α (CCL3 and RANTES (CCL5, in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.

  13. Identification of IDO-positive and IDO-negative human dendritic cells after activation by various proinflammatory stimuli.

    Science.gov (United States)

    Von Bubnoff, Dagmar; Scheler, Marina; Wilms, Helene; Fimmers, Rolf; Bieber, Thomas

    2011-06-15

    Dendritic cells (DCs) can induce tolerance or immunity. We identified and characterized an IDO-expressing and an IDO-negative human DC population after stimulation by various proinflammatory stimuli. IDO expression was strongly dependent on the maturation status of the cells (CD83-positive cells only). The two DC subpopulations remained IDO positive and IDO negative, respectively, over a time period of at least 48 h. IDO enzyme activity of human DCs was highest during stimulation by strongly maturation-inducing TLR ligands such as highly purified LPS (TLR4 ligand) or polyriboinosinic-polyribocytidilic acid (TLR3 ligand); factors of the adaptive immune system such as IFN-γ, a mixture of cytokines, and IFN-α had lesser stimulatory capacity for IDO induction and activity. After stimulation with CD40L, IDO-positive DCs expressed significantly increased levels of B7 family molecules such as CD40, CD80, CD86, ICOS ligand, as well as PD-L1 (B7-H1) and PD-L2 (B7-DC) compared with the IDO-negative DC subset. At the same time, the inhibitory receptors Ig-like transcripts 3 and 4 were significantly downregulated on IDO-positive cells. Functionally, IDO-positive DCs produced significantly more IL-1β and IL-15 and less IL-10 and IL-6 than the IDO-negative subset after CD40L stimulation. These results show that IDO expression is associated with a distinctive phenotype and functional capacity in mature DCs. It seems likely that the IDO-positive DC subset possesses a regulatory function and might skew a T cell response toward tolerance.

  14. Ginseng Rb fraction protects glia, neurons and cognitive function in a rat model of neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Kangning Xu

    Full Text Available The loss and injury of neurons play an important role in the onset of various neurodegenerative diseases, while both microgliosis and astrocyte loss or dysfunction are significant causes of neuronal degeneration. Previous studies have suggested that an extract enriched panaxadiol saponins from ginseng has more neuroprotective potential than the total saponins of ginseng. The present study investigated whether a fraction of highly purified panaxadiol saponins (termed as Rb fraction was protective for both glia and neurons, especially GABAergic interneurons, against kainic acid (KA-induced excitotoxicity in rats. Rats received Rb fraction at 30 mg/kg (i.p., 40 mg/kg (i.p. or saline followed 40 min later by an intracerebroventricular injection of KA. Acute hippocampal injury was determined at 48 h after KA, and impairment of hippocampus-dependent learning and memory as well as delayed neuronal injury was determined 16 to 21 days later. KA injection produced significant acute hippocampal injuries, including GAD67-positive GABAergic interneuron loss in CA1, paralbumin (PV-positive GABAergic interneuron loss, pyramidal neuron degeneration and astrocyte damage accompanied with reactive microglia in both CA1 and CA3 regions of the hippocampus. There was also a delayed loss of GAD67-positive interneurons in CA1, CA3, hilus and dentate gyrus. Microgliosis also became more severe 21 days later. Accordingly, KA injection resulted in hippocampus-dependent spatial memory impairment. Interestingly, the pretreatment with Rb fraction at 30 or 40 mg/kg significantly protected the pyramidal neurons and GABAergic interneurons against KA-induced acute excitotoxicity and delayed injury. Rb fraction also prevented memory impairments and protected astrocytes from KA-induced acute excitotoxicity. Additionally, microglial activation, especially the delayed microgliosis, was inhibited by Rb fraction. Overall, this study demonstrated that Rb fraction protected both

  15. Initial fate of prions upon peripheral infection: half-life, distribution, clearance, and tissue uptake

    Science.gov (United States)

    Urayama, Akihiko; Morales, Rodrigo; Niehoff, Michael L.; Banks, William A.; Soto, Claudio

    2011-01-01

    Prion diseases are infectious neurodegenerative disorders associated with the misfolded prion protein (PrPSc), which appears to be the sole component of the infectious agent (termed prion). To produce disease, prions have to be absorbed into the body and reach sufficient quantities in the brain. Very little is known about the biological mechanisms controlling the initial fate of prions. Here, we studied the systemic pharmacokinetics and biodistribution of PrPSc in vivo. After an intravenous injection of highly purified radiolabeled or native unlabeled PrPSc, the protein was eliminated rapidly from the serum (half-life of 3.24 h), mostly through tissue uptake. The quantity of intact PrPSc reaching the brain was ∼0.2% of the injected dose per gram of brain tissue (ID/g). The highest levels were found in liver (∼20% ID/g), spleen (∼13% ID/g), and kidney (∼7.4% ID/g). Cell surface PrPC does not appear to play a role in PrPSc pharmacokinetics, since the infectious protein distributed similarly in wild-type and PrP-null mice. To measure tissue uptake kinetics and biodistribution accurately, vascular space in tissues was measured with radioactively labeled albumin coinjected with radioactively labeled PrPSc. Our results provide a fundamental pharmacokinetic characterization of PrPSc in vivo, which may be relevant to estimate tissue risks and mechanisms of prion neuroinvasion and to identify novel therapeutic strategies.—Urayama, A., Morales, R., Niehoff, M. L., Banks, W. A., Soto, C. Initial fate of prions upon peripheral infection: half-life, distribution, clearance, and tissue uptake PMID:21555356

  16. Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Amin, Anthony; Zaccardi, Joe; Mullen, Stanley; Olland, Stephane; Orlowski, Mark; Feld, Boris; Labonte, Patrick; Mak, Paul

    2003-01-01

    A class of disulfide constrained peptides containing a core motif FPWG was identified from a screen of phage displayed library using the HCV RNA-dependent RNA polymerase (NS5B) as a bait. Surface plasmon resonance studies showed that three highly purified synthetic constrained peptides bound to immobilized NS5B with estimated K d values ranging from 30 to 60 μM. In addition, these peptides inhibited the NS5B activity in vitro with IC 50 ranging from 6 to 48 μM, whereas in contrast they had no inhibitory effect on the enzymatic activities of calf thymus polymerase α, human polymerase β, RSV polymerase, and HIV reverse transcriptase in vitro. Two peptides demonstrated conformation-dependent inhibition since their synthetic linear versions were not inhibitory in the NS5B assay. A constrained peptide with the minimum core motif FPWG retained selective inhibition of NS5B activity with an IC 50 of 50 μM. Alanine scan analyses of a representative constrained peptide, FPWGNTW, indicated that residues F1 and W7 were critical for the inhibitory effect of this peptide, although residues P2 and N5 had some measurable inhibitory effect as well. Further analyses of the mechanism of inhibition indicated that these peptides inhibited the formation of preelongation complexes required for the elongation reaction. However, once the preelongation complex was formed, its activity was refractory to peptide inhibition. Furthermore, the constrained peptide FPWGNTW inhibited de novo initiated RNA synthesis by NS5B from a poly(rC) template. These data indicate that the peptides confer selective inhibition of NS5B activity by binding to the enzyme and perturbing an early step preceding the processive elongation step of RNA synthesis

  17. Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells.

    Science.gov (United States)

    Tsang, Jason C H; Yu, Yong; Burke, Shannon; Buettner, Florian; Wang, Cui; Kolodziejczyk, Aleksandra A; Teichmann, Sarah A; Lu, Liming; Liu, Pentao

    2015-09-21

    Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment. In this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a (+/+) and Bcl11a (-/-)). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a (-/-) HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a (-/-) HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a (-/-) HSCs are myeloerythroid-restricted and defective in self-renewal. Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment.

  18. Angiopoietin-like protein 3 promotes preservation of stemness during ex vivo expansion of murine hematopoietic stem cells.

    Science.gov (United States)

    Farahbakhshian, Elnaz; Verstegen, Monique M; Visser, Trudi P; Kheradmandkia, Sima; Geerts, Dirk; Arshad, Shazia; Riaz, Noveen; Grosveld, Frank; van Til, Niek P; Meijerink, Jules P P

    2014-01-01

    Allogeneic hematopoietic stem cell (HSC) transplantations from umbilical cord blood or autologous HSCs for gene therapy purposes are hampered by limited number of stem cells. To test the ability to expand HSCs in vitro prior to transplantation, two growth factor cocktails containing stem cell factor, thrombopoietin, fms-related tyrosine kinase-3 ligand (STF) or stem cell factor, thrombopoietin, insulin-like growth factor-2, fibroblast growth factor-1 (STIF) either with or without the addition of angiopoietin-like protein-3 (Angptl3) were used. Culturing HSCs in STF and STIF media for 7 days expanded long-term repopulating stem cells content in vivo by ∼6-fold and ∼10-fold compared to freshly isolated stem cells. Addition of Angptl3 resulted in increased expansion of these populations by ∼17-fold and ∼32-fold, respectively, and was further supported by enforced expression of Angptl3 in HSCs through lentiviral transduction that also promoted HSC expansion. As expansion of highly purified lineage-negative, Sca-1+, c-Kit+ HSCs was less efficient than less pure lineage-negative HSCs, Angptl3 may have a direct effect on HCS but also an indirect effect on accessory cells that support HSC expansion. No evidence for leukemia or toxicity was found during long-term follow up of mice transplanted with ex vivo expanded HSCs or manipulated HSC populations that expressed Angptl3. We conclude that the cytokine combinations used in this study to expand HSCs ex vivo enhances the engraftment in vivo. This has important implications for allogeneic umbilical cord-blood derived HSC transplantations and autologous HSC applications including gene therapy.

  19. The effect of gamma irradiation on some carotenoid compounds of paprika

    International Nuclear Information System (INIS)

    Zachariev, Gy.; Kiss, I.

    1983-01-01

    Highly purified crystalline carotenoids (capsanthin, capsorubin, cryptoxanthin and zeaxanthin) were isolated from the pericarp of ripen paprika. The carotenoids were irradiated in a 60 Co source in benzene and aqueous solutions to study their radiolysis. In 100 μM benzene solutions at a dose of 0.91 kGy the following G-values were determined: beta-carotene 0.51, capsanthin 0.44, capsorubin 0.41, zeaxanthin 0.32, cryptoxanthin 0.15. In water at a 0.30 kGy dose (0.91 kGy is very destructive) the obtained G-values were: capsanthin 2.57, capsorubin: 2.31, zeaxanthin 2.34, beta-carotene 1.75, crytpoxanthin 1.54. Ascorbic acid reduced the radiolysis of carotenoids in water. 100 μM benzene solutions of capsanthin and zeaxanthin were irradiated at 1.2 kGy and 1.5 kGy doses, resp. The irradiated solutions were column-chromatographed and the separated bands were investigated by spectrophotometric and chemical methods. It was established that 40% of the capsanthin and 56% of the cryptoxanthin was decomposed to UV absorbing, not yet identified compounds. The remainder consisted in 69-72% of the original all-trans carotenoids. Among the radiation products cis-trans isomers and monoepoxy derivatives of the respective carotenoids could be identified. These amounted only to 0.3-2.2% of the all-trans carotenoids that survived the damage of irradiation. (author)

  20. Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and Starchless Chlamydomonas reinhardtii▿ †

    Science.gov (United States)

    Wang, Zi Teng; Ullrich, Nico; Joo, Sunjoo; Waffenschmidt, Sabine; Goodenough, Ursula

    2009-01-01

    When the unicellular green soil alga Chlamydomonas reinhardtii is deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid “popped-cell” microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ∼50% saturated (C16 and C18) fatty acids and ∼50% unsaturated fatty acids, half of which are in the form of oleic acid (C18:1). The FFA are ∼50% C16 and ∼50% C18. The LB-derived TAG yield from a liter of sta6 cells at 107 cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation. PMID:19880756

  1. Infection of human islets of Langerhans with two strains of Coxsackie B virus serotype 1: assessment of virus replication, degree of cell death and induction of genes involved in the innate immunity pathway.

    Science.gov (United States)

    Anagandula, Mahesh; Richardson, Sarah J; Oberste, M Steven; Sioofy-Khojine, Amir-Babak; Hyöty, Heikki; Morgan, Noel G; Korsgren, Olle; Frisk, Gun

    2014-08-01

    Type 1 diabetes mellitus is believed to be triggered, in part, by one or more environmental factors and human enteroviruses (HEVs) are among the candidates. Therefore, this study has examined whether two strains of HEV may differentially affect the induction of genes involved in pathways leading to the synthesis of islet hormones, chemokines and cytokines in isolated, highly purified, human islets. Isolated, purified human pancreatic islets were infected with strains of Coxsackievirus B1.Viral replication and the degree of CPE/islet dissociation were monitored. The expression of insulin, glucagon, CXCL10, TLR3, IF1H1, CCL5, OAS-1, IFNβ, and DDX58 was analyzed. Both strains replicated in islets but only one of strain caused rapid islet dissociation/CPE. Expression of the insulin gene was reduced during infection of islets with either viral strain but the gene encoding glucagon was unaffected. All genes analyzed which are involved in viral sensing and the development of innate immunity were induced by Coxsackie B viruses, with the notable exception of TLR3. There was no qualitative difference in the expression pattern between each strain but the magnitude of the response varied between donors. The lack of virus induced expression of TLR3, together with the differential regulation of IF1H1, OAS1 and IFNβ, (each of which has polymorphic variants influence the predisposition to type 1 diabetes), that might result in defective clearance of virus from islet cells. The reduced expression of the insulin gene and the unaffected expression of the gene encoding glucagon by Coxsackie B1 infection is consistent with the preferential β-cell tropism of the virus. © 2013 Wiley Periodicals, Inc.

  2. Human periodontal ligament stem cells suppress T-cell proliferation via down-regulation of non-classical major histocompatibility complex-like glycoprotein CD1b on dendritic cells.

    Science.gov (United States)

    Shin, C; Kim, M; Han, J-A; Choi, B; Hwang, D; Do, Y; Yun, J-H

    2017-02-01

    Periodontal ligament stem cells (PDLSCs) from the periodontal ligament tissue were recently identified as mesenchymal stem cells (MSCs). The capabilities of PDLSCs in periodontal tissue or bone regeneration have been reported, but their immunomodulatory role in T-cell immune responses via dendritic cells (DCs), known as the most potent antigen-presenting cell, has not been studied. The aim of this study is to understand the immunological function of homogeneous human STRO-1 + CD146 + PDLSCs in DC-mediated T-cell immune responses to modulate the periodontal disease process. We utilized highly purified (> 95%) human STRO-1 + CD146 + PDLSCs and human bone marrow mesenchymal stem cells (BMSCs). Each stem cell was co-cultured with human monocyte-derived DCs in the presence of lipopolysaccharide isolated from Porphyromonas gingivalis, a major pathogenic bacterium responsible for periodontal disease, in vitro to examine the immunological effect of each stem cell on DCs and DC-mediated T-cell proliferation. We discovered that STRO-1 + CD146 + PDLSCs, as well as BMSCs, significantly decreased the level of non-classical major histocompatibility complex glycoprotein CD1b on DCs, resulting in defective T-cell proliferation, whereas most human leukocyte antigens and the co-stimulatory molecules CD80 and CD86 in/on DCs were not significantly affected by the presence of BMSCs or STRO-1 + CD146 + PDLSCs. This study unveiled an immunomodulatory role of STRO-1 + CD146 + PDLSCs in negatively regulating DC-mediated T-cell immune responses, demonstrating their potential to be utilized in promising new stem cell therapies. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Preparation of function-enhanced vegetable oils

    Directory of Open Access Journals (Sweden)

    Hiroshi Maeda

    2016-01-01

    Full Text Available Background: Previously, we (HM found that most commercially available edible oils, which were processed by hexane extraction followed by a number of purification steps, were extremely low in anti-peroxy radical (ROO., or radical scavenging activity. This is a great contrast to the respective virgin oils as exemplified by extra-virgin olive oil or crude rape seed oil [1-4] (Figure 1. Therefore, such highly purified oils will became prooxidant and less desirable food components in terms of health oriented diet. Oxidized oils may eventually cause DNA cleavages, modification of proteins, RNA, and lipids, as well as cellular damage, or promote inflammation and carcinogenesis at later time [5-9]. These commercial oils of low antioxidant activity may be improved by adding functionally effective antioxidative components, by using dried vegetable-waste such as tomato-juice-waste-residues and wine-ferment-waste-residues. Their antioxiative components will be transferred into the functionally poor grade edible oils, and consequently, one can improve the quality of such functionally poor oils and thereby contributing human health [2,8,9]. The purpose of this paper is to report a practical procedure to fortify functionally low grade conventional edible oils to functionally enriched edible oils using dried vegetable-waste residues such as tomato juice waste, and wine-ferment-residues, or other vegetable-waste residues. Methods: (1 Preparation and measurements of lycopene and carotenoid enriched oils. To 5.0g or 1.0g of the dried residue of tomato juice waste, 100ml of commercial rape seed (canola oil was added respectively. Each mixture was incubated at room temperature in dark for several weeks. Amount of lycopene and carotenoids extracted into the oil was monitored by increase of absorption (400-550nm and fluorescence at 470nm of carotenoid. Grape-juice ferment (wine waste was similarly prepared after hot air drying, and immersed in canola oil. (2

  4. Reduction of the HIV-1 reservoir in resting CD4+ T-lymphocytes by high dosage intravenous immunoglobulin treatment: a proof-of-concept study

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    Karlsson Annika C

    2009-07-01

    Full Text Available Abstract Background The latency of HIV-1 in resting CD4+ T-lymphocytes constitutes a major obstacle for the eradication of virus in patients on antiretroviral therapy (ART. As yet, no approach to reduce this viral reservoir has proven effective. Methods Nine subjects on effective ART were included in the study and treated with high dosage intravenous immunoglobulin (IVIG for five consecutive days. Seven of those had detectable levels of replication-competent virus in the latent reservoir and were thus possible to evaluate. Highly purified resting memory CD4+ T-cells were activated and cells containing replication-competent HIV-1 were quantified. HIV-1 from plasma and activated memory CD4+ T-cells were compared with single genome sequencing (SGS of the gag region. T-lymphocyte activation markers and serum interleukins were measured. Results The latent HIV-1 pool decreased with in median 68% after IVIG was added to effective ART. The reservoir decreased in five, whereas no decrease was found in two subjects with detectable virus. Plasma HIV-1 RNA ≥ 2 copies/mL was detected in five of seven subjects at baseline, but in only one at follow-up after 8–12 weeks. The decrease of the latent HIV-1 pool and the residual plasma viremia was preceded by a transitory low-level increase in plasma HIV-1 RNA and serum interleukin 7 (IL-7 levels, and followed by an expansion of T regulatory cells. The magnitude of the viral increase in plasma correlated to the size of the latent HIV-1 pool and SGS of the gag region showed that viral clones from plasma clustered together with virus from activated memory T-cells, pointing to the latent reservoir as the source of HIV-1 RNA in plasma. Conclusion The findings from this uncontrolled proof-of-concept study suggest that the reservoir became accessible by IVIG treatment through activation of HIV-1 gene expression in latently-infected resting CD4+ T-cells. We propose that IVIG should be further evaluated as an adjuvant

  5. Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors.

    Science.gov (United States)

    Amable, Paola Romina; Carias, Rosana Bizon Vieira; Teixeira, Marcus Vinicius Telles; da Cruz Pacheco, Italo; Corrêa do Amaral, Ronaldo José Farias; Granjeiro, José Mauro; Borojevic, Radovan

    2013-06-07

    Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 10(6) to 1.9 × 10(6) platelets/μl). Platelets were highly purified, because only platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF. Our study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool

  6. A serum-free, purified vero cell rabies vaccine is safe and as immunogenic as the reference vaccine Verorab for pre-exposure use in healthy adults: results from a randomized controlled phase-II trial.

    Science.gov (United States)

    Pichon, Sylvie; Guinet-Morlot, Françoise; Minutello, Maria; Donazzolo, Yves; Rouzier, Regine; Chassard, Didier; Fitoussi, Serge; Hou, Victor

    2013-04-26

    Verorab was licensed in 1985 for both pre- and post-exposure prophylaxis of rabies. The next generation purified Vero cell rabies vaccine (PVRV-NG) is a highly purified vaccine. We performed a phase II clinical study in adults in France to assess its immunological non-inferiority and clinical safety for pre-exposure prophylaxis. In a randomized phase-II trial, 384 healthy adult subjects were randomized (2:1) to receive a three-dose primary series of PVRV-NG or Verorab. One year later, the PVRV-NG group received a PVRV-NG booster while the Verorab group participants were randomized to receive a booster of PVRV-NG or Verorab for. Rabies virus neutralizing antibodies (RVNA) were evaluated on days 0, 28 (subgroup), 42, months 6, 12 and 12+14 days. Safety was evaluated for seven days after each dose. Adverse event between doses, until 28 days after the final dose was recorded. Serious adverse events were recorded up to 6 months after the last dose. The criterion for non-inferiority was met in the per-protocol analysis set and confirmed in the full analysis set (FAS). In the FAS, 99.6% and 100% of subjects had RVNA titers ≥0.5 IU/mL in PVRV-NG and Verorab groups, respectively. While RVNA levels gradually decreased over the 12-month period, at 6 and 12 months after vaccination >89% and >77%, respectively, in both groups had RVNA titers ≥0.5 IU/mL. The PVRV-NG booster induced a strong response, irrespective of the vaccine given for the primary series. PVRV-NG was safe and well tolerated and its safety profile was similar to Verorab for unsolicited adverse events and solicited systemic reactions. The incidence of solicited injection-site reactions was lower with PVRV-NG than with Verorab after the primary series and the booster dose. PVRV-NG was shown to be at least as immunogenic as Verorab and to present a similar safety profile. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors

    Science.gov (United States)

    2013-01-01

    Introduction Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. Methods PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Results Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of

  8. Gene expression profiles of primary HPV16- and HPV18-infected early stage cervical cancers and normal cervical epithelium: identification of novel candidate molecular markers for cervical cancer diagnosis and therapy.

    Science.gov (United States)

    Santin, Alessandro D; Zhan, Fenghuang; Bignotti, Eliana; Siegel, Eric R; Cané, Stefania; Bellone, Stefania; Palmieri, Michela; Anfossi, Simone; Thomas, Maria; Burnett, Alexander; Kay, Helen H; Roman, Juan J; O'Brien, Timothy J; Tian, Erming; Cannon, Martin J; Shaughnessy, John; Pecorelli, Sergio

    2005-01-20

    With the goal of identifying genes with a differential pattern of expression between invasive cervical carcinomas (CVX) and normal cervical keratinocytes (NCK), we used oligonucleotide microarrays to interrogate the expression of 14,500 known genes in 11 primary HPV16 and HPV18-infected stage IB-IIA cervical cancers and four primary normal cervical keratinocyte cultures. Hierarchical cluster analysis of gene expression data identified 240 and 265 genes that exhibited greater than twofold up-regulation and down-regulation, respectively, in primary CVX when compared to NCK. Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), mesoderm-specific transcript, forkhead box M1, v-myb myeloblastosis viral oncogene homolog (avian)-like2 (v-Myb), minichromosome maintenance proteins 2, 4, and 5, cyclin B1, prostaglandin E synthase (PTGES), topoisomerase II alpha (TOP2A), ubiquitin-conjugating enzyme E2C, CD97 antigen, E2F transcription factor 1, and dUTP pyrophosphatase were among the most highly overexpressed genes in CVX when compared to NCK. Down-regulated genes in CVX included transforming growth factor beta 1, transforming growth factor alpha, CFLAR, serine proteinase inhibitors (SERPING1 and SERPINF1), cadherin 13, protease inhibitor 3, keratin 16, and tissue factor pathway inhibitor-2 (TFPI-2). Differential expression of some of these genes including CDKN2A/p16, v-Myb, PTGES, and TOP2A was validated by quantitative real-time PCR. Flow cytometry on primary CVX and NCK and immunohistochemical staining of formalin fixed paraffin-embedded tumor specimens from which primary CVX cultures were derived as well as from a separate set of invasive cervical cancers confirmed differential expression of the CDKN2A/p16 and PTGES markers on CVX versus NCK. These results identify several genes that are coordinately disregulated in cervical cancer, likely representing common signaling pathways triggered by HPV transformation. Moreover, these data obtained with highly purified primary tumor

  9. Mechanisms of Egg Yolk Formation and Implications on Early Life History of White Perch (Morone americana).

    Science.gov (United States)

    Schilling, Justin; Loziuk, Philip L; Muddiman, David C; Daniels, Harry V; Reading, Benjamin J

    2015-01-01

    The three white perch (Morone americana) vitellogenins (VtgAa, VtgAb, VtgC) were quantified accurately and precisely in the liver, plasma, and ovary during pre-, early-, mid-, and post-vitellogenic oocyte growth using protein cleavage-isotope dilution mass spectrometry (PC-IDMS). Western blotting generally mirrored the PC-IDMS results. By PC-IDMS, VtgC was quantifiable in pre-vitellogenic ovary tissues and VtgAb was quantifiable in pre-vitellogenic liver tissues however, neither protein was detected by western blotting in these respective tissues at this time point. Immunohistochemistry indicated that VtgC was present within pre-vitellogenic oocytes and localized to lipid droplets within vitellogenic oocytes. Affinity purification coupled to tandem mass spectrometry using highly purified VtgC as a bait protein revealed a single specific interacting protein (Y-box binding protein 2a-like [Ybx2a-like]) that eluted with suramin buffer and confirmed that VtgC does not bind the ovary vitellogenin receptors (LR8 and Lrp13). Western blotting for LR8 and Lrp13 showed that both receptors were expressed during vitellogenesis with LR8 and Lrp13 expression highest in early- and mid-vitellogenesis, respectively. The VtgAa within the ovary peaked during post-vitellogenesis, while VtgAb peaked during early-vitellogenesis in both white perch and the closely related striped bass (M. saxatilis). The VtgC was steadily accumulated by oocytes beginning during pre-vitellogenesis and continued until post-vitellogenesis and its composition varies widely between striped bass and white perch. In striped bass, the VtgC accounted for 26% of the vitellogenin-derived egg yolk, however in the white perch it comprised only 4%. Striped bass larvae have an extended developmental window and these larvae have yolk stores that may enable them to survive in the absence of food for twice as long as white perch after hatch. Thus, the VtgC may play an integral role in providing nutrients to late stage

  10. Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: a powerful quality-control tool.

    Science.gov (United States)

    Frankaer, Christian G; Moroz, Olga V; Turkenburg, Johan P; Aspmo, Stein I; Thymark, Majbritt; Friis, Esben P; Stahl, Kenny; Nielsen, Jens E; Wilson, Keith S; Harris, Pernille

    2014-04-01

    A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.

  11. Identification and characterization of cross-reactive natural rubber latex and Ficus benjamina allergens.

    Science.gov (United States)

    Chen, Z; Düser, M; Flagge, A; Maryska, S; Sander, I; Raulf-Heimsoth, M; Baur, X

    2000-12-01

    An association between allergy to Ficus benjamina and natural rubber latex (NRL) has been suspected based on clinical and immunological observations. The responsible cross-reactive allergens have not been identified yet. This study was undertaken to investigate the cross-reactivity between hevein (Hev b 6.02, 4.7 kD), a major allergen of NRL, and F. benjamina and identify its counterpart in F. benjamina. 89 serum samples from subjects allergic to NRL were used in the study. Skin prick tests were performed with highly purified hevein and sap extract of F. benjamina. Specific IgE antibodies to NRL, F. benjamina and Hev b 6.02 were determined by the Pharmacia CAP method. Cross-reactivity among these allergens was investigated by means of CAP and immunoblot inhibition experiments. Two-dimensional gel electrophoresis separation and protein microsequencing were performed to identify the cross-reactive allergens in F. benjamina. 67 out of 89 (75%) sera showed elevated IgE to hevein. Specific IgE to Ficus were found in 22 (24.7%) sera, and with 1 exception, all these sera also had IgE to Hev b 6.02. Results of CAP inhibition assays using 11 sera showing IgE to both Hev b 6.02 and Ficus demonstrated that the IgE to Ficus could be completely inhibited by Hev b 6.02 in 6 of 11 sera. Immunoblots and immunoblot inhibition assays revealed that a protein of about 45 kD in F. benjamina is strongly recognized by serum IgE. In addition, the IgE-binding reactivity to this 45-kD protein could be completely inhibited by preincubation of the sera with Hev b 6.02. N-terminal protein sequencing of 14 amino acids indicated that this 45-kD protein has a hevein-like domain at the N-terminal region and may belong to the endochitinase family. Latex-allergic patients are at higher risk of becoming sensitized to Ficus. Hev b 6.02 in latex is a major cross-reactive allergen and its counterpart in F. benjamina is an acidic protein with a molecular weight of about 45 kD and a hevein-like N

  12. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

    Energy Technology Data Exchange (ETDEWEB)

    Dick, Robert A.; Datta, Siddhartha A.K.; Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M. (NCI); (Cornell); (CM); (NIST)

    2016-05-06

    Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

    Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our

  13. Dual ferroic properties of hexagonal ferrite ceramics BaFe{sub 12}O{sub 19} and SrFe{sub 12}O{sub 19}

    Energy Technology Data Exchange (ETDEWEB)

    Kostishyn, V.G. [National University of Science and Technology MISIS, Leninskiy Aven. 4, Moscow 119049 (Russian Federation); Panina, L.V., E-mail: lpanina@plymouth.ac.uk [National University of Science and Technology MISIS, Leninskiy Aven. 4, Moscow 119049 (Russian Federation); Timofeev, A.V.; Kozhitov, L.V.; Kovalev, A.N. [National University of Science and Technology MISIS, Leninskiy Aven. 4, Moscow 119049 (Russian Federation); Zyuzin, A.K. [Science and Technology Institute of Interbranch Information, Sorge, St. 22, Moscow 125252 (Russian Federation)

    2016-02-15

    Dual ferroic properties of a strong magnetism and large ferroelectricity have been observed in barium BaFe{sub 12}O{sub 19} and strontium SrFe{sub 12}O{sub 19} hexaferrite ceramics. The samples were fabricated by a modified ceramic technique from highly purified raw materials with addition of boron oxide allowing the control of grain size and enhancement of bulk resistivity. Whereas the samples of PbFe{sub 12}O{sub 19} fabricated by the same technological method did not have sufficient electric resistivity to support an electric field and did not exhibit the ferroelectric properties. The structure of the samples examined by X-ray diffraction is consistent with a single hexagonal phase. The grains are randomly oriented with the average grain size of 300–400 nm coated with boron oxide. The magnetic properties are characterised by standard ferrimagnetic behavior with the Neel temperature of about 450 °C. Large spontaneous polarization was observed with the maximal values of 45–50 μC/cm{sup 2} under an applied electric field of 100–300 kV/m. A strong coupling between magnetic and electric ordering was confirmed by measuring the magnetoelectric (ME) parameter and magnetodielectric ratio. These ME characteristics are more advanced than those for well-known room temperature multiferroic BiFeO{sub 3}. Furthermore, by applying an electric field, the manipulation of magnetization in the range of up to 9% was observed, which is even greater than in some substituted hexaferrites with a non-collinear magnetic structure. The obtained results on electrical polarization are similar to the values reported for the corresponding hexaferrites sintered by polymer precursor technique. This suggests a promising potential of M-type hexaferrite ceramics in devices utilizing magnetoelectric coupling. - Highlights: • Ba(Sr)Fe{sub 12}O{sub 19}-hexaferrites show large room-temperature multiferroic properties. • Small addition of B{sub 2}O{sub 3} increases the hexaferrite

  14. Laboratory assessment of Activated Protein C Resistance/Factor V-Leiden and performance characteristics of a new quantitative assay.

    Science.gov (United States)

    Amiral, Jean; Vissac, Anne Marie; Seghatchian, Jerard

    2017-12-01

    Activated Protein C Resistance is mainly associated to a factor V mutation (RQ506), which induces a deficient inactivation of activated factor V by activated protein C, and is associated to an increased risk of venous and arterial thrombosis in affected individuals, caused by the prolonged activated factor V survival. Its prevalence is mainly in Caucasians (about 5%), and this mutation is absent in Africans and Asians. Presence of Factor V-Leiden is usually evidenced with clotting methods, using a two-step APTT assay performed without or with APC: prolongation of blood coagulation time is decreased if this factor is present. The R506Q Factor V-Leiden mutation is now usually characterized using molecular biology, and this technique tends to become the first intention assay for characterization of patients. Both techniques are qualitative, and allow classifying tested individuals as heterozygotes or homozygotes for the mutation, when present. A new quantitative assay for Factor V-Leiden, using a one-step clotting method, has been developed, and designed with highly purified human coagulation proteins. Clotting is triggered with human Factor Xa, in presence of calcium and phospholipids (mixture which favours APC action over clotting process). Diluted tested plasma, is supplemented with a clotting mixture containing human fibrinogen, prothrombin, and protein S at a constant concentration. APC is added, and clotting is initiated with calcium. Calibration is performed with a pool of plasmas from patients carrying the R506Q Factor V mutation, and its mixtures with normal plasma. Homozygous patients have clotting times of about 70sec. Factor V-Leiden concentration is usually >75% in homozygous patients, 30-60% in heterozygous patients and below 5% in normal. The assay is insensitive to clotting factor deficiencies (II, VII, VIII: C, IX, X), dicoumarol or heparin therapies, and has no interference with lupus anticoagulant (LA). This new assay for Factor V-Leiden can be

  15. Efficacy of artisanal preparations of cannabidiol for the treatment of epilepsy: Practical experiences in a tertiary medical center.

    Science.gov (United States)

    Porcari, Giulia S; Fu, Cary; Doll, Emily D; Carter, Emma G; Carson, Robert P

    2018-03-01

    Medically refractory epilepsy continues to be a challenge worldwide, and despite an increasing number of medical therapies, approximately 1 in 3 patients continues to have seizures. Cannabidiol (CBD), one of many constituents of the Cannabis sativa or marijuana plant, has received renewed interest in the treatment of epilepsy. While highly purified CBD awaits Food and Drug Administration (FDA) approval, artisanal formulations of CBD are readily available and are seeing increased use in our patient population. Although randomized controlled trials of CBD are ongoing and promising, data regarding artisanal formulations of CBD are minimal and largely anecdotal. Here, we report a retrospective study to define the efficacy of artisanal CBD preparations in children with epilepsy. Given the known interaction between CBD and clobazam, we also conducted a subgroup comparison to determine if clobazam use was related to any beneficial effects of CBD. Additionally, we compared response rates with CBD and with clobazam alone within an overlapping patient cohort. A pediatric cohort with epilepsy of 108 patients was identified through a medical record search for patients using CBD oil. The addition of CBD resulted in 39% of patients having a >50% reduction in seizures, with 10% becoming seizure-free. The responder rate for clobazam was similar. No patients achieved CBD monotherapy, although the weaning of other antiepileptic drugs (AEDs) became possible in 22% of patients. A comparable proportion had AED additions during CBD therapy. With concomitant use of clobazam, 44% of patients had a 50% reduction in seizures upon addition of CBD compared with 33% in the population not taking clobazam; this difference was not statistically significant. The most common reported side effect of CBD was sedation in less than 4% of patients, all of whom were also taking clobazam. Increased alertness and improved verbal interactions were reported in 14% of patients in the CBD group and 8% of

  16. Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha

    International Nuclear Information System (INIS)

    Valtieri, M.; Venturelli, D.; Care, A.; Fossati, C.; Pelosi, E.; Labbaye, C.; Mattia, G.; Gewirtz, A.M.; Calabretta, B.; Peschle, C.

    1991-01-01

    These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited. Using purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase

  17. Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution.

    Directory of Open Access Journals (Sweden)

    Juan Du

    Full Text Available Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and

  18. PLASMINOGEN ACTIVATOR OF YERSINIA PESTIS

    Directory of Open Access Journals (Sweden)

    V. V. Evseeva

    2015-01-01

    Full Text Available Plague has been the cause of three pandemics and has led to the death of millions of people. Plague is a typical zoonosis caused by Yersinia pestis that circulates in populations of wild rodents inhabiting natural plague foci on all continents except for Australia. Transmission of plague is provided by flea bites. Circulation of Y. pestis in natural plague foci is supported by a numerous of pathogenicity factors. This review explores one of them, plasminogen activator Pla. This protein is one of representatives of omptins, a family of enterobacterial outer membrane proteases that are responsible for colonization of specific organs or even infection generalization as a result of successful overcoming of the host innate immunity. The review reflects the history of its discovery and studying of its genetic control, biosynthesis, isolation and purification, physicochemical properties. Highly purified preparations of plasminogen activator are deficient in enzymatic activities but renaturation in the presence of Y. pestis lipooligosaccharide restores enzymatic properties of Pla. This pathogenicity factor is absent in representatives of the most ancient phylogenetic group of the plague pathogen, bv. caucasica, while the ancestor of other groups of Y. pestis subsp. microtus obtained in result of horizontal transfer Pla isoform with characteristics similar to properties of omptins from the less virulent enterobacteria. After that in the course of microevolution the “classic” isoform of Pla with increased protease activity was selected that is typical of all highly virulent for humans strains of Y. pestis subsp. pestis. The “classic” isoform of Pla Y. pestis is functionally similar to mammalian plasminogen activators transforming plasminogen into plasmin with the help of limited proteolysis. Pla protease activating plasminogen and also degrading the main plasmin inhibitor — α2-antiplasmin and, respectively, determining Y. pestis ability to lyse

  19. Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: implications for autologous bone marrow purging

    International Nuclear Information System (INIS)

    Gulliya, K.S.; Pervaiz, S.

    1989-01-01

    Laser photoradiation therapy was tested in an in vitro model for its efficacy in the elimination of non-Hodgkin's lymphoma cells. Results show that at 31.2 J/cm2 of laser light in the presence of 20 micrograms/mL of merocyanine 540 (MC540) there was greater than 5 log reduction in Burkitt's lymphoma (Daudi) cells. Similar tumor cell kill was obtained for leukemia (HL-60) cells at a laser light dose of 93.6 J/cm2. However, to obtain the same efficiency of killing for histiocytic lymphoma (U-937) cells, a higher dose of MC540 (25 micrograms/mL) was required. Clonogenic tumor stem cell colony formation was reduced by greater than 5 logs after laser photoradiation therapy. Under identical conditions for each cell line the percent survival for granulocyte-macrophage colony-forming units (CFU-GM, 45.9%, 40%, 17.5%), granulocyte/erythroid/macrophage/megakaryocyte (GEMM, 40.1%, 20.1%, 11.5%), colony-forming units (CFU-C, 16.2%, 9.1%, 1.8%), and erythroid burst-forming units (BFU-E, 33.4%, 17.8%, 3.9%) was significantly higher than the tumor cells. Mixing of gamma ray-irradiated normal marrow cells with tumor cells (1:1 and 10:1 ratio) did not interfere with the elimination of tumor cells. The effect of highly purified recombinant interferon alpha (rIFN) on laser photoradiation therapy of tumor cells was also investigated. In the presence of rIFN (30 to 3,000 U/mL), the viability of leukemic cells was observed to increase from 0% to 1.5% with a concurrent decrease in membrane polarization, suggesting an increase in fluidity of cell membrane in response to rIFN. However, at higher doses of rIFN (6,000 to 12,000 U/mL) this phenomenon was not observed. The viability of lymphoma cells remained unaffected at all doses of rIFN tested

  20. Awakened by cellular stress: isolation and characterization of a novel population of pluripotent stem cells derived from human adipose tissue.

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    Saleh Heneidi

    Full Text Available Advances in stem cell therapy face major clinical limitations, particularly challenged by low rates of post-transplant cell survival. Hostile host factors of the engraftment microenvironment such as hypoxia, nutrition deprivation, pro-inflammatory cytokines, and reactive oxygen species can each contribute to unwanted differentiation or apoptosis. In this report, we describe the isolation and characterization of a new population of adipose tissue (AT derived pluripotent stem cells, termed Multilineage Differentiating Stress-Enduring (Muse Cells, which are isolated using severe cellular stress conditions, including long-term exposure to the proteolytic enzyme collagenase, serum deprivation, low temperatures and hypoxia. Under these conditions, a highly purified population of Muse-AT cells is isolated without the utilization of cell sorting methods. Muse-AT cells grow in suspension as cell spheres reminiscent of embryonic stem cell clusters. Muse-AT cells are positive for the pluripotency markers SSEA3, TR-1-60, Oct3/4, Nanog and Sox2, and can spontaneously differentiate into mesenchymal, endodermal and ectodermal cell lineages with an efficiency of 23%, 20% and 22%, respectively. When using specific differentiation media, differentiation efficiency is greatly enhanced in Muse-AT cells (82% for mesenchymal, 75% for endodermal and 78% for ectodermal. When compared to adipose stem cells (ASCs, microarray data indicate a substantial up-regulation of Sox2, Oct3/4, and Rex1. Muse-ATs also exhibit gene expression patterns associated with the down-regulation of genes involved in cell death and survival, embryonic development, DNA replication and repair, cell cycle and potential factors related to oncogenecity. Gene expression analysis indicates that Muse-ATs and ASCs are mesenchymal in origin; however, Muse-ATs also express numerous lymphocytic and hematopoietic genes, such as CCR1 and CXCL2, encoding chemokine receptors and ligands involved in stem cell

  1. Lipopolysaccharide induces IFN-γ production in human NK cells

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    Leonid M Kanevskiy

    2013-01-01

    Full Text Available NK cells have been shown to play a regulatory role in sepsis. According to the current view, NK cells become activated via macrophages or dendritic cells primed by lipopolysaccharide (LPS. Recently TLR4 gene expression was detected in human NK cells suggesting the possibility of a direct action of LPS on NK cells. In this study, effects of LPS on NK cell cytokine production and cytotoxicity were studied using highly purified human NK cells. LPS induced IFN-γ production in the presence of IL-2 in cell populations containing >98% CD56+ cells. Surprisingly, in the same experiments LPS decreased NK cell degranulation. No significant expression of markers related to blood dendritic cells, monocytes or T or B lymphocytes in the NK cell preparations was observed; the portions of HLA-DRbright, CD14+, CD3+ and CD20+ cells amounted to less than 0.1% within the cell populations. No more than 0.2% of NK cells were shown to be slightly positive for surface TLR4 in our experimental system, although intracellular staining revealed moderate amounts of TLR4 inside the NK cell population. These cells were negative for surface CD14, the receptor participating in LPS recognition by TLR4. Incubation of NK cells with IL-2 or/and LPS did not lead to an increase in TLR4 surface expression. TLR4–CD56+ NK cells isolated by cell sorting secreted IFN-γ in response to LPS. Antibody to TLR4 did not block the LPS-induced increase in IFN-γ production. We have also shown that Re-form of LPS lacking outer core oligosaccharide and O-antigen induces less cytokine production in NK cells than full length LPS. We speculate that the polysaccharide fragments of LPS molecule may take part in LPS-induced IFN-γ production by NK cells. Collectively our data suggest the existence of a mechanism of LPS direct action on NK cells distinct from established TLR4-mediated signaling.

  2. Large-scale production of poly(3-hydroxyoctanoic acid) by Pseudomonas putida GPo1 and a simplified downstream process.

    Science.gov (United States)

    Elbahloul, Yasser; Steinbüchel, Alexander

    2009-02-01

    The suitability of Pseudomonas putida GPo1 for large-scale cultivation and production of poly(3-hydroxyoctanoate) (PHO) was investigated in this study. Three fed-batch cultivations of P. putida GPo1 at the 350- or 400-liter scale in a bioreactor with a capacity of 650 liters were done in mineral salts medium containing initially 20 mM sodium octanoate as the carbon source. The feeding solution included ammonium octanoate, which was fed at a relatively low concentration to promote PHO accumulation under nitrogen-limited conditions. During cultivation, the pH was regulated by addition of NaOH, NH(4)OH, or octanoic acid, which was used as an additional carbon source. Partial O(2) pressure (pO(2)) was adjusted to 20 to 40% by controlling the airflow and stirrer speed. Under the optimized conditions, P. putida GPo1 was able to grow to cell densities as high as 18, 37, and 53 g cells (dry mass) (CDM) per liter containing 49, 55, and 60% (wt/wt) of PHO, respectively. The resulting 40 kg CDM from these three cultivations was used directly for extraction of PHO. Three different methods of extraction of PHO were applied. From these, only acetone extraction showed better performance and resulted in 94% recovery of the PHO contents of cells. A novel mixture of precipitation solvents composed of 70% (vol/vol) methanol and 70% (vol/vol) ethanol was identified in this study. The ratio of PHO concentrate to the mixture was 0.2:1 (vol/vol) and allowed complete precipitation of PHO as white flakes. However, at a ratio of 1:1 (vol/vol) of the solvent mixture to PHO concentrate, a highly purified PHO was obtained. Precipitation yielded a dough-like polymeric material which was cast into thin layers and then shredded into small strips to allow evaporation of the remaining solvents. Gas chromatographic analysis revealed a purity of about 99% +/- 0.2% (wt/wt) of the polymer, which consisted mainly of 3-hydroxyoctanoic acid (96 mol%).

  3. Characteristics of myeloid differentiation and maturation pathway derived from human hematopoietic stem cells exposed to different linear energy transfer radiation types.

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    Monzen, Satoru; Yoshino, Hironori; Kasai-Eguchi, Kiyomi; Kashiwakura, Ikuo

    2013-01-01

    Exposure of hematopoietic stem/progenitor cells (HSPCs) to ionizing radiation causes a marked suppression of mature functional blood cell production in a linear energy transfer (LET)- and/or dose-dependent manner. However, little information about LET effects on the proliferation and differentiation of HSPCs has been reported. With the aim of characterizing the effects of different types of LET radiations on human myeloid hematopoiesis, in vitro hematopoiesis in Human CD34(+) cells exposed to carbon-ion beams or X-rays was compared. Highly purified CD34(+) cells exposed to each form of radiation were plated onto semi-solid culture for a myeloid progenitor assay. The surviving fractions of total myeloid progenitors, colony-forming cells (CFC), exposed to carbon-ion beams were significantly lower than of those exposed to X-rays, indicating that CFCs are more sensitive to carbon-ion beams (D(0) = 0.65) than to X-rays (D(0) = 1.07). Similar sensitivities were observed in granulocyte-macrophage and erythroid progenitors, respectively. However, the sensitivities of mixed-type progenitors to both radiation types were similar. In liquid culture for 14 days, no significant difference in total numbers of mononuclear cells was observed between non-irradiated control culture and cells exposed to 0.5 Gy X-rays, whereas 0.5 Gy carbon-ion beams suppressed cell proliferation to 4.9% of the control, a level similar to that for cells exposed to 1.5 Gy X-rays. Cell surface antigens associated with terminal maturation, such as CD13, CD14, and CD15, on harvest from the culture of X-ray-exposed cells were almost the same as those from the non-irradiated control culture. X-rays increased the CD235a(+) erythroid-related fraction, whereas carbon-ion beams increased the CD34(+)CD38(-) primitive cell fraction and the CD13(+)CD14(+/-)CD15(-) fraction. These results suggest that carbon-ion beams inflict severe damage on the clonal growth of myeloid HSPCs, although the intensity of cell surface

  4. Cost-effectiveness analysis on the use of rFSH + rLH for the treatment of anovulation in hypogonadotropic hypogonadal women

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    Papaleo E

    2014-06-01

    Full Text Available Enrico Papaleo,1 Carlo Alviggi,2 Giorgio Lorenzo Colombo,3,4 Claudio Pisanelli,5,6 Claudio Ripellino,7 Salvatore Longobardi,8 Pier Luigi Canonico91Centro Scienze della Natalità, Gynecological-Obstetrics Department, San Raffaele Hospital, Vita-Salute San Raffaele, Milan, Italy; 2Department of Neuroscience, Reproductive Sciences and Odontostomatology, University “Federico II” of Naples, Naples, Italy; 3Department of Drug Sciences, University of Pavia, Pavia, Italy; 4SAVE Studi Analisi Valutazioni Economiche, Milan, Italy; 5ACO San Filippo Neri, Rome, Italy; 6Società Italiana Di Farmacia Ospedaliera, Milan, Italy; 7CSD Medical Research Srl, Milan, Italy; 8Medical Department, Merck Serono SpA, Rome, Italy; 9Department of Pharmaceutical Sciences, University of Piemonte Orientale, Novara, Italy Background: Hypogonadotropic hypogonadal women are characterized by ovarian functionality deficiency, caused by low concentrations of follicle-stimulating hormone (FSH and luteinizing hormone (LH. To recover reproduction functionality, recommended therapies for ovarian induction involve injections of FSH and LH medications. Objective: Since important differences exist between recombinant and urinary gonadotropin therapies in terms of efficacy and cost, the objective of this study was to develop a cost-effectiveness model to compare recombinant FSH (rFSH + recombinant LH (rLH and highly purified human menopausal gonadotropin (HP-HMG. Methods: A Markov model was developed, considering three cycles of therapy; probability of pregnancy and miscarriage were considered, and the efficacy was evaluated in terms of pregnancy occurrence. The perspective of the model was that of the Italian Health Service, so only direct cost (drugs, specialist visits, patient examinations, and hospitalizations were included. Results: rFSH + rLH is associated with a higher total cost (€3,453.50 and higher efficacy (0.87 compared with HP-HMG (€2,719.70 and 0.50. rFSH + r

  5. CYP1A1 based on metabolism of xenobiotics by cytochrome P450 regulates chicken male germ cell differentiation.

    Science.gov (United States)

    Li, Dong; Wang, Man; Cheng, Shaoze; Zhang, Chen; Wang, Yilin; Zhang, Wenhui; Zhao, Ruifeng; Sun, Changhua; Zhang, Yani; Li, Bichun

    2017-04-01

    This study aimed to explore the regulatory mechanism of metabolism of xenobiotics by cytochrome P450 during the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) and consummate the induction differentiation system of chicken embryonic stem cells (cESCs) into SSCs in vitro. We performed RNA-Seq in highly purified male ESCs, male primordial germ cells (PGCs), and SSCs that are associated with the male germ cell differentiation. Thereinto, the metabolism of xenobiotics by cytochrome P450 was selected and analyzed with Venny among male ESC vs male PGC, male PGC vs SSC, and male ESC vs SSC groups and several candidates differentially expressed genes (DEGs) were excavated. Finally, quantitative real-time PCR (qRT-PCR) detected related DEGs under the condition of retinoic acid (RA) induction in vitro, and the expressions were compared with RNA-Seq. By knocking down CYP1A1, we detected the effect of CYP1