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Sample records for lysate antigen ortho

  1. [The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

    PubMed

    Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

    2014-08-01

    The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

  2. Polymer nanoparticles containing tumor lysates as antigen delivery vehicles for dendritic cell-based antitumor immunotherapy.

    PubMed

    Prasad, Shashi; Cody, Virginia; Saucier-Sawyer, Jennifer K; Saltzman, W Mark; Sasaki, Clarence T; Edelson, Richard L; Birchall, Martin A; Hanlon, Douglas J

    2011-02-01

    Encapsulation of tumor-associated antigens in polymer nanoparticles (NP) is a promising approach to enhance efficiency of antigen delivery for anti-tumor vaccines. Head and neck squamous carcinoma (HNSCC) cell lines were initially used to generate tumor-associated antigens (TAA)-containing poly (lactic-co-glycolic acid) (PLGA) NP; encapsulation efficiency and release kinetics were profiled. Findings were adopted to entrap fresh tumor lysate from five patients with advanced HNSCC. To test the hypothesis that NP enhance antigen presentation, dendritic cell (DC) produced from patient blood monocyte precursors were loaded with either the un-encapsulated or NP-encapsulated versions of tumor lysates. These were used to stimulate freshly-isolated autologous CD8+ T cells. In four of five patients, anti-tumor CD8+ T cells showed significantly increased immunostimulatory IFN-γ (p=0.071) or decreased immmunoinhibitory IL-10 production (p=0.0004) associated with NP-mediated antigen delivery. The observations represent an enabling step in the production of clinically-translatable, inexpensive, highly-efficient, and personalized polymer-based immunotherapy for solid organ malignancies. Enhancing the antigen presentation may be a viable approach to increase the efficiency of tumor cell directed cytotoxicity via immune mechanisms. This study presents an example for this using head and neck cancer cell lines and nanotechnology-based encapsulated antigen presentation to dendritic cells. The observed CD8+ T-cell response was significantly enhanced. This method may pave the way to a highly efficient cancer cell elimination method with minimal to no toxicity. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Blastomyces dermatitidis: Antibody Detection in Sera from Dogs with Blastomycosis with Yeast Lysate Antigens Produced from Human and Dog Isolates.

    PubMed

    Mondada, Katie; Fullmer, Jessie; Hungerford, Eric; Novack, Katrina; Vickers, Kristen; Scalarone, Gene

    2014-01-01

    Dogs are common hosts to the fungal organism Blastomyces dermatitidis, which causes the systemic disease blastomycosis. The goal of our study was to compare the reactivity of two B. dermatitidis yeast lysate antigens prepared from dog isolates (ERC-2, Wisconsin; T-58, Tennessee) and two lysate antigens prepared from human isolates (B5931 and B5896, Minnesota) against 48 serum specimens from dogs with confirmed blastomycosis using the indirect enzyme-linked immunosorbent assay (ELISA). Secondarily, we used three different ELISA substrates (Ultra TMB: A, SureBlue: B, and SureBlue Reserve: C) to compare the effectiveness of each substrate. Mean absorbance values ranged from 0.446 (B) to 0.651 (C) for the B5931 antigen and from 0.393 (B) to 0.540 (C) for the ERC-2 antigen in Trial 1. In Trial 2, the absorbance values ranged from 0.628 (B) to 0.909 (A) for the B5896 antigen and from 0.828 (B) to 1.375 (C) for the T-58 antigen. In Trial 1, the lysate antigen prepared from the human isolate B5931 exhibited the highest absorbance value and in Trial 2 the lysate prepared from the dog isolate T-58 was the most reactive. The overall results thus indicated that the T-58 lysate was the optimal reagent when used to detect antibody with the Sure-Blue Reserve substrate. Our laboratory is continuing to study B. dermatitidis antigen and substrate combinations for the reliable immunodiagnosis of blastomycosis in humans and animals.

  4. A midgut lysate of the Riptortus pedestris has antibacterial activity against LPS O-antigen-deficient Burkholderia mutants.

    PubMed

    Jang, Ho Am; Seo, Eun Sil; Seong, Min Young; Lee, Bok Luel

    2017-02-01

    Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O-antigen. The LPS O-antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild-type Burkholderia. To determine why LPS O-antigen-deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O-antigen-deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O-antigen-deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O-antigen-deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N-terminal sequence of the protein was determined to be a soybean Kunitz-type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O-antigen deficient (rough-type) Burkholderia.

  5. Role of adherent spleen cells in the induction of cytotoxic activity by Toxoplasma lysate antigen.

    PubMed

    Miyahara, K; Tose, S; Sakurai, H; Igarashi, I; Saito, A; Hirose, T; Suzuki, N

    1992-08-01

    In order to identify mechanisms responsible for the anti-tumor effects of Toxoplasma lysate antigen (TLA), we used an in vitro 51Cr release assay to study the functional properties of plastic-adherent cells during induction of splenic cytotoxic activity by TLA. Cytotoxic activity of non-adherent cells was measured in all experiments after a 6 days incubation. Induction of cytotoxic non-adherent cells by TLA required the presence of plastic-adherent spleen cells. In contrast, rhIL-2 alone was able to induce transformation of cytotoxic non-adherent cells from non-adherent spleen cells. Contact between adherent and non-adherent spleen cells was necessary for successful induction of cytotoxic non-adherent cells by TLA. Treatment of spleen cells with anti-macrophage serum prevented induction of cytotoxic activity by TLA. Biologically active IL-2 was not detected in culture supernatants of spleen cells exposed to TLA. These findings suggest that contact between TLA-sensitized non-adherent cells and macrophages is necessary for induction of cytotoxic cells in the presence of TLA. This contact, however, is not necessary for generation of IL-2-induced killer cells.

  6. Therapeutic effects of Toxoplasma lysate antigen on 20-methylcholanthrene-induced BALB/c mouse tumors.

    PubMed

    Miyahara, K; Honda, U; Tose, S; Sakurai, H; Igarashi, I; Saito, A; Hirose, T; Suzuki, N

    1992-02-01

    Therapeutic effects of Toxoplasma lysate antigen (TLA) were studied in mice bearing the tumor in the second passage of 20-methylcholanthrene (MC)-induced tumor cells. Intramuscular administration of TLA 7 days after the tumor-cell inoculation caused apparent inhibition of the tumor growth on day 14. The second treatment facilitated the therapeutic effects. Intravenous transfer of spleen cells prepared from TLA-sensitized mice into tumor-bearing mice also represented the growth inhibitory effects. Prominent effects were seen when the transferred cells were prepared 5 days after sensitization of donor animals. The inhibitory effects were absent in the groups transferred only the adherent cells or the non-adherent cells prepared from sensitized mice. The strongest inhibitory effect was observed in the group to which both adherent and non-adherent spleen cells were transferred simultaneously from sensitized mice. In in vitro experiments, spleen cells obtained from sensitized mice showed cytolytic effect on P-815 or YAC-1 cells after the secondary stimulation in vitro with TLA. Large non-adherent cells containing densely packed granules were induced when cultured with the adherent cells obtained from sensitized mice. These results revealed that TLA can inhibit the growth of the chemically-induced transplantable tumors by activation of adherent and non-adherent spleen cells.

  7. Characteristics of cytotoxic cells induced by Toxoplasma lysate antigen in mouse spleen.

    PubMed

    Miyahara, K; Matsumoto, T; Tose, S; Sakurai, H; Igarashi, I; Saito, A; Hirose, T; Suzuki, N

    1992-02-01

    Spleen cells from Toxoplasma lysate antigen (TLA)-sensitized BALB/c mice showed the strong cytotoxic activity against both natural killer (NK)-sensitive cells (YAC-1 and RL male-1) and NK-insensitive cells (P-815), when incubated with TLA or recombinant human IL-2 (rhIL-2). The increment of TLA concentration in culture medium increased the cytotoxic activity. Treatment of effector cells; spleen cells from TLA-sensitized mice incubated with TLA, with anti-asialo GM1 or anti-Thy-1 plus complement inhibited the cytotoxic activity of effector cells, whereas treatment with anti-mouse Lyt-2.2 serum plus complement had no effect on the cytotoxic activity. Treatment of spleen cells from TLA-sensitized mice with anti-asialo GM1 and/or anti-Thy-1 plus complement inhibited cytotoxic activities of effector cells. These results suggested that spleen cells sensitized with TLA both in vivo and in vitro were asialo GM1 positive and Thy-1 positive, and the majority of cytotoxic cells induced by TLA were similar to lymphokine-activated killer (LAK) cells induced by IL-2.

  8. Immune response and functional role of antibodies raised in heifers against a Staphylococcus aureus CP5 lysate and recombinant antigens vaccine formulated with Iscom Matrix adjuvant.

    PubMed

    Camussone, C M; Pujato, N; Renna, M S; Veaute, C M; Morein, B; Marcipar, I S; Calvinho, L F

    2014-12-15

    Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or inactivated whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 lysate vaccine adjuvanted with Iscom Matrix to generate a longer lasting specific antibody response in blood and milk, with improved opsonic capacity, compared with a S. aureus CP5 whole-cell formulation. The aim of the present study was to obtain an experimental immunogen composed of lysed cells of a CP5 S. aureus strain supplemented with recombinant clumping factor A, fibronectin binding protein A and β-toxin formulated with Iscom Matrix, characterize the immune response generated when immunizing pregnant heifers and assess the functional role of antibodies raised against this immunogen in experimental models. Both a lysate vaccine and a lysate+recombinant antigens vaccine elicited antibodies that promoted neutrophil phagocytosis and inhibited internalization into mammary epithelial cells, in vitro. Incorporation of defined antigenic molecules to the lysate formulation elicited a strong specific humoral immune response against both lysate and recombinant antigens and was associated with higher expression of regulatory and pro-inflammatory cytokines. In addition, antibodies were efficient for blocking S. aureus binding to bovine fibrinogen and fibronectin, and neutralizing β-toxin effect in vitro, placing these antigens as candidates to be included in a formulation directed to prevent staphylococcal bovine mastitis. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Differential capacity of chaperone-rich lysates in cross-presenting human endogenous and exogenous melanoma differentiation antigens.

    PubMed

    Bleifuss, Elke; Bendz, Henriette; Sirch, Birgit; Thompson, Sylvia; Brandl, Anna; Milani, Valeria; Graner, Michael W; Drexler, Ingo; Kuppner, Maria; Katsanis, Emmanuel; Noessner, Elfriede; Issels, Rolf-Dieter

    2008-12-01

    The goal of immune-based tumor therapies is the activation of immune cells reactive against a broad spectrum of tumor-expressed antigens. Vaccines based on chaperone proteins appear promising as these proteins naturally exist as complexes with various protein fragments including those derived from tumor-associated antigens. Multi-chaperone systems are expected to have highest polyvalency as different chaperones can carry distinct sets of antigenic fragments. A free-solution isoelectric focusing (FS-IEF) technique was established to generate chaperone-rich cell lysates (CRCL). Results from murine systems support the contention that CRCL induce superior anti-tumor responses than single chaperone vaccines. We established an in vitro model for human melanoma to evaluate the capacity of CRCL to transfer endogenously expressed tumor antigens to the cross-presentation pathway of dendritic cells (DC) for antigen-specific T cell stimulation. CRCL prepared from human melanoma lines contained the four major chaperone proteins Hsp/Hsc70, Hsp90, Grp94/gp96 and calreticulin. The chaperones within the melanoma cell-derived CRCL were functionally active in that they enhanced cross-presentation of exogenous peptides mixed into the CRCL preparation. Superior activity was observed for Hsp70-rich CRCL obtained from heat-stressed melanoma cells. Despite the presence of active chaperones, melanoma cell-derived CRCL failed to transfer endogenously expressed melanoma-associated antigens to DC for cross-presentation and cytotoxic T cell (CTL) recognition, even after increasing intracellular protein levels of tumor antigen or chaperones. These findings reveal limitations of the CRCL approach regarding cross-presentation of endogenously expressed melanoma-associated antigens. Yet, CRCL may be utilized as vehicles to enhance the delivery of exogenous antigens for DC-mediated cross-presentation and T cell stimulation.

  10. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    PubMed

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Incorporation of antigens from whole cell lysates and purified virions from MP12 into fluorescence microsphere immunoassays for the detection of antibodies against Rift Valley fever virus

    USDA-ARS?s Scientific Manuscript database

    Background: The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of Rift Valley fever virus (RVFV) IgG and IgM antibodies by incorporation of antigens from whole cell lysates and purified virions from MP12. Methods and Findings: Vir...

  12. Detection of Antiendothelial Cell Antibodies by an Enzyme-Linked Immunosorbent Assay Using Antigens from Cell Lysate: Minimal Interference with Antinuclear Antibodies and Rheumatoid Factors

    PubMed Central

    Drouet, Christian; Nissou, Marie-France; Ponard, Denise; Arvieux, Josiane; Dumestre-Pérard, Chantal; Gaudin, Philippe; Imbert, Bernard; Massot, Christian; Sarrot-Reynauld, Françoise

    2003-01-01

    The objective of the present work was to set up a routine test adapted to screening for antiendothelial cell antibodies (AECAs) in serum samples with minimal interference from antinuclear antibodies (ANAs) or rheumatoid factors (RFs). We compared the titers of AECAs titrated following two enzyme-linked immunosorbent assays (ELISAs): (i) an ELISA with ethanol-fixed EA.hy926 monolayers as the antigenic substrate and (ii) an ELISA with nucleus-depleted lysates prepared from EA.hy926 cells and normalized for protein (1.0 to 1.7 mg/ml) and DNA (≤0.1 μg/ml) contents as a surrogate substrate (postnuclear supernatant ELISA [PNS-ELISA]). The AECA titers in 51 serum samples, including 28 samples containing ANAs, were compared. A significantly positive correlation (r = 0.77; P < 0.001) between the two series was shown only for the ANA-negative serum samples. Conversely, ANAs or RFs in samples were shown not to interfere in tests for AECAs by the PNS-ELISA. AECAs recognize their antigenic targets in postnuclear supernatants, which is representative of the endothelial antigenic content, with improvement of the reliability of the assay, a prerequisite to application of the assay for their evaluation in clinical practice. PMID:12965929

  13. Chaperone-rich tumor cell lysate-mediated activation of antigen-presenting cells resists regulatory T cell suppression.

    PubMed

    Larmonier, Nicolas; Cantrell, Jessica; Lacasse, Collin; Li, Gang; Janikashvili, Nona; Situ, Elaine; Sepassi, Marjan; Andreansky, Samita; Katsanis, Emmanuel

    2008-04-01

    CD4(+)CD25(+) regulatory T lymphocytes (Tregs) critically contribute to the mechanisms of cancer-induced tolerance. These cells suppress anti-tumoral CD8(+) and CD4(+) T lymphocytes and can also restrain the function of APCs. We have previously documented the immunostimulatory effects of a chaperone-rich cell lysate (CRCL) anti-cancer vaccine. Tumor-derived CRCL induces tumor immunity in vivo, partly by promoting dendritic cell (DC) and macrophage activation. In the current study, we evaluated the effects of CD4(+)CD25(+)forkhead box P3(+) Tregs isolated from mice bearing 12B1 bcr-abl(+) leukemia on DC and macrophages that had been activated by 12B1-derived CRCL. CRCL-activated DC and macrophages resisted Treg suppression, as the production of proinflammatory cytokines, the activation of transcription factor NF-kappaB, and their immunostimulatory potential was unaffected by Tregs. Our results thus highlight CRCL as a powerful adjuvant endowed with the capacity to overcome tumor-induced Treg-inhibitory effects on APCs.

  14. Evaluation of In-House ELISA Using Trypanosoma cruzi Lysate and Recombinant Antigens for Diagnosis of Chagas Disease and Discrimination of Its Clinical Forms

    PubMed Central

    Longhi, Silvia A.; Brandariz, Silvia B.; Lafon, Sonia O.; Niborski, Leticia L.; Luquetti, Alejandro O.; Schijman, Alejandro G.; Levin, Mariano J.; Gómez, Karina A.

    2012-01-01

    The aim of this work was to investigate the potential usefulness of Trypanosoma cruzi lysate, recombinant protein JL7, and peptides P013, R13, JL18, JL19, and P0β as serological markers for human Chagas disease. We analyzed 228 sera from Brazilian Chagas disease patients classified into four clinical groups and 108 from non-chagasic patients. We defined the diagnostic sensitivity, specificity, and Kappa index measured by enzyme-linked immunosorbent assay (ELISA). As previously described, the highest values of diagnostic parameters were achieved for T. cruzi lysate and JL7; peptide P013 showed high specificity but low sensitivity. The other peptides resulted in lower sensitivity and specificity in our ELISA than T. cruzi lysate and JL7 protein. Antibodies against JL7 protein were mainly detected in sera from patients with severe chagasic cardiomyopathy, compared with those from the indeterminate form, whereas peptides failed to discriminate between the clinical forms of the disease. PMID:22855757

  15. Tumor cell lysates as immunogenic sources for cancer vaccine design

    PubMed Central

    González, Fermín E; Gleisner, Alejandra; Falcón-Beas, Felipe; Osorio, Fabiola; López, Mercedes N; Salazar-Onfray, Flavio

    2015-01-01

    Autologous dendritic cells (DCs) loaded with tumor-associated antigens (TAAs) are a promising immunological tool for cancer therapy. These stimulate the antitumor response and immunological memory generation. Nevertheless, many patients remain refractory to DC approaches. Antigen (Ag) delivery to DCs is relevant to vaccine success, and antigen peptides, tumor-associated proteins, tumor cells, autologous tumor lysates, and tumor-derived mRNA have been tested as Ag sources. Recently, DCs loaded with allogeneic tumor cell lysates were used to induce a potent immunological response. This strategy provides a reproducible pool of almost all potential Ags suitable for patient use, independent of MHC haplotypes or autologous tumor tissue availability. However, optimizing autologous tumor cell lysate preparation is crucial to enhancing efficacy. This review considers the role of cancer cell-derived lysates as a relevant source of antigens and as an activating factor for ex vivo therapeutic DCs capable of responding to neoplastic cells. These promising therapies are associated with the prolonged survival of advanced cancer patients. PMID:25625929

  16. A Proteomic Characterization of NTHi lysates

    PubMed Central

    Preciado, Diego; Poley, Marian; Tsai, Stephanie; Tomney, Amarel; Brown, Kristy; Val, Stephanie

    2015-01-01

    Background Non-typeable Haemophilus influenzae (NTHi) is a ubiquitous bacterial pathogen which accounts for a majority of human upper respiratory tract infections. Laboratory lysate preparations from this bacterium are commonly utilized to investigate the promulgation of inflammatory responses in respiratory and middle ear epithelium both in vivo and in vitro. We undertook an unbiased proteomics based analysis of NTHi lysate preps to: a) identify abundant bacterial proteins present in these lysates that could play a role in NTHi biological effects and b) determine the protein content variability in different lysate prep batches from the same NTHI strain. Study Design Proteomic analysis of laboratory NTHi lysate preparations from clinical strain 12. Methods NTHi lysates were denatured, gel-fractionated, digested by trypsin and the generated peptides were identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS). Western blot analyses for the important proinflammatory enhancer, outer membrane protein 6 (OMP6), was performed to validate the MS findings. Luciferase assays for NF-kB activation were used to measure the pro-inflammatory biologic effects from each NTHi lysate preparation. Results The MS identified 793 unique NTHi proteins. Most common and abundant proteins found have been described to either contribute to biofilm formation, elude the innate immune system, or activate epithelial pro-inflammatory pathways such as Toll Like Receptor 2 (TLR-2) signaling and NF-kB transcription factor. Strong positive signal for OMP6 was found in each of the NTHi lysate preparations. Significant NF-kB promoter response activation as expected with NTHi stimulation over control was also noted for each NTHi lysate preparation. Conclusions Proteomics was a successful technique to broadly define the protein content of NTHi lysates. This is the first report of the proteome of NTHi lysates widely used in laboratories to study the biological effect of NTHi. Despite

  17. ortho-Anisidine

    Integrated Risk Information System (IRIS)

    ortho - Anisidine ; CASRN 90 - 04 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  18. Cladribine. Ortho Biotech Inc.

    PubMed

    Tortorella, C; Rovaris, M; Filippi, M

    2001-12-01

    Cladribine, an adenosine deaminase inhibitor, has been developed and launched by Ortho Biotech in collaboration with The Scripps Research Institute for the treatment of several neoplasms, including acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, cutaneos T-cell lymphoma, hairy-cell leukemia and non-Hodgkin's lymphoma. It was first launched in the US in February 1993. Ortho Biotech and The Scripps Research Institute have since been developing the compound for its potential use in multiple sclerosis (MS). In 1997, Ortho filed air NDA in the US for the use of cladribine in the treatment of relapsing-remitting and secondary progressive MS. An FDA drug advisory committee was planning to meet in January 1999 to discuss the NDA. However, Ortho cancelled the meeting. Following an FDA inspection during December 1998 and January 1999, the Scripps Clinic received a warning letter from the FDA in April 1999 regarding violations in the clinical studies of cladribine for MS, and Ortho withdrew the NDA after concluding that further clinical studies would be necessary. Cladribine has been known since the 1960s as an intermediate for the synthesis of 2-deoxynucleotides and its potential for the treatment of leukemia was disclosed in 1984. The Scripps Research Institute and the Johnson & Johnson group hold several patents claiming preparation methods (US 05208327), and additional indications, such as multiple sclerosis (WO-09316706) and rheumatoid arthritis (US-05310732). The associated patent, WO-09323508, is the only one among those patents that claims the use of unmodified cladribine for the treatment of leukemia, but it focuses particularly on a specific form of the disease, chronic myelogenous leukemia. Analysts at UBS Warburg predicted in October 2001, that the product would make US sales of $50 million in 2004 for its MS indication.

  19. Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544.

    PubMed

    Jain, Lata; Rawat, Mayank; Prajapati, Awadhesh; Tiwari, Ashok Kumar; Kumar, Bablu; Chaturvedi, V K; Saxena, H M; Ramakrishnan, Sarvanan; Kumar, Jatin; Kerketta, Priscilla

    2015-09-01

    The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage 'ϕLd' was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 10(8) CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ∼16, 19 and 23 kDa could be identified as immuno-dominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 μg protein and 60 μg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner.

  20. Compaction agent clarification of microbial lysates

    NASA Technical Reports Server (NTRS)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  1. Compaction agent clarification of microbial lysates

    NASA Technical Reports Server (NTRS)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  2. Detection of multiple cytokines by protein arrays from cell lysate and tissue lysate.

    PubMed

    Lin, Ying; Huang, Ruochun; Cao, Xuan; Wang, Shen-Ming; Shi, Qing; Huang, Ruo-Pan

    2003-02-01

    Previously we demonstrated that multiple cytokines could be simultaneously detected using an antibody-based protein array system with high sensitivity and specificity from conditioned medium and serum. Here, we created a higher density array system to simultaneously detect 35 cytokines from cell lysates and tissue lysates. This assay combines the advantages of the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL), and high-throughput of microspot. In this system, capture antibodies dissolved in methanol were spotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated with tissue lysates or cell lysates. After removing unbound proteins by extensive washing, the membranes were exposed to horseradish peroxidase (HRP)-conjugated antibody(ies). The signals were visualized with an ECL system. High specificity, sensitivity, and accuracy of this approach were demonstrated. This approach can be used in any general laboratory setting without any sophisticated equipment. It should be feasible to extend this concept to develop a high-throughput protein array system. Combining nitrocellulose membrane-based and PVDF membrane-based approaches, the human cytokine array system can be applied to detect multiple cytokine expression from cell lysate, tissue lysate, serum, plasma, and conditioned medium. Future applications of this new approach include direct protein expression profiling, immunological disease diagnostics, and discovery of new biomarkers.

  3. RNase activity in erythroid cell lysates

    PubMed Central

    Burka, Edward R.

    1969-01-01

    The characteristics of degradation of reticulocyte ribonucleic acid (RNA) and ribosomes were studied in a whole erythroid cell lysate system. The process followed Michaelis-Menten kinetics, and indicated that RNA degradation in the erythroid cell is mediated by an enzyme previously isolated from reticulocyte hemolysates. Erythroid cell RNase activity had a temperature optimum of 50°C, a pH optimum of 7.0, was not energy dependent, was heat labile at physiologic pH, and was inhibited by Mg++, Ca++, and exposure to bentonite and deoxycholate. Free sulfhydryl groups were not essential for RNase activity. Of the substrates occurring naturally within the erythroid cell, isolated ribosomal RNA was most susceptible to the action of the enzyme, intact ribosomes least susceptible, and transfer RNA intermediate between them. Natural substrates were degraded completely to nucleotides in cell lysates. Competitive inhibition studies indicate that one enzyme system is capable of degrading both RNA and ribosomes, although the existence of more than one enzyme has not been excluded. Erythroid cell lysates quickly broke down polyribosomes into single ribosomes. The more rapid degradation of ribosomes, as compared with transfer RNA, which occurs in vivo, as opposed to findings in vitro, suggests that there is a special intracellular mechanism responsible for ribosome degradation in the maturing erythroid cell. Images PMID:5822581

  4. Tumor lysate-loaded biodegradable microparticles as cancer vaccines

    PubMed Central

    Joshi, Vijaya B.; Geary, Sean M.; Gross, Brett P; Wongrakpanich, Amaraporn; Norian, Lyse A.; Salem, Aliasger K.

    2014-01-01

    Cancer vaccines that use tumor lysate (TL) as a source of tumor-associated antigens (TAAs) have significant potential for generating therapeutic anti-tumor immune responses. Vaccines encompassing TL bypass the limitations of single antigen vaccines by simultaneously stimulating immunity against multiple TAAs, thereby broadening the repertoire of TAA-specific T cell clones available for activation. Administration of TL in particulate form, such as when encapsulated in biodegradable microparticles, increases its immunostimulatory capacity and produces more robust immune responses than when TL is given in soluble form. These effects can be further enhanced by co-administering TL with adjuvants. A number of recent studies using polymeric microparticle delivery of TL, with or without adjuvants, have produced promising results in preclinical studies. In this review, we will discuss current experimental approaches involving TL being pursued in the oncoimmunology field, and comment on strategies such as combining specific chemotherapeutic agents with TL microparticle delivery that may eventually lead to improved survival outcomes for cancer patients. PMID:24219096

  5. The Microsoft Global Ortho Program

    NASA Astrophysics Data System (ADS)

    Walcher, W.; Leberl, F.; Gruber, M.

    2012-07-01

    Wide area and thus continental mapping extending beyond national borders is a novel concept in civilian photogrammetry. The Microsoft Global Ortho Program was launched in the Spring of 2009 as a result of Microsoft's need for global geo-data at a high geometric resolution and radiometric excellence. By fall of 2012 more than 10 million km2 of the USA and 14 European countries will have been covered by seamless 30 cm GSD color-, 60 cm GSD false-color infrared ortho-mosaics and a 1 meter GSD digital surface model. The ortho-maps are being published to Microsoft's Bing Maps Internet mapping portal. The Global Ortho Program was designed for highly and unprecedented automated mapping of essentially entire continents. In 2011, exclusive of flight operations, the product output per person has been measured in excess of 275,000 square km per year. We describe research efforts that made this achievement possible. Those include a specially designed aerial sensor (Ultracam G), logistics simulation for fight planning and optimization, in-flight blur detection and subsequent automatic blur removal, modeling and removal of atmospheric and environmental conditions, automated shear detection and DTM refinement, an IT architecture to process >200,000 aerial images/day, and for creating over 1,000,000 km2 ortho-imagery and DSM data in 24 hours. While addressing these issues, we provide ideas how this might affect the future of spatial infrastructure initiatives.

  6. Murine Dendritic Cells Pulsed with Whole Tumor Lysates Mediate Potent Antitumor Immune Responses in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Fields, R. C.; Shimizu, K.; Mule, J. J.

    1998-08-01

    The highly efficient nature of dendritic cells (DC) as antigen-presenting cells raises the possibility of uncovering in tumor-bearing hosts very low levels of T cell reactivity to poorly immunogenic tumors that are virtually undetectable by other means. Here, we demonstrate the in vitro and in vivo capacities of murine bone marrow-derived, cytokine-driven DC to elicit potent and specific anti-tumor responses when pulsed with whole tumor lysates. Stimulation of naive spleen-derived T cells by tumor lysate-pulsed DC generated tumor-specific proliferative cytokine release and cytolytic reactivities in vitro. In addition, in two separate strains of mice with histologically distinct tumors, s.c. injections of DC pulsed with whole tumor lysates effectively primed these animals to reject subsequent lethal challenges with viable parental tumor cells and, important to note, also mediated significant reductions in the number of metastases established in the lungs. Tumor rejection depended on host-derived CD8+ T cells and, to a lesser extent, CD4+ T cells. Spleens from mice that had rejected their tumors contained specific precursor cytotoxic T lymphocytes. The use of whole tumor lysates as a source of tumor-associated antigen(s) for pulsing of DC circumvents several limitations encountered with other methods as well as provides certain distinct advantages, which are discussed. These data serve as rationale for our recent initiation of a phase I clinical trial of immunization with autologous tumor lysate-pulsed DC in adult and pediatric cancer patients.

  7. Immunological Characterization of Whole Tumour Lysate-Loaded Dendritic Cells for Cancer Immunotherapy

    PubMed Central

    Ottobrini, Luisa; Biasin, Mara; Borelli, Manuela; Lucignani, Giovanni; Trabattoni, Daria; Clerici, Mario

    2016-01-01

    Introduction Dendritic cells play a key role as initiators of T-cell responses, and even if tumour antigen-loaded dendritic cells can induce anti-tumour responses, their efficacy has been questioned, suggesting a need to enhance immunization strategies. Matherials & Methods We focused on the characterization of bone marrow-derived dendritic cells pulsed with whole tumour lysate (TAA-DC), as a source of known and unknown antigens, in a mouse model of breast cancer (MMTV-Ras). Dendritic cells were evaluated for antigen uptake and for the expression of MHC class I/II and costimulatory molecules and markers associated with maturation. Results Results showed that antigen-loaded dendritic cells are characterized by a phenotypically semi-mature/mature profile and by the upregulation of genes involved in antigen presentation and T-cell priming. Activated dendritic cells stimulated T-cell proliferation and induced the production of high concentrations of IL-12p70 and IFN-γ but only low levels of IL-10, indicating their ability to elicit a TH1-immune response. Furthermore, administration of Antigen loaded-Dendritic Cells in MMTV-Ras mice evoked a strong anti-tumour response in vivo as demonstrated by a general activation of immunocompetent cells and the release of TH1 cytokines. Conclusion Data herein could be useful in the design of antitumoral DC-based therapies, showing a specific activation of immune system against breast cancer. PMID:26795765

  8. Stability of DNA Origami Nanoarrays in Cell Lysate

    PubMed Central

    Mei, Qian; Wei, Xixi; Su, Fengyu; Liu, Yan; Youngbull, Cody; Johnson, Roger; Lindsay, Stuart; Yan, Hao; Meldrum, Deirdre

    2012-01-01

    Scaffolded DNA origami, a method to create self-assembled nanostructures with spatially addressable features, has recently been used to develop water-soluble molecular chips for label-free RNA detection, platforms for deterministic protein positioning, and single molecule reaction observatories. These applications highlight the possibility of exploiting the unique properties and biocompatibility of DNA nanostructures in live, cellular systems. Herein, we assembled several DNA origami nanostructures of differing shape, size and probes, and investigated their interaction with lysate obtained from various normal and cancerous cell lines. We separated and analyzed the origami–lysate mixtures using agarose gel electrophoresis and recovered the DNA structures for functional assay and subsequent microscopic examination. Our results demonstrate that DNA origami nanostructures are stable in cell lysate and can be easily separated from lysate mixtures, in contrast to natural, single- and double-stranded DNA. Atomic force microscope (AFM) and transmission electron microscope (TEM) images show that the DNA origami structures are fully intact after separation from cell lysates and hybridize to their targets, verifying the superior structural integrity and functionality of self-assembled DNA origami nanostructures relative to conventional oligonucleotides. The stability and functionality of DNA origami structures in cell lysate validate their use for biological applications, for example, as programmable molecular rafts or disease detection platforms. PMID:21366226

  9. Adenovirus particle quantification in cell lysates using light scattering.

    PubMed

    Hohl, Adrian; Ramms, Anne Sophie; Dohmen, Christian; Mantwill, Klaus; Bielmeier, Andrea; Kolk, Andreas; Ruppert, Andreas; Nawroth, Roman; Holm, Per Sonne

    2017-08-15

    Adenoviral vector production for therapeutic applications is a well-established routine process. However, current methods for measurement of adenovirus particle titers as a quality characteristic require highly purified virus preparations. While purified virus is typically obtained in the last step of downstream purification, rapid and reliable methods for adenovirus particle quantification in intermediate products and crude lysates to allow for optimization and validation of cell cultures and intermediate downstream processing steps are currently not at hand. Light scattering is an established process to measure virus particles' size. Though, due to cell impurities adequate quantification of adenovirus particles in cell lysates by light scattering has been impossible until today. This report describes a new method using light scattering to measure virus concentration in non-purified cell lysates. Here we report application of light scattering, a routine method to measure virus particle size, to virus quantification in enzymatically conditioned crude lysates. Samples are incubated with phospholipase A2 and benzonase and filtered through 0.22 µm filter cartridge prior to quantification by light scattering. Our results show that this treatment provides a precise method for fast and easy determination of total adenovirus particle numbers in cell lysates and is useful to monitor virus recovery throughout all downstream processing.

  10. Directed ortho,ortho'-dimetalation of hydrobenzoin: Rapid access to hydrobenzoin derivatives useful for asymmetric synthesis.

    PubMed

    Cho, Inhee; Meimetis, Labros; Belding, Lee; Katz, Michael J; Dudding, Travis; Britton, Robert

    2011-01-01

    A variety of ortho,ortho'-disubstituted hydrobenzoin derivatives are readily accessible through a directed ortho,ortho'-dimetalation strategy in which the alcohol functions in hydrobenzoin are deprotonated by n-BuLi and the resulting lithium benzyl alkoxides serve as directed metalation groups. The optimization and scope of this reaction are discussed, and the utility of this process is demonstrated in the one-pot preparation of a number of chiral diols as well as a short synthesis of the chiral ligand Vivol.

  11. Generation of a pool of human platelet lysate and efficient use in cell culture.

    PubMed

    Schallmoser, Katharina; Strunk, Dirk

    2013-01-01

    Human platelets represent a promising source of bioactive substances as growth factors not just for in vivo wound healing and tissue repair, but also for the expansion of human stem and progenitor cells in vitro. The replacement of fetal bovine serum (FBS) as a standard culture supplement by human platelet-derived growth factors now allows for the GMP-compliant implementation of various cell therapeutics in the growing field of regenerative medicine.For this purpose a protocol for the preparation of human platelet lysate (HPL) by several freeze-thaw cycles has been developed, resulting in platelet fragmentation and the release of stored growth factors. By pooling up to 15 U of HPL derived from individual blood donors, a virtually standardized product is achieved. The depletion of platelet particles and fragments in a final centrifugation step reduces the risk of alloimmunization against platelet antigens and the formation of aggregates in cell culture.The successful application of pooled human platelet lysate (pHPL) as a culture medium supplement for the ex vivo propagation of human mesenchymal stem/progenitor cells (MSPCs) and endothelial colony forming progenitor cells (ECFCs) indicates the feasibility of this animal serum-free source of growth factors. Further studies will evaluate efficacy and safety of pHPL.

  12. Ability of gonococcal and meningococcal lipooligosaccharides to clot Limulus amebocyte lysate.

    PubMed Central

    Roth, R I; Yamasaki, R; Mandrell, R E; Griffiss, J M

    1992-01-01

    We investigated whether the striking difference in severity of coagulopathy observed between bacterial sepsis involving Neisseria meningitidis and Neisseria gonorrhoeae species is related to species-dependent abilities to directly activate coagulation. Using lipooligosaccharide (LOS)-activated gelation of Limulus amebocyte lysate, we compared the relative abilities of outer membrane LOS of 10 N. meningitidis and 10 N. gonorrhoeae strains to initiate coagulation. A wide range of procoagulant potencies was observed for each species, and there was significant overlap of potencies between species. Relative biological activities did not correlate with the oligosaccharide components as defined by LOS molecular weight or specific antigenic epitopes. Purified lipid A of two LOS strains of different potency demonstrated relative procoagulant biological activities similar to those of their parent LOSs. When these lipid A preparations were further separated by thin-layer chromatography, the most polar component of each lipid A possessed the majority of the procoagulant activity. We concluded that the ability of neisserial LOS to initiate coagulation of Limulus lysate is a property of the lipid A portion of the molecule and is most likely determined by fine structural differences in the lipid A which are independent of species. Images PMID:1541549

  13. Molecularly engineered poly(ortho ester) microspheres for enhanced delivery of DNA vaccines

    NASA Astrophysics Data System (ADS)

    Wang, Chun; Ge, Qing; Ting, David; Nguyen, David; Shen, Hui-Rong; Chen, Jianzhu; Eisen, Herman N.; Heller, Jorge; Langer, Robert; Putnam, David

    2004-03-01

    Genetic vaccination using plasmid DNA presents a unique opportunity for achieving potent immune responses without the potential limitations of many conventional vaccines. Here we report the design of synthetic biodegradable polymers specifically for enhancing DNA vaccine efficacy in vivo. We molecularly engineered poly(ortho ester) microspheres that are non-toxic to cells, protect DNA from degradation, enable uptake by antigen-presenting cells, and release DNA rapidly in response to phagosomal pH. One type of microsphere of poly(ortho esters) that releases DNA vaccines in synchrony with the natural development of adaptive immunity, elicited distinct primary and secondary humoral and cellular immune responses in mice, and suppressed the growth of tumour cells bearing a model antigen. This polymer microparticulate system could, with further study, have implications for advancing the clinical utility of DNA vaccines as well as other nucleic-acid-based therapeutics against viral infections and cancer.

  14. May We Strengthen the Human Natural Defenses with Bacterial Lysates?

    PubMed Central

    2010-01-01

    During the last twenty years bacterial lysates have gained a new interest and their use has obtained a progressively larger consensus in the medical practice. They are commonly used as immunomodulators, in order to up-regulate immune responses against infectious damages. As a matter of fact, the role of these lysate seems relevant in upper and lower respiratory tract infections prevention, frequently observed both in paediatric and elder ages, and which represent a relevant problem also in terms of socio-economical implications. The effects of bacterial lysates as immunostimulatory agents have become the central point of many studies. The aim of those in vivo and in vitro studies was to understand and evaluate the capacity of this kind of treatments to create a better answer of the immune system against microbial infections, eventually leading to a reduction in their number. All the in vivo and in vitro findings analyzed support the evidence that bacterial lysates are powerful inducers of a specific immune response against bacterial infections. Both in paediatric and adult clinical trials, a positive trend has been found in terms of overall reduction of infection rates and duration, beneficial effect on symptoms, reduction in antibiotics use and possibility to improve the patient's quality of life in several diseases. Further well-designed trials in terms of blinding and randomization procedures and including a higher number of patients, selected according to the disease and its severity, are needed. PMID:23282746

  15. Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

    PubMed Central

    Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

    1994-01-01

    We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system. PMID:7496936

  16. Direct ortho-arylation of ortho-substituted benzoic acids: overriding Pd-catalyzed protodecarboxylation.

    PubMed

    Arroniz, Carlos; Ironmonger, Alan; Rassias, Gerry; Larrosa, Igor

    2013-02-15

    ortho-Arylation of ortho-substituted benzoic acids is a challenging process due to the tendency of the reaction products toward Pd-catalyzed protodecarboxylation. A simple method for preventing decarboxylation in sterically hindered benzoic acids is reported. The method described represents a reliable and broadly applicable entry to 2-aryl-6-substituted benzoic acids.

  17. Ortho and parahydrogen in interstellar material

    NASA Technical Reports Server (NTRS)

    Reeves, R. R.; Harteck, P.

    1979-01-01

    The ortho/para molecular hydrogen ratio in the interstellar medium is considered. It is shown that the ortho/para ratio will be 3:1 in practically all chemical reactions, even at relatively low temperatures. Two examples of exothermic processes that will result in the formation of a 3:1 ortho:para ratio, corresponding to a high-temperature equilibrium, are examined: H2 formation via three-body or surface recombination and catalytic recombination involving electrons and H(-) ions. Gas-phase scrambling ion reactions are also discussed, and it is suggested that virtually all the H2 equilibrated via scrambling reactions involving H(+) and H3(+) ions should exist as parahydrogen in the J ? 0 quantum state. Arguments are given that deuterium cannot interfere with the long scrambling chain that results in parahydrogen formation.

  18. Vaccination with melanoma lysate-pulsed dendritic cells, of patients with advanced colorectal carcinoma: report from a phase I study.

    PubMed

    Burgdorf, S K; Fischer, A; Claesson, M H; Kirkin, A F; Dzhandzhugazyan, K N; Rosenberg, J

    2006-06-01

    Immune therapy have shown new and exciting perspectives for cancer treatment. Aim of our study was to evaluate toxicity and possible adverse effects from vaccination of patients with advanced colorectal cancer with autologous dendritic cells (DC) pulsed with lysate from a newly developed melanoma cell line, DDM-1.13. Six patients were enrolled in the phase I trial. Autologous DCs were generated in vitro from peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were pulsed with melanoma cell lysate from a cloned and selected melanoma cell line enriched in expression of MAGE-A antigens and deficient in expression of melanoma differentiation antigens: tyrosinase, MART-1 and gp100. Vaccinations were administered intradermally on the proximal thigh with a total of five given vaccines at 2 weeks intervals. Each vaccine contained 3-5 x 10(6) DCs. Five of the six patients received all five vaccines. The treatment was well tolerated in all patients without any observed vaccine-correlated adverse effects. Treatment with this DC-based cancer vaccine proved safe and non-toxic.

  19. The effect of subarachnoid erythrocyte lysate on brain injury: a preliminary study

    PubMed Central

    Zhang, Zi-Huan; Han, Yan-Ling; Wang, Chun-Xi; Zhou, Chen-Hui; Wu, Ling-Yun; Zhang, Hua-Sheng; Chen, Qiang; Fan, Jie-Mei; Zhou, Meng-Liang; Hang, Chun-Hua

    2016-01-01

    Abundant erythrocytes remain and lyse partially in the subarachnoid space after severe subarachnoid haemorrhage (SAH). But the effect of subarachnoid erythrocyte lysate on brain injury is still not completely clear. In this study, autologous erythrocytes (the non-lysate group) and their lysate (the lysate group) were injected separately into the cistern magna of rabbits to induce a model of experimental SAH, although the control group received isotonic sodium chloride solution instead of erythrocyte solution. Results showed that vasospasm of the basilar artery was observed at 72 h after experimental SAH, but there was no significant difference between the non-lysate group and the lysate group. Brain injury was more severe in the lysate group than in the non-lysate group. Meanwhile, the levels of peroxiredoxin 2 (Prx2), IL-6 and TNF-α in brain cortex and in CSF were significantly higher in the lysate group than those in the non-lysate group. These results demonstrated that brain injury was more likely to be caused by erythrocyte lysate than by intact erythrocytes in subarachnoid space, and inflammation response positively correlated with Prx2 expression might be involved in mechanism of brain injury after SAH. PMID:27279653

  20. Anaphylaxis following cystoscopy with equipment sterilized with Cidex OPA (ortho-phthalaldehyde): a review of two cases.

    PubMed

    Cooper, Daniel E; White, Andrew A; Werkema, Angelina N; Auge, Brain K

    2008-09-01

    Ortho-phthalaldehyde (Cidex OPA) is a commonly used solution for rapid sterilization of flexible endoscopic equipment. We report two cases of anaphylaxis following cystoscopy with endoscopes sterilized with this agent. Only a handful of such reactions have been reported in the published literature, the majority of which are in the bladder cancer population undergoing surveillance cystoscopy. We reviewed the clinical presentation of two cases of anaphylaxis following flexible cystoscopy with instruments sterilized with ortho-phthalaldehyde. We further describe their subsequent evaluation by an allergy and immunology specialist who performed skin testing to confirm a suspected ortho-phthalaldehyde allergy. Both patients were skin test positive to ortho-phthalaldehyde antigen. As a result, sterilization techniques for our flexible endoscopes has been altered. To date, no further anaphylactic reactions have occurred in our bladder cancer patients, including the two cases presented herein following subsequent cystoscopic evaluations. Ortho-phthalaldehyde-sterilized cystoscopes have been associated with anaphylactic reactions in a small number of patients who have undergone repeated cystoscopy. The manufacturer has already made recommendations to avoid this agent in bladder cancer patients. It may be prudent to extend this practice to other populations undergoing repeat cystoscopy.

  1. Carbon Nanotubes Activate Limulus Amebocyte Lysate Coagulation by Interface Adsorption.

    PubMed

    Yang, Man; Nie, Xin; Meng, Jie; Liu, Jian; Sun, Zhiwei; Xu, Haiyan

    2017-03-15

    Limulus amebocyte lysate (LAL) assay is worldwide requested in the assessment of endotoxin contamination for biomaterials. As carbon nanotubes are one major nanomaterial with multiple potentials in biomedical application, here we investigate whether oxidized multiwalled carbon nanotubes (O-MWCNT) interferes the assessment by LAL assays. We showed that the endotoxin free O-MWCNT dispersing in aqueous solutions could activate both the gel-clotting and the end-point chromogenic LAL assay by converting coagulogen into coagulin through interfacial interactions between O-MWCNT and enzymes in the assays. In conclusion, the O-MWCNT could induce false positive results by activating the enzyme cascade of LAL.

  2. Serospecific antigens of Legionella pneumophila.

    PubMed Central

    Otten, S; Iyer, S; Johnson, W; Montgomery, R

    1986-01-01

    Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria. Images PMID:3017918

  3. Optimization of Stability, Encapsulation, Release, and Cross-Priming of Tumor Antigen-Containing PLGA Nanoparticles

    PubMed Central

    Prasad, Shashi; Cody, Virginia; Saucier-Sawyer, Jennifer K.; Fadel, Tarek R.; Edelson, Richard L.; Birchall, Martin A.

    2014-01-01

    Purpose In order to investigate Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as potential vehicles for efficient tumor antigen (TA) delivery to dendritic cells (DC), this study aimed to optimize encapsulation/release kinetics before determining immunogenicity of antigen-containing NP. Methods Various techniques were used to liberate TA from cell lines. Single (gp100) and multiple (B16-tumor lysate containing gp100) antigens were encapsulated within differing molecular weight PLGA co-polymers. Differences in morphology, encapsulation/release and biologic potency were studied. Findings were adopted to encapsulate fresh tumor lysate from patients with advanced tumors and compare stimulation of tumor infiltrating lymphocytes (TIL) against that achieved by soluble lysate. Results Four cycles of freeze-thaw + 15 s sonication resulted in antigen-rich lysates without the need for toxic detergents or protease inhibitors. The 80KDa polymer resulted in maximal release of payload and favorable production of immunostimulatory IL-2 and IFN-γ. NP-mediated antigen delivery led to increased IFN-γ and decreased immunoinhibitory IL-10 synthesis when compared to soluble lysate. Conclusions Four cycles of freeze-thaw followed by 15 s sonication is the ideal technique to obtain complex TA for encapsulation. The 80KDa polymer has the most promising combination of release kinetics and biologic potency. Encapsulated antigens are immunogenic and evoke favorable TIL-mediated anti-tumor responses. PMID:22798259

  4. Two new ortho benzoquinones from Uncaria rhynchophylla.

    PubMed

    Zhang, Qian; Chen, Lei; Hu, Le-Jian; Liu, Wen-Yuan; Feng, Feng; Qu, Wei

    2016-03-01

    The present study was designed to determine the chemical constituents of the stems and hooks of Uncaria rhynchophylla. The chemical constituents were isolated and purified from CH2Cl2 fraction by chromatography. Their structures were elucidated by spectroscopic analyses. Their cytotoxicity was tested using MTT method. Two new ortho benzoquinones, 3-diethylamino-5-methoxy-1, 2-benzoquinone (1) and 3-ethylamino-5-methoxy-1, 2-benzoquinone (2), together with a known compound isorhynchophyllic acid (3) were isolated from U. rhynchophylla. These compounds were evaluated for their cytotoxicity against cancer cells A549, HepG2 and A2780. Compounds 1 and 2 were new ortho benzoquinones and showed weak antiproliferative activities on A549, HepG2 and A2780 cells. Compound 3 significantly inhibited the proliferation of A549, HepG2 and A2780 cells with IC50 values being 5.8, 12.8 and 11.8 µmol·L(-1), respectively.

  5. In vitro nonsense suppression in [psi+] and [psi-] cell-free lysates of Saccharomyces cerevisiae.

    PubMed Central

    Tuite, M F; Cox, B S; McLaughlin, C S

    1983-01-01

    An homologous in vitro assay for yeast nonsense suppressors was used to examine the effect of the cytoplasmically inherited genetic determinant [psi] on the efficiency of in vitro nonsense suppression. The efficiency of all three types of yeast tRNA-mediated nonsense suppressor (ochre, amber, and UGA) is much greater in cell-free lysates prepared from a sup+ [psi+] strain than in lysates prepared from an isogeneic sup+ [psi-] strain. Lysates prepared from a [psi-] strain, into which the [psi+] determinant was reintroduced by kar1-mediated cytoduction, support efficient suppression. Evidence is also presented that [psi-] lysates contain an inhibitor of in vitro nonsense suppression. Images PMID:6344070

  6. Pharmacokinetic overview of Ortho Evra/Evra.

    PubMed

    Abrams, Larry S; Skee, Donna; Natarajan, Jaya; Wong, Frankie A

    2002-02-01

    The pharmacokinetics of norelgestromin, the primary active metabolite of norgestimate, plus ethinyl estradiol (EE), delivered by the once-weekly contraceptive patch (Ortho Evra/Evra), have been studied in eight trials. This overview summarizes the relevant pharmacokinetic data for the contraceptive patch. Review article. The amount of norelgestromin and EE absorbed from the patch is proportional to patch size: the 20-cm(2) patch (Ortho Evra) delivers norelgestromin, 150 microg/d, and EE, 20 microg/d, to the systemic circulation. After single and multiple applications of the contraceptive patch, daily serum concentrations (area under the serum concentration-versus-time curve) of norelgestromin and EE were within the ranges generally seen with oral norgestimate, 250 microg/EE 35 microg (Ortho-Cyclen/Cilest), but without the peaks and troughs characteristic of oral dosing. Moreover, the contraceptive patch maintains serum concentrations of norelgestromin and EE within these ranges for up to 10 days, suggesting that clinical efficacy would be maintained even if a scheduled change is missed for as long as two full days. Regardless of the location of patch application (abdomen, buttock, upper outer arm, or torso [excluding breasts]) and even under conditions of heat, humidity, exercise, and cool-water immersion, efficacious concentrations of norelgestromin and EE are achieved. Coadministration of the patch with tetracycline did not affect the pharmacokinetics of norelgestromin and EE. The contraceptive patch exhibits an excellent pharmacokinetic profile, maintaining efficacious serum hormone concentrations under varying conditions.

  7. Native antigen fractionation protein microarrays for biomarker discovery.

    PubMed

    Caiazzo, Robert J; O'Rourke, Dennis J; Barder, Timothy J; Nelson, Bryce P; Liu, Brian C-S

    2011-01-01

    In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.

  8. Peroxide-Free Pd(II)-Catalyzed Ortho Aroylation and Ortho Halogenation of Directing Arenes.

    PubMed

    Santra, Sourav Kumar; Banerjee, Arghya; Mohanta, Prakash Ranjan; Patel, Bhisma K

    2016-07-15

    A Pd(II)-catalyzed peroxide-free ortho aroylation of directing arenes has been developed via cross dehydrogenative coupling (CDC) in the presence of the terminal oxidant Cu(OAc)2·H2O. Ortho aroylation of directing arenes proceeds via decarbonylation of the in situ generated phenyl glyoxal, which is obtained from 2-acetoxyacetophenone in the presence of the oxidant Cu(OAc)2·H2O. However, changing the oxidant to CuX2 (X = Cl, Br) provided exclusive di-ortho-halogenated 2-arylbenzothiazoles. During the halogenation, CuX2 served the dual role of a halogen source as well as a co-oxidant.

  9. New, sensitive rocket immunoelectrophoretic assay for measurement of the reaction between endotoxin and Limulus amoebocyte lysate.

    PubMed Central

    Baek, L

    1983-01-01

    To study the active proteins which participate in the reaction of Limulus amoebocyte lysate (LAL) with lipopolysaccharide, antibody was raised in rabbits against LAL. When LAL was run against rabbit antiserum in crossed immunoelectrophoresis, a complex precipitin pattern appeared. Profound changes took place after reaction of LAL with lipopolysaccharide. The most distinct change was the complete disappearance of the cathodic migrating protein coagulogen, because the antigenicity of coagulogen was lost. Based on this observation, a new rocket immunoelectrophoretic method was developed to detect the disappearance of coagulogen after reaction of LAL with lipopolysaccharide. This assay method was used on clinical specimens (cerebrospinal fluid, plasma, ascites, and urine). It was used as a qualitative test, when a single sample is tested, or as a quantitative assay, when a number of sample dilutions were tested. The new method showed a higher degree of accuracy and sensitivity in comparison with the tube test and it can be used for both research and diagnostic purposes. Images PMID:6348072

  10. Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates.

    PubMed

    Zolg, J W; Lanciotti, R S; Wendlinger, M; Meyer, W A

    1990-09-01

    Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.

  11. Membrane protein production in Escherichia coli cell-free lysates.

    PubMed

    Henrich, Erik; Hein, Christopher; Dötsch, Volker; Bernhard, Frank

    2015-07-08

    Cell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.

  12. Role of ortho-retronasal olfaction in mammalian cortical evolution.

    PubMed

    Rowe, Timothy B; Shepherd, Gordon M

    2016-02-15

    Fossils of mammals and their extinct relatives among cynodonts give evidence of correlated transformations affecting olfaction as well as mastication, head movement, and ventilation, and suggest evolutionary coupling of these seemingly separate anatomical regions into a larger integrated system of ortho-retronasal olfaction. Evidence from paleontology and physiology suggests that ortho-retronasal olfaction played a critical role at three stages of mammalian cortical evolution: early mammalian brain development was driven in part by ortho-retronasal olfaction; the bauplan for neocortex had higher-level association functions derived from olfactory cortex; and human cortical evolution was enhanced by ortho-retronasal smell. © 2015 Wiley Periodicals, Inc.

  13. Oral administration of Lactobacillus plantarum lysates attenuates the development of atopic dermatitis lesions in mouse models.

    PubMed

    Kim, Hangeun; Kim, Hye Rim; Kim, Na-Ra; Jeong, Bong Jun; Lee, Jong Suk; Jang, Soojin; Chung, Dae Kyun

    2015-01-01

    Lactobacillus plantarum is a well-documented probiotic that has been used in clinical trials for the regulation of the immune system and treatment of gastrointestinal diseases. In this study, we evaluated the effects of L. plantarum cell lysates on the immune regulation through the in vitro and in vivo studies. L. plantarum lysates were prepared by sonication method, and we observed that the repetition of disruption step increased indicator components within the bacterial lysates. Indicator components might affect TNF-α production. L. plantarum lysates did not induce TNF-α production, while LPS-induced TNF-α production was dramatically inhibited in a sonication-dependent manner in THP-1 cells. Oral administration of L. plantarum lysates effectively attenuated the horny layer formation and decreased epidermal thickening in NC/Nga mice skin. The damage to barrier function after the 8 weeks oral administration was reduced by L. plantarum lysates as compared to that in the atopic dermatitis (AD) mice. Further study revealed that L. plantarum lysates polarized Th1 response via induction of IL-12 and IFN-γ production and inhibition of IL-4 and IgE production in NC/Nga mice. Together, our results suggest that L. plantarum lysates are remarkable material for host homeostasis and it could be used for the treatment of inflammatory diseases.

  14. ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR.

    PubMed

    Alissandratos, Apostolos; Caron, Karine; Loan, Thomas D; Hennessy, James E; Easton, Christopher J

    2016-12-16

    E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.

  15. The E. coli S30 lysate proteome: A prototype for cell-free protein production.

    PubMed

    Foshag, Daniel; Henrich, Erik; Hiller, Ekkehard; Schäfer, Miriam; Kerger, Christian; Burger-Kentischer, Anke; Diaz-Moreno, Irene; García-Mauriño, Sofía M; Dötsch, Volker; Rupp, Steffen; Bernhard, Frank

    2017-09-21

    Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a "black box". Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. ORTHO- ELIMINATION OF TRACKING SYSTEM CLOCK ERRORS

    NASA Technical Reports Server (NTRS)

    Wu, J. T.

    1994-01-01

    ORTHO is part of the Global Positioning System (GPS) being developed by the U.S. Air Force, a navigational system that will use 18 NAVSTAR satellites to broadcast navigation messages and achieve worldwide coverage. The normal positioning technique uses one receiver which receives signals from at least four GPS satellites. For higher accuracy work it is often necessary to use a differential technique in which more than one receiver is used. The geodetic measurement has all receivers on the ground and allows the determination of the relative locations of the ground sites. The main application of the ORTHO program is in the elimination of clock errors in a GPS based tracking system. The measured distance (pseudo-range) from a GPS receiver contains errors due to differences in the receiver and satellite clocks. The conventional way of eliminating clock errors is to difference pseudo-ranges between different GPS satellites and receivers. The Householder transformation used in this program performs a function similar to the conventional single differencing or double differencing. This method avoids the problem of redundancy and correlation encountered in a differencing scheme. It is able to keep all information contained in the measurements within the scope of a least square estimation. For multiple transmitter and receiver GPS tracking network, this method is in general more accurate than the differencing technique. This program assumes that the non-clock measurement partial derivatives for the particular application are computed earlier by another program. With the partial derivatives and information to identify the transmitters and receivers as the input, the program performs the Householder transformation on the partial derivatives. The transformed partials are output by the program and may be used as an input to the filter program in the subsequent estimation process. Clock partial derivatives are generated internally and are not part of the input to the program

  17. ORTHO- ELIMINATION OF TRACKING SYSTEM CLOCK ERRORS

    NASA Technical Reports Server (NTRS)

    Wu, J. T.

    1994-01-01

    ORTHO is part of the Global Positioning System (GPS) being developed by the U.S. Air Force, a navigational system that will use 18 NAVSTAR satellites to broadcast navigation messages and achieve worldwide coverage. The normal positioning technique uses one receiver which receives signals from at least four GPS satellites. For higher accuracy work it is often necessary to use a differential technique in which more than one receiver is used. The geodetic measurement has all receivers on the ground and allows the determination of the relative locations of the ground sites. The main application of the ORTHO program is in the elimination of clock errors in a GPS based tracking system. The measured distance (pseudo-range) from a GPS receiver contains errors due to differences in the receiver and satellite clocks. The conventional way of eliminating clock errors is to difference pseudo-ranges between different GPS satellites and receivers. The Householder transformation used in this program performs a function similar to the conventional single differencing or double differencing. This method avoids the problem of redundancy and correlation encountered in a differencing scheme. It is able to keep all information contained in the measurements within the scope of a least square estimation. For multiple transmitter and receiver GPS tracking network, this method is in general more accurate than the differencing technique. This program assumes that the non-clock measurement partial derivatives for the particular application are computed earlier by another program. With the partial derivatives and information to identify the transmitters and receivers as the input, the program performs the Householder transformation on the partial derivatives. The transformed partials are output by the program and may be used as an input to the filter program in the subsequent estimation process. Clock partial derivatives are generated internally and are not part of the input to the program

  18. Exosomes: novel effectors of human platelet lysate activity.

    PubMed

    Torreggiani, E; Perut, F; Roncuzzi, L; Zini, N; Baglìo, S R; Baldini, N

    2014-09-22

    Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

  19. Protein microarrays using liquid phase fractionation of cell lysates.

    PubMed

    Yan, Fang; Sreekumar, Arun; Laxman, Bharathi; Chinnaiyan, Arul M; Lubman, David M; Barder, Timothy J

    2003-07-01

    We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves a pI-based fractionation using chromatofocusing in the first dimension followed by nonporous reversed-phase high-performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero-diagnostic markers for prostate cancer. Importantly, this method allows for use of post-translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.

  20. Effect of platelet lysate on human cells involved in different phases of wound healing.

    PubMed

    Barsotti, Maria Chiara; Chiara Barsotti, Maria; Losi, Paola; Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

    2013-01-01

    Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

  1. Effects of oral intake of kimchi-derived Lactobacillus plantarum K8 lysates on skin moisturizing.

    PubMed

    Kim, Hangeun; Kim, Hye Rim; Jeong, Bong Jun; Lee, Seung Su; Kim, Tae-Rahk; Jeong, Ji Hye; Lee, Miyeong; Lee, Sinai; Lee, Jong Suk; Chung, Dae Kyun

    2015-01-01

    Skin is the soft outer covering of vertebrates that provides protection from pathogenic infection, physical damage, or UV irradiation, and controls body temperature and water content. In this study, we examined the effects of oral intake of kimchi-derived Lactobacillus plantarum K8 lysates on skin moisturizing. In an in vitro study, we observed that the hyaluronic acid content increased in HaCaT cells treated with L. plantarum K8 lysates. Oral administration of L. plantarum K8 lysates effectively attenuated the horny layer formation and decreased epidermal thickening in DNCB-treated SKH-1 hairless mice skin. The damage to barrier function was reduced after 8 weeks of oral administration of L. plantarum K8 lysates as compared with that in the atopic dermatitis mice. For the test with volunteers, we manufactured experimental candy containing 2.1% L. plantarum K8 lysates, while control candy did not contain bacterial lysate. A significant increase in hydration in the experimental candy-administered group as compared with the control candy-administered group was observed on the face after 4 and 8 weeks, and on the forearm after 4 weeks. Decreases in horny layer thickness and TEWL value were observed on the face and forearm of the experimental group. Together, the in vitro cell line and in vivo mouse studies revealed that L. plantarum K8 lysates have a moisturizing effect. A clinical research study with healthy volunteers also showed an improvement in barrier repair and function when volunteers took L. plantarum K8 lysates-containing candy. Thus, our results suggest that L. plantarum K8 lysates may help to improve skin barrier function.

  2. A mixture of bacterial mechanical lysates is more efficient than single strain lysate and of bacterial-derived soluble products for the induction of an activating phenotype in human dendritic cells.

    PubMed

    Morandi, Barbara; Agazzi, Alessia; D'Agostino, Antonella; Antonini, Francesca; Costa, Gregorio; Sabatini, Federica; Ferlazzo, Guido; Melioli, Giovanni

    2011-07-01

    Dendritic cells (DCs), following an optimal maturation, are able to drive an efficient immune-response. For this, both co-stimulatory molecules (CD80 and CD86), activation molecules (CD83) and peptide presenting molecules (HLA) are over-expressed. The in vitro treatment of immature DC with fragments of bacterial strains, obtained by using a mechanical lysis as well as with bacterial-derived molecules (such as lipopolysaccharide and protido-glycan), induced the maturation of DCs and the secretion of a panel of cytokines and chemokines. Of note, ex vivo treated circulating DCs and plasmacytoid DCs were also activated by these bacterial bodies. However, while the particulate fraction of single bacterial strains or soluble bacterial-derived molecules induced a sub-optimal maturation (as evaluated by the expression of an activating phenotype on DCs and the amount of cytokine secretion), the addition of the mixture of the particulate fractions of the different bacterial strains was able to mediate an optimal maturation. These results were also confirmed by using the secretion of both cytokines and chemokines as markers of DC activation. All these findings suggest that the particulate fraction of bacterial lysate mixtures, because of their ability to interact with different surface structures, might be exploited not only as an immunogen, but also as an adjuvant treatment to boost an immune-response to poorly "antigenic" proteins, such as cancer antigens or allergens.

  3. Characterization of Limulus amoebocyte lysate-reactive material from hollow-fiber dialyzers.

    PubMed Central

    Pearson, F C; Bohon, J; Lee, W; Bruszer, G; Sagona, M; Jakubowski, G; Dawe, R; Morrison, D; Dinarello, C

    1984-01-01

    Hollow-fiber hemodialyzers containing cellulose-based membranes have been shown to produce positive results with the Limulus amoebocyte lysate test. This study was undertaken to determine whether endotoxin was causing the reaction. Rinses from 45 parallel-plate and hollow-fiber dialyzers from eight different manufacturers were tested before and after treatment with cellulase, using three lysates and four Limulus amoebocyte lysate methods. In addition, four in vitro cellular methods--human leukocytic pyrogen, lymphocytic activating factor, peritoneal macrophage, and arginase release--were used to evaluate endotoxin activity. The substance causing the reaction was identified by chromatographic methods. Results indicate that the Limulus amoebocyte lysate reactive material is cellulose derived and not pyrogenic. PMID:6517586

  4. Multiplexed specific label-free detection of NCI-H358 lung cancer cell line lysates with silicon based photonic crystal microcavity biosensors.

    PubMed

    Chakravarty, Swapnajit; Lai, Wei-Cheng; Zou, Yi; Drabkin, Harry A; Gemmill, Robert M; Simon, George R; Chin, Steve H; Chen, Ray T

    2013-05-15

    We experimentally demonstrate label-free photonic crystal (PC) microcavity biosensors in silicon-on-insulator (SOI) to detect the epithelial-mesenchymal transition (EMT) transcription factor, ZEB1, in minute volumes of sample. Multiplexed specific detection of ZEB1 in lysates from NCI-H358 lung cancer cells down to an estimated concentration of 2 cells per micro-liter is demonstrated. L13 photonic crystal microcavities, coupled to W1 photonic crystal waveguides, are employed in which resonances show high Q in the bio-ambient phosphate buffered saline (PBS). When the sensor surface is derivatized with a specific antibody, the binding of the corresponding antigen from a complex whole-cell lysate generates a change in refractive index in the vicinity of the photonic crystal microcavity, leading to a change in the resonance wavelength of the resonance modes of the photonic crystal microcavity. The shift in the resonance wavelength reveals the presence of the antigen. The sensor cavity has a surface area of ∼11μm(2). Multiplexed sensors permit simultaneous detection of many binding interactions with specific immobilized antibodies from the same bio-sample at the same instant of time. Specificity was demonstrated using a sandwich assay which further amplifies the detection sensitivity at low concentrations. The device represents a proof-of-concept demonstration of label-free, high throughput, multiplexed detection of cancer cells with specificity and sensitivity on a silicon chip platform.

  5. Vaccine efficacy of a cell lysate with recombinant baculovirus-expressed feline infectious peritonitis (FIP) virus nucleocapsid protein against progression of FIP.

    PubMed

    Hohdatsu, Tsutomu; Yamato, Hiroshi; Ohkawa, Tasuku; Kaneko, Miyuki; Motokawa, Kenji; Kusuhara, Hajime; Kaneshima, Takashi; Arai, Setsuo; Koyama, Hiroyuki

    2003-12-02

    The Type II feline infectious peritonitis virus (FIPV) infection of feline macrophages is enhanced by a monoclonal antibody (MAb) to the S protein of FIPV. This antibody-dependent enhancement (ADE) activity increased with the MAb that showed a neutralizing activity with feline kidney cells, suggesting that there was a distinct correlation between ADE activity and the neutralizing activity. The close association between enhancing and neutralizing epitopes is an obstacle to developing a vaccine containing only neutralizing epitopes without enhancing epitopes. In this study, we immunized cats with cell lysate with recombinant baculovirus-expressed N protein of the Type I FIPV strain KU-2 with an adjuvant and investigated its preventive effect on the progression of FIP. Cats immunized with this vaccine produced antibodies against FIPV virion-derived N protein but did not produce virus-neutralizing antibodies. A delayed type hypersensitivity skin response to N protein was observed in these vaccinated cats, showing that cell mediated immunity against the FIPV antigen was induced. When these vaccinated cats were challenged with a high dose of heterologous FIPV, the survival rate was 75% (6/8), while the survival rate in the control group immunized with SF-9 cell-derived antigen was 12.5% (1/8). This study showed that immunization with the cell lysate with baculovirus-expressed N protein was effective in preventing the progression of FIP without inducing ADE of FIPV infection in cats.

  6. Multiplexed Specific Label-Free Detection of NCI-H358 Lung Cancer Cell Line Lysates with Silicon Based Photonic Crystal Microcavity Biosensors

    PubMed Central

    Chakravarty, Swapnajit; Lai, Wei-Cheng; Zou, Yi; Drabkin, Harry A.; Gemmill, Robert M.; Simon, George R.; Chin, Steve H.; Chen, Ray T.

    2012-01-01

    We experimentally demonstrate label-free photonic crystal (PC) microcavity biosensors in silicon-on-insulator (SOI) to detect the epithelial-mesenchymal transition (EMT) transcription factor, ZEB1, in minute volumes of sample. Multiplexed specific detection of ZEB1 in lysates from NCI-H358 lung cancer cells down to an estimated concentration of 2 cells per micro-liter is demonstrated. L13 photonic crystal microcavities, coupled to W1 photonic crystal waveguides, are employed in which resonances show high Q in the bio-ambient phosphate buffered saline (PBS). When the sensor surface is derivatized with a specific antibody, the binding of the corresponding antigen from a complex whole-cell lysate generates a change in refractive index in the vicinity of the photonic crystal microcavity, leading to a change in the resonance wavelength of the resonance modes of the photonic crystal microcavity. The shift in the resonance wavelength reveals the presence of the antigen. The sensor cavity has a surface area of ~11 μm2. Multiplexed sensors permit simultaneous detection of many binding interactions with specific immobilized antibodies from the same bio-sample at the same instant of time. Specificity was demonstrated using a sandwich assay which further amplifies the detection sensitivity at low concentrations. The device represents a proof-of-concept demonstration of label-free, high throughput, multiplexed detection of cancer cells with specificity and sensitivity on a silicon chip platform. PMID:23274197

  7. Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Jonsdottir-Buch, Sandra Mjoll; Lieder, Ramona; Sigurjonsson, Olafur Eysteinn

    2013-01-01

    Background Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells. Methodology/Principal Findings In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed. Conclusion/Significance Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets. PMID:23874839

  8. Dendritic cell immunotherapy for urological cancers using cryopreserved allogeneic tumour lysate-pulsed cells: a phase I/II study.

    PubMed

    Pandha, Hardev S; John, Robert J; Hutchinson, James; James, Nick; Whelan, Mike; Corbishley, Catherine; Dalgleish, Angus G

    2004-08-01

    To assess the feasibility, toxicity and immunogenicity of dendritic cell (DC)-based immunotherapy in patients with advanced urological cancers. Patients with hormone-refractory prostate cancer (11) and metastatic renal cell carcinoma (five) received 1-3 x 10(6) intradermal allogeneic tumour lystate-pulsed DCs fortnightly for six vaccinations then monthly until disease progression. Intradermal keyhole limpet haemocyanin was injected near the DCs as the adjuvant. DC vaccine was prepared from buffy coats, then lysate-pulsed, cryopreserved in aliquots, and tested for phenotypic expression and activity in an allogeneic mixed lymphocyte reaction before clinical use. There was no evidence of significant toxicity from vaccine or adjuvant. Delayed-type hypersensitivity skin testing and biopsy revealed a cellular infiltrate to intradermal re-challenge to tumour lysate and adjuvant in almost all patients. In addition, there was increased expression of T helper type 1 cytokines, interferon-gamma-expressing T cell by ELISPOT analysis, but also interleukin-10 in a few patients. Vaccination resulted in a reduction in the level of prostate-specific antigen (PSA) in one patient, a reduction in PSA velocity in a further man and an increased PSA doubling time in six. Two of five patients with renal cell carcinoma had stabilization of disease. The cryopreservation and repeated administration of DC vaccine was feasible and not toxic. There was evidence of induction of both humoral and cellular immunity to vaccine and adjuvant in most patients. The use of sequential aliquots of identical cryopreserved vaccine will ensure quality control and greatly facilitate future clinical studies in terms of consistency of vaccine administered and the provision of primed DCs for in vitro assessment of response.

  9. The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation.

    PubMed

    Moreira Teixeira, Liliana S; Leijten, Jeroen C H; Wennink, Jos W H; Chatterjea, Anindita G; Feijen, Jan; van Blitterswijk, Clemens A; Dijkstra, Pieter J; Karperien, Marcel

    2012-05-01

    In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells.

  10. Organic extraction of bone lysates improves DNA purification with silica beads.

    PubMed

    Desmyter, Stijn; De Cock, Greet; Moulin, Sabine; Noël, Fabrice

    2017-04-01

    In our standard protocol for DNA extraction from skeletal remains of unidentified bodies, bone lysates resulting from decalcification and Proteinase K treatment were purified with the DNA IQ™ Casework Pro Kit for Maxwell(®)16 automate (Promega, WI). Despite its success in the majority of cases, the DNA purification with paramagnetic silica beads failed in some challenging samples. This failure in DNA recovery was often associated with filter clogging during the required volume reduction of the lysate to enable loading on the automate. Two modifications to the standard method were tested for a more efficient filtering and purification. Adding collagenase to the lysate reduced the filter lead time but did not enhance DNA yield, while organic extraction of the crude lysate solved the filter clogging and resulted in successful DNA purification. The modified method in which a phenol treated lysate was loaded on the automate resulted in successful STR-profiling of the skeletal remains of all 13 unidentified bodies tested, which showed a wide variety in post mortem interval and preservation conditions. The variation in DNA yield between the 28 samples tested showed the importance of bone type selection and multiple sampling in successful STR-profiling of skeletal remains. Despite the disadvantages inherent to phenol, the organic extraction of crude bone lysates enhanced the efficiency of DNA purification with paramagnetic silica beads. The combined method of organic extraction and purification with silica beads resulted in STR-profiling of challenging bone samples.

  11. JL1, a novel differentiation antigen of human cortical thymocyte

    PubMed Central

    1993-01-01

    Expression of a novel thymocyte differentiation antigen, JL1, defined by a monoclonal antibody (mAb) developed against human thymocytes showed a specificity for stage II double positive (CD4+CD8+) human cortical thymocytes. This antigen was not expressed at detectable levels on medullary thymocytes, mature peripheral leukocytes, bone marrow cells or on other types of tissues elsewhere in the human body. Immunohistologic analysis revealed that JL1 had a clear pattern of distribution on cortical thymocytes. Immunoprecipitation of 125I- labeled cell lysates from human thymocytes and Molt-4 leukemic cell line with anti-JL1 mAb yielded a 120-130-kD single chain glycoprotein. When immunoprecipitation of cell lysate was done after endoglycosidase F treatment, JL1 antigen was still detected by antibody but the band showed a reduction in apparent molecular mass of approximately 5 kD. This suggests that, although JL1 molecule contains carbohydrate group, this does not form a critical part of the antigenic determinant for anti-JL1 antibody. JL1 antigen appears to be the first double positive, stage-specific differentiation antigen of human thymocyte reported so far. This antigen would be a useful marker for lymphoblastic malignancy of stage II thymocyte origin and it may be involved in the thymocyte education process. PMID:8376947

  12. Ortho-mode Vivaldi antennas for agile polarization systems

    NASA Astrophysics Data System (ADS)

    Macedo, A. D.; Mosso, M. M.

    The design criteria and assembly techniques for the ortho-mode Vivaldi (OMV) antenna system are described. The system is composed of a set of two orthogonal linearly polarized elements that are fed by an ortho-mode tee. The performances of two prototype OMV antennas system are examined and the applicability OMV is noted. Diagrams of the two prototypes and graphs of their performance are provided.

  13. Cooling by conversion of para to ortho-hydrogen

    NASA Technical Reports Server (NTRS)

    Sherman, A. (Inventor)

    1983-01-01

    The cooling capacity of a solid hydrogen cooling system is significantly increased by exposing vapor created during evaporation of a solid hydrogen mass to a catalyst and thereby accelerating the endothermic para-to-ortho transition of the vapor to equilibrium hydrogen. Catalyst such as nickel, copper, iron or metal hydride gels of films in a low pressure drop catalytic reactor are suitable for accelerating the endothermic para-to-ortho conversion.

  14. Synergistic combination of murine bone marrow-derived dendritic cells loaded ex vivo with whole tumor lysate and systemic chemotherapy mediates antitumor immune responses in vivo.

    PubMed

    Salem, Mohamed L; Nassef, Mohamed; Gomaa, Soha; Essa, Ibrahim

    2017-09-01

    In order to get mature dendritic cells (DC) that is a crucial prerequisite for success in tumor immunotherapy protocols. Herein, we assumed that administration of murine bone marrow (BM)-derived DC (BM-DC), loaded ex vivo with whole Ehrlich ascites carcinoma (EAC) lysate, in the context of systemic chemotherapy cyclophosphamide (CTX) to induce antitumor immune responses, may be a good strategy to improve the presentation of tumor-specific antigens to the immune system. In the first series of experiments, BM cells generated either from BM of naïve mice or from BM of EAC-bearing mice were cultured in the presence of GM-CSF and IL-4 for 6days. At day 7, cells were loaded for 48h with one of the following maturation agents: EAC lysate (1mg/ml), poly-inosinic: polycytidylic acid [poly(I:C)] (25μg/ml) or mRNA encoding human telomerase reverse transcriptase (hTERT-mRNA) (2μg/ml). In the second series of experiments, EAC-bearing mice were intraperitoneally (i.p.) injected with CTX followed by i.p. vaccination with DC, loaded ex vivo with EAC lysate. DC yield and the phenotypic expression of maturity-related surface markers of DC (i.e. CD11b and CD11c) in both series of experiments were investigated. As a result, a significant decrease in the number of DC generated from poly(I:C)-supplemented BM culture from EAC-bearing mice has been detected. Loading of BM cells with poly(I:C), EAC lysate or hTERT-mRNA could induce the expression of CD11b and CD11c. Additionally, vaccination of EAC-bearing mice with DC loaded ex vivo with EAC lysate following CTX treatment, resulted in increases in the percentage of multiple populations of CD11b(+)CD11c(+) in BM, spleen and peripheral blood (PB). To conclude, further researches to clarify the mechanism involved in DC maturation are crucial not only to comprehend DC biology but also to optimize DC immunotherapy protocols. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  15. Ortho- and para-hydrogen in neutron thermalization

    SciTech Connect

    Daemen, L. L.; Brun, T. O.

    1998-01-01

    The large difference in neutron scattering cross-section at low neutron energies between ortho- and para-hydrogen was recognized early on. In view of this difference (more than an order of magnitude), one might legitimately ask whether the ortho/para ratio has a significant effect on the neutron thermalization properties of a cold hydrogen moderator. Several experiments performed in the 60`s and early 70`s with a variety of source and (liquid hydrogen) moderator configurations attempted to investigate this. The results tend to show that the ortho/para ratio does indeed have an effect on the energy spectrum of the neutron beam produced. Unfortunately, the results are not always consistent with each other and much unknown territory remains to be explored. The problem has been approached from a computational standpoint, but these isolated efforts are far from having examined the ortho/para-hydrogen problem in neutron moderation in all its complexity. Because of space limitations, the authors cannot cover, even briefly, all the aspects of the ortho/para question here. This paper will summarize experiments meant to investigate the effect of the ortho/para ratio on the neutron energy spectrum produced by liquid hydrogen moderators.

  16. Investigating Birth Control: Comparing Oestrogen Levels in Patients Using the Ortho Evra[R] Patch versus the Ortho-Cyclen[R] Pill

    ERIC Educational Resources Information Center

    Laurent, Theresa A.

    2008-01-01

    Recent drug studies have investigated the incidence of blood clots among patients using the Ortho Evra[R] birth control patch. In this article, the author describes an investigation of oestrogen levels in the body resulting from the application of the Ortho Evra[R] birth control patch versus daily use of Ortho-Cyclen[R] birth control pills.…

  17. Investigating Birth Control: Comparing Oestrogen Levels in Patients Using the Ortho Evra[R] Patch versus the Ortho-Cyclen[R] Pill

    ERIC Educational Resources Information Center

    Laurent, Theresa A.

    2008-01-01

    Recent drug studies have investigated the incidence of blood clots among patients using the Ortho Evra[R] birth control patch. In this article, the author describes an investigation of oestrogen levels in the body resulting from the application of the Ortho Evra[R] birth control patch versus daily use of Ortho-Cyclen[R] birth control pills.…

  18. Palladium-catalyzed/norbornene-mediated ortho-amination/N-tosylhydrazone insertion reaction: an approach to the synthesis of ortho-aminated vinylarenes.

    PubMed

    Zhou, Ping-Xin; Ye, Yu-Ying; Ma, Jun-Wei; Zheng, Lan; Tang, Qian; Qiu, Yi-Feng; Song, Bo; Qiu, Zi-Hang; Xu, Peng-Fei; Liang, Yong-Min

    2014-07-18

    ortho-Aminated vinylarene derivatives were obtained via a reaction of aryl iodides, N-benzoyloxyamines, and N-tosylhydrazones. This approach involves a palladium-catalyzed, norbornene-mediated ortho-amination/N-tosylhydrazone insertion reaction. In this transformation, one C-N bond and one C-C bond are formed and an amine group is introduced at the ortho position successfully.

  19. The Domestication of ortho-Quinone Methides

    PubMed Central

    2015-01-01

    Conspectus An ortho-quinone methide (o-QM) is a highly reactive chemical motif harnessed by nature for a variety of purposes. Given its extraordinary reactivity and biological importance, it is surprising how few applications within organic synthesis exist. We speculate that their widespread use has been slowed by the complications that surround the preparation of their precursors, the harsh generation methods, and the omission of this stratagem from computer databases due to its ephemeral nature. About a decade ago, we discovered a mild anionic triggering procedure to generate transitory o-QMs at low temperature from readily available salicylaldehydes, particularly OBoc derivatives. This novel reaction cascade included both the o-QM formation and the subsequent consumption reaction. The overall transformation was initiated by the addition of the organometallic reagent, usually a Grignard reagent, which resulted in the formation of a benzyloxy alkoxide. Boc migration from the neighboring phenol produced a magnesium phenoxide that we supposed underwent β-elimination of the transferred Boc residue to form an o-QM for immediate further reactions. Moreover, the cascade proved controllable through careful manipulation of metallic and temperature levers so that it could be paused, stopped, or restarted at various intermediates and stages. This new level of domestication enabled us to deploy o-QMs for the first time in a range of applications including diastereocontrolled reactions. This sequence ultimately could be performed in either multipot or single pot processes. The subsequent reaction of the fleeting o-QM intermediates included the 1,4-conjugate additions that led to unbranched or branched ortho-alkyl substituted phenols and Diels–Alder reactions that provided 4-unsubstituted or 4-substituted benzopyrans and chroman ketals. The latter cycloadducts were obtained for the first time with outstanding diastereocontrol. In addition, the steric effects of the newly

  20. In Vitro Amplification of Misfolded Prion Protein Using Lysate of Cultured Cells

    PubMed Central

    Mays, Charles E.; Yeom, Jihyun; Kang, Hae-Eun; Bian, Jifeng; Khaychuk, Vadim; Kim, Younghwan; Bartz, Jason C.; Telling, Glenn C.; Ryou, Chongsuk

    2011-01-01

    Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrPC). PrPSc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA. PMID:21464935

  1. Isolation, Bioactivity, and Production of ortho-Hydroxydaidzein and ortho-Hydroxygenistein

    PubMed Central

    Chang, Te-Sheng

    2014-01-01

    Daidzein and genistein are two major components of soy isoflavones. They exist abundantly in plants and possess multiple bioactivities. In contrast, ortho-hydroxydaidzein (OHD) and ortho-hydroxygenistein (OHG), including 6-hydroxydaidzein (6-OHD), 8-hydroxydaidzein (8-OHD), 3′-hydroxydaidzein (3′-OHD), 6-hydroxygenistein (6-OHG), 8-hydroxygenistein (8-OHG), and 3′-hydroxygenistein (3′-OHG), are rarely found in plants. Instead, they are usually isolated from fermented soybean foods or microbial fermentation broth feeding with soybean meal. Accordingly, the bioactivity of OHD and OHG has been investigated less compared to that of soy isoflavones. Recently, OHD and OHG were produced by genetically engineering microorganisms through gene cloning of cytochrome P450 (CYP) enzyme systems. This success opens up bioactivity investigation and industrial applications of OHD and OHG in the future. This article reviews isolation of OHD and OHG from non-synthetic sources and production of the compounds by genetically modified microorganisms. Several bioactivities, such as anticancer and antimelanogenesis-related activities, of OHD and OHG, are also discussed. PMID:24705463

  2. Supramolecular layers and versatile packing modes: The solid state behavior of ortho, ortho-linked bisphenols

    NASA Astrophysics Data System (ADS)

    Augustin, André U.; Katzsch, Felix; Prior, Stephen H.; Gruber, Tobias

    2017-05-01

    A series of ortho-ortho-linked bisphenols featuring electron-withdrawing groups (EWGs) attached to the phenolic rings is reported. Their respective molecular structures and packing behaviors have been studied by X-ray diffraction, comparatively discussed and put into relation with the unsubstituted mother compound. Except for the mother compound, the molecular structures of all bisphenols studied here exhibit distorted aromatic moieties. Hence, the substituents studied here prevent proximal positioning of phenolic units and the formation of strong Osbnd H⋯O hydrogen bonds. In the packing of the underivatized bisphenol we found a strand-like molecular arrangement featuring strong Osbnd H⋯O hydrogen bonds and extensive edge-to-face contacts (Csbnd H⋯π) between the bisphenol molecules. The introduction of EWGs to the aromatic moieties changes these intermolecular interactions into face-to-face contacts resulting either in bisphenol stacks or handshake-like motifs between two bisphenol molecules. In both cases, the Csbnd H⋯π interactions are more or less replaced by Csbnd H⋯O contacts as the prevalent non-covalent interaction. In the packing of two nitro bisphenols in their DMSO inclusion compounds an exciting layered arrangement is observed, which also matches with the pronounced foliated habitus of their crystals. Additionally, proton NMR was used to establish the binding coefficients between the respective bisphenols and DMSO in solution.

  3. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens

    SciTech Connect

    Tomavo, S.; Schwarz, R.T.; Dubremetz, J.F. )

    1989-10-01

    The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with {sup 3}H-fatty acids, ({sup 3}H)ethanolamine, and ({sup 3}H)carbohydrates. Treatment of {sup 3}H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment.

  4. Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

    PubMed Central

    Stahl, S; MacKay, P; Magazin, M; Bruce, S A; Murray, K

    1982-01-01

    Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples. Images PMID:7041126

  5. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens.

    PubMed Central

    Tomavo, S; Schwarz, R T; Dubremetz, J F

    1989-01-01

    The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with 3H-fatty acids, [3H]ethanolamine, and [3H]carbohydrates. Treatment of 3H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment. Images PMID:2531282

  6. Detection of the MW Transition Between Ortho and Para States

    NASA Astrophysics Data System (ADS)

    Kanamori, Hideto; Dehghani, Zeinab Tafti; Mizoguchi, Asao; Endo, Yasuki

    2017-06-01

    Thorough the detailed analysis of the hyperfine resolved rotational transitions, we have been pointed out that there exists not a little interaction between ortho and para states in the molecular Hamiltonian of S_2Cl_2. Using the ortho-para mixed molecular wavefunctions derived from the Hamiltonian, we calculated the transition moment and frequency of the ortho-para forbidden transitions in the cm- and mm-wave region, and picked up some promising candidate transitions for the spectroscopic detection. In the experiment, the S_2Cl_2 vapor with Ar buffer gas in a supersonic jet condition was used with FTMW spectrometer at National Chiao Tung University. As a result, seven hyperfine resolved rotational transitions in the cm-wave region were detected as the ortho-para transition at the predicted frequency within the experimental error range. The observed intensity was 10^{-3} smaller than that of an allowed transition, which is also consistent with the prediction. This is the first time the electric dipole transition between ortho and para states has been detected in a free isolated molecule. A. Mizoguchi, S. Ota, H. Kanamori, Y. Sumiyoshi, and Y. Endo, J. Mol. Spectrosc, 250, 86 (2008) Z. T. Dehghani, S. Ota, A. Mizoguchi and H. Kanamori, J. Phys. Chem. A, 117(39), 10041, (2013)

  7. Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

    PubMed

    Canonico, B; Campana, R; Luchetti, F; Arcangeletti, M; Betti, M; Cesarini, E; Ciacci, C; Vittoria, E; Galli, L; Papa, S; Baffone, W

    2014-08-01

    Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

  8. Dendritic cells pulsed with glioma lysates induce immunity against syngeneic intracranial gliomas and increase survival of tumor-bearing mice.

    PubMed

    Pellegatta, S; Poliani, P L; Corno, D; Grisoli, M; Cusimano, M; Ubiali, F; Baggi, F; Bruzzone, M G; Finocchiaro, G

    2006-07-01

    In recent years, the use of dendritic cells (DC), the most powerful antigen presenting cells, has been proposed for the creation of vaccines against gliomas. This approach has been demonstrated to be safe and non-toxic in phase I or I-II trials (2, 3). Immunotherapy plays a central role in the search for new treatments for glioblastoma multiforme (GBM). In particular, several phase I studies have been performed using DC pulsed by GBM proteins as a vaccine for patients with relapsing GBM. The studies demonstrated that DC vaccination is safe and may produce a significant increase in overall survival. As the first step in the preparation of appropriate conditions for a clinical evaluation in Italy, we have performed pre-clinical experiments on immune-competent mice injected intra-cerebrally with syngeneic GL261GBM cells and treated subcutaneously and intra-tumorally with DC loaded with a GL261 homogenate. These results show that vaccination with DC pulsed with a tumor lysate increases considerably survival in mice bearing intracranial glioblastomas and supports the development of DC-based clinical trials for patients with glioblastomas that do not respond to standard therapies.

  9. Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells.

    PubMed

    González, Fermín E; Ortiz, Carolina; Reyes, Montserrat; Dutzan, Nicolás; Patel, Vyomesh; Pereda, Cristián; Gleisner, Maria A; López, Mercedes N; Gutkind, J Silvio; Salazar-Onfray, Flavio

    2014-07-01

    We have previously reported a novel method for the production of tumour-antigen-presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas.

  10. Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

    PubMed Central

    Even-Desrumeaux, Klervi; Baty, Daniel; Chames, Patrick

    2010-01-01

    Antibodies microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnosis and proteome analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecule for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs produced in crude bacterial lysates to generate proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers. PMID:20859568

  11. Multicomponent Condensation Reactions via ortho-Quinone Methides.

    PubMed

    Allen, Emily E; Zhu, Calvin; Panek, James S; Schaus, Scott E

    2017-03-30

    Iron(III) salts promote the condensation of aldehydes or acetals with electron-rich phenols to generate ortho-quinone methides that undergo Diels-Alder condensations with alkenes. The reaction sequence occurs in a single vessel to afford benzopyrans in up to 95% yield. The reaction was discovered while investigating a two-component strategy using 2-(hydroxy(phenyl)methyl)phenols to access the desired ortho-quinone methide in a Diels-Alder condensation. The two-component condensation also afforded the corresponding benzopyran products in yields up to 97%. Taken together, the two- and three-component strategies using ortho-quinone methide intermediates provide efficient access to benzopyrans in good yields and selectivities.

  12. Tautomeric design of ortho-hydroxyheterocyclic Schiff bases

    NASA Astrophysics Data System (ADS)

    Kwocz, Agnieszka; Kochel, Andrzej; Chudoba, Dorota; Filarowski, Aleksander

    2015-01-01

    The article reports on the synthesis and crystallographic and theoretical studies on various heterocyclic derivatives of ortho-hydroxy Schiff bases. The prevailing of one of the two (enolimine - OH and ketoamine - NH) tautomeric forms has been stated in studied compounds depending on the substitutes in heterocyclic formation, the nitrogen and carbon atoms of the imine group. A specific situation has been shown in (1E)-1-(5-chloro-2-hydroxy-3-nitrophenyl)ethanone oxime compound. The potentials on the proton transfer of the hydrogen bridges in these compounds have been found out. Grounded on the obtained potential curves the influence of the protonation on nitrogen in the pyridoxal derivative of the studied ortho-hydroxy Schiff bases has been analysed. The most efficient method of the impact (by means of various substitutes) on the intramolecular tautomeric equilibrium in ortho-hydroxy Schiff bases has been presented.

  13. Th2 Allergic Immune Response to Inhaled Fungal Antigens is Modulated By TLR-4-Independent Bacterial Products

    PubMed Central

    Allard, Jenna B.; Rinaldi, Lisa; Wargo, Matt; Allen, Gilman; Akira, Shizuo; Uematsu, Satoshi; Poynter, Matthew E.; Hogan, Deborah A.; Rincon, Mercedes; Whittaker, Laurie A.

    2009-01-01

    SUMMARY Allergic airway disease is characterized by eosinophilic inflammation, mucus hypersecretion and increased airway resistance. Fungal antigens are ubiquitous within the environment and are well know triggers of allergic disease. Bacterial products are also frequently encountered within the environment and may alter the immune response to certain antigens. The consequence of simultaneous exposure to bacterial and fungal products on the lung adaptive immune response has not been explored. Here we show that oropharyngeal aspiration of fungal lysates (Candida albicans, Aspergillus fumigatus) promotes airway eosinophilia, secretion of Th2 cytokines and mucus cell metaplasia. In contrast, oropharyngeal exposure to bacterial lysates (Pseudomonas aeruginosa) promotes airway inflammation characterized by neutrophils, Th1 cytokine secretion and no mucus production. More importantly, administration of bacterial lysates together with fungal lysates deviates the adaptive immune response to a Th1 type associated with neutrophilia and diminished mucus production. The immunomodulatory effect that bacterial lysates have on the response to fungi is TLR4-independent but MyD88 dependent. Thus, different types of microbial products within the airway can alter the host's adaptive immune response, and potentially impact the development of allergic airway disease to environmental fungal antigens. PMID:19224641

  14. Ortho and para-armalcolite samples in Apollo 17.

    NASA Technical Reports Server (NTRS)

    Haggerty, S. E.

    1973-01-01

    Two paragenetically contrasting forms of armalcolite are present in basalts from the Apollo 17 Taurus-Littrow landing site. These armalcolites differ in optical properties, in crystal habit and in their distribution between coarse and fine grained rocks. It is proposed to call the two armalcolite forms ortho-armalcolite and para-armalcolite. Texural relationships and the evidence of experimental melting show that ortho-armalcolite is always the first crystalline phase to appear from unusually titanium rich magmas. The origin of para-armalcolite is not yet fully understood.

  15. Production Ratio for Para- and Ortho-Ps in Photodetachment of Ps^-

    NASA Astrophysics Data System (ADS)

    Igarashi, Akinori

    2017-01-01

    Para- and ortho-Ps atoms are formed in the photodetachment of positronium negative ion. Since the lifetime against the pair annihilation is much shorter for para-Ps( ns) than for ortho-Ps( ns), the production ratio of para- and ortho-Ps atoms is important for the photodetachment experiments. We have derived the ratio explicitly.

  16. Platelet lysate and chondroitin sulfate loaded contact lenses to heal corneal lesions.

    PubMed

    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Delfino, Alessio; Riva, Federica; Icaro Cornaglia, Antonia; Marrubini, Giorgio; Musitelli, Giorgio; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

    2016-07-25

    Hemoderivative tear substitutes contain various ephiteliotrophic factors, such as growth factors (GF), involved in ocular surface homeostasis without immunogenic properties. The aim of the present work was the loading of platelet lysate into contact lenses to improve the precorneal permanence of platelet lysate growth factors on the ocular surface to enhance the treatment of corneal lesions. To this purpose, chondroitin sulfate, a sulfated glycosaminoglycan, which is normally present in the extracellular matrix, was associated with platelet lysate. In fact, chondroitin sulfate is capable of electrostatic interaction with positively charged growth factors, in particular, with bFGF, IGF, VEGF, PDGF and TGF-β, resulting in their stabilization and reduced degradation in solution. In the present work, various types of commercially available contact lenses have been loaded with chondroitin sulfate or chondroitin sulfate in association with platelet lysate to achieve a release of growth factors directly onto the corneal surface lesions. One type of contact lenses (PureVision(®)) showed in vitro good proliferation properties towards corneal cells and were able to enhance cut closure in cornea constructs.

  17. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    PubMed

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-11-05

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

  18. Mitochondrial lysates induce inflammation and Alzheimer's disease-relevant changes in microglial and neuronal cells.

    PubMed

    Wilkins, Heather M; Carl, Steven M; Weber, Sam G; Ramanujan, Suruchi A; Festoff, Barry W; Linseman, Daniel A; Swerdlow, Russell H

    2015-01-01

    Neuroinflammation occurs in Alzheimer's disease (AD). While AD genetic studies implicate inflammation-relevant genes and fibrillar amyloid-β protein promotes inflammation, our understanding of AD neuroinflammation nevertheless remains incomplete. In this study we hypothesized damage-associated molecular pattern (DAMP) molecules arising from mitochondria, intracellular organelles that resemble bacteria, could contribute to AD neuroinflammation. To preliminarily test this possibility, we exposed neuronal and microglial cell lines to enriched mitochondrial lysates. BV2 microglial cells treated with mitochondrial lysates showed decreased TREM2 mRNA, increased TNFα mRNA, increased MMP-8 mRNA, increased IL-8 mRNA, redistribution of NFκB to the nucleus, and increased p38 MAPK phosphorylation. SH-SY5Y neuronal cells treated with mitochondrial lysates showed increased TNFα mRNA, increased NFκB protein, decreased IκBα protein, increased AβPP mRNA, and increased AβPP protein. Enriched mitochondrial lysates from SH-SY5Y cells lacking detectable mitochondrial DNA (ρ0 cells) failed to induce any of these changes, while mtDNA obtained directly from mitochondria (but not PCR-amplified mtDNA) increased BV2 cell TNFα mRNA. These results indicate at least one mitochondrial-derived DAMP molecule, mtDNA, can induce inflammatory changes in microglial and neuronal cell lines. Our data are consistent with the hypothesis that a mitochondrial-derived DAMP molecule or molecules could contribute to AD neuroinflammation.

  19. Hypusine formation in protein by a two-step process in cell lysates.

    PubMed

    Murphey, R J; Gerner, E W

    1987-11-05

    The putative protein synthesis initiation factor eukaryotic initiation factor 4D (eIF-4D) is post-translationally modified by the polyamine spermidine, forming the rare amino acid hypusine from a lysine residue. The hypusine precursor, deoxyhypusine, was formed in crude cell lysates at pH 9.5 and converted to hypusine at pH 7.1. The modification occurred in eIF-4D, since the isoelectric points and molecular weights of the proteins modified in intact cells and lysates were indistinguishable. Only lysates from cells treated with alpha-difluoromethylornithine, to deplete endogenous polyamine pools, supported the formation of deoxyhypusine, suggesting that unmodified eIF-4D accumulated in spermidine deficient cells. Guazatine, an inhibitor of enzymes which form delta 1-pyrroline from spermidine, blocked deoxyhypusine formation in lysates by nearly 70% at 100 microM and completely at 1 mM. Other mammalian amine oxidase inhibitors had little or no effect on this reaction. Thus, deoxyhypusine formation in eIF-4D is catalyzed by a guazatine-sensitive enzyme with a basic pH optimum.

  20. A sandwich assay for quantitative detection of transcription factors in cell lysate.

    PubMed

    Fang, Zhiyuan; Zhang, Wenjuan; Ge, Chenchen; Liu, Jie; Lie, Puchang; Zeng, Lingwen

    2012-09-21

    A double-stranded DNA (dsDNA) mediated sandwich assay was developed for quantitative detection of transcription factors. The detection limit for human recombinant c-jun protein is 2.5 ng, and for c-jun protein the limit is as low as 0.625 μg of cell lysate.

  1. Whole Tumor Antigen Vaccines: Where Are We?

    PubMed Central

    Chiang, Cheryl Lai-Lai; Coukos, George; Kandalaft, Lana E.

    2015-01-01

    With its vast amount of uncharacterized and characterized T cell epitopes available for activating CD4+ T helper and CD8+ cytotoxic lymphocytes simultaneously, whole tumor antigen represents an attractive alternative source of antigens as compared to tumor-derived peptides and full-length recombinant tumor proteins for dendritic cell (DC)-based immunotherapy. Unlike defined tumor-derived peptides and proteins, whole tumor lysate therapy is applicable to all patients regardless of their HLA type. DCs are essentially the master regulators of immune response, and are the most potent antigen-presenting cell population for priming and activating naïve T cells to target tumors. Because of these unique properties, numerous DC-based immunotherapies have been initiated in the clinics. In this review, we describe the different types of whole tumor antigens that we could use to pulse DCs ex vivo and in vivo. We also discuss the different routes of delivering whole tumor antigens to DCs in vivo and activating them with toll-like receptor agonists. PMID:26343191

  2. Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types.

    PubMed

    Shanskii, Ya D; Sergeeva, N S; Sviridova, I K; Kirakozov, M S; Kirsanova, V A; Akhmedova, S A; Antokhin, A I; Chissov, V I

    2013-11-01

    We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum.

  3. Improved Preparation of Halopropyl Bridged Carboxylic Ortho Esters

    USDA-ARS?s Scientific Manuscript database

    Protection of a carboxylic acid function as a bridged ortho ester derivative enables the use of strongly basic conditions in the synthetic strategy because the protons, alpha to the previous carbonyl carbon, are less acidic. Protected 3-halopropionic acid can behave like an alkyl halide making them...

  4. Improved preparation of haloalkyl bridged carboxylic ortho esters

    USDA-ARS?s Scientific Manuscript database

    Protection of a carboxylic acid function as a bridged ortho ester derivative enables the use of strong basic conditions in the synthetic strategy. For example, a protected 3-halopropionic acid can behave like an alkyl halide because the protons, alpha to the halide function, are less acidic. Ester...

  5. FipsOrtho: A Spell Checker for Learners of French

    ERIC Educational Resources Information Center

    L'Haire, Sebastien

    2007-01-01

    This paper presents FipsOrtho, a spell checker targeted at learners of French, and a corpus of learners' errors which has been gathered to test the system and to get a sample of specific language learners' errors. Spell checkers are a standard feature of many software products, however they are not designed for specific language learners' errors.…

  6. Silver-catalysed protodecarboxylation of ortho-substituted benzoic acids.

    PubMed

    Cornella, Josep; Sanchez, Carolina; Banawa, David; Larrosa, Igor

    2009-12-14

    Catalytic amounts of Ag(I) salts in DMSO have been found to promote the protodecarboxylation of a wide variety of ortho-substituted benzoic acids under mild conditions and in excellent yields, highlighting a possible role for silver in decarboxylative cross-couplings.

  7. FipsOrtho: A Spell Checker for Learners of French

    ERIC Educational Resources Information Center

    L'Haire, Sebastien

    2007-01-01

    This paper presents FipsOrtho, a spell checker targeted at learners of French, and a corpus of learners' errors which has been gathered to test the system and to get a sample of specific language learners' errors. Spell checkers are a standard feature of many software products, however they are not designed for specific language learners' errors.…

  8. Cooling by Para-to-Ortho-Hydrogen Conversion

    NASA Technical Reports Server (NTRS)

    Sherman, A.; Nast, T.

    1983-01-01

    Catalyst speeds conversion, increasing capacity of solid hydrogen cooling system. In radial-flow catalytic converter, para-hydrogen is converted to equilibrium mixture of para-hydrogen and ortho-hydrogen as it passes through porous cylinder of catalyst. Addition of catalyst increases capacity of hydrogen sublimation cooling systems for radiation detectors.

  9. ANTIGENIC MODULATION

    PubMed Central

    Old, Lloyd J.; Stockert, Elisabeth; Boyse, Edward A.; Kim, Jae Ho

    1968-01-01

    Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions. PMID:5636556

  10. Unimolecular thermal fragmentation of ortho-benzene.

    SciTech Connect

    Zhang, X.; Maccarone, A. T.; Nimlos, M. R.; Kato, S.; Bierbaum, V. M.; Ellison, G. B.; Ruscic, B.; Simmonett, A. C.; Allen, W. D.; Schaefer, H. F.; Chemistry; Univ. of Colorado; NREL; Univ. of Georgia

    2007-01-01

    The ortho-benzyne diradical, o-C{sub 6}H{sub 4} has been produced with a supersonic nozzle and its subsequent thermal decomposition has been studied. As the temperature of the nozzle is increased, the benzyne molecule fragments o-C{sub 6}H{sub 4}{sup +} {Delta} {yields} products. The thermal dissociation products were identified by three experimental methods: (i) time-of-flight photoionization mass spectrometry, (ii) matrix-isolation Fourier transform infrared absorption spectroscopy, and (iii) chemical ionization mass spectrometry. At the threshold dissociation temperature, o-benzyne cleanly decomposes into acetylene and diacetylene via an apparent retro-Diels-Alder process: o-C{sub 6}H{sub 4}{sup +}{Delta}{yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH. The experimental {Delta}{sub rxn}H{sub 298}(o-C{sub 6}H{sub 4} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH) is found to be 57 {+-} 3 kcal mol{sup -1}. Further experiments with the substituted benzyne, 3,6-(CH{sub 3}){sub 2}-o-C{sub 6}H{sub 2}, are consistent with a retro-Diels-Alder fragmentation. But at higher nozzle temperatures, the cracking pattern becomes more complicated. To interpret these experiments, the retro-Diels-Alder fragmentation of o-benzyne has been investigated by rigorous ab initio electronic structure computations. These calculations used basis sets as large as [C(7s6p5d4f3g2h1i)/H(6s5p4d3f2g1h)] (cc-pV6Z) and electron correlation treatments as extensive as full coupled cluster through triple excitations (CCSDT), in cases with a perturbative term for connected quadruples [CCSDT(Q)]. Focal point extrapolations of the computational data yield a 0 K barrier for the concerted, C{sub 2v}-symmetric decomposition of o-benzyne, E{sub b}(o-C{sub 6}H{sub 4} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH) = 88.0 {+-} 0.5 kcal mol{sup -1}. A barrier of this magnitude is consistent with the experimental results. A careful assessment of the

  11. Unimolecular Thermal Fragmentation of Ortho-Benzyne

    SciTech Connect

    Zhang, X.; Maccarone, A. T.; Nimlos, M. R.; Kato, S.; Bierbaum, V. M.; Ellison, G. B.; Ruscic, B.; Simmonett, A. C.; Allen, W. D.; Schaefer, H. F., III

    2007-01-01

    The ortho-benzyne diradical, o-C{sub 6}H{sub 4} has been produced with a supersonic nozzle and its subsequent thermal decomposition has been studied. As the temperature of the nozzle is increased, the benzyne molecule fragments: o-C{sub 6}H{sub 4} + {Delta} {yields} products. The thermal dissociation products were identified by three experimental methods: (i) time-of-flight photoionization mass spectrometry, (ii) matrix-isolation Fourier transform infrared absorption spectroscopy, and (iii) chemical ionization mass spectrometry. At the threshold dissociation temperature, o-benzyne cleanly decomposes into acetylene and diacetylene via an apparent retro-Diels-Alder process: o-C{sub 6}H{sub 4} + {Delta} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH. The experimental {Delta}{sub rxn}H{sub 298}(o-C{sub 6}H{sub 4} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH) is found to be 57 {+-} 3 kcal mol{sup -1}. Further experiments with the substituted benzyne, 3,6-(CH{sub 3}){sub 2}-o-C{sub 6}H{sub 2}, are consistent with a retro-Diels-Alder fragmentation. But at higher nozzle temperatures, the cracking pattern becomes more complicated. To interpret these experiments, the retro-Diels-Alder fragmentation of o-benzyne has been investigated by rigorous ab initio electronic structure computations. These calculations used basis sets as large as [C(7s6p5d4f3g2h1i)/H(6s5p4d3f2g1h)] (cc-pV6Z) and electron correlation treatments as extensive as full coupled cluster through triple excitations (CCSDT), in cases with a perturbative term for connected quadruples [CCSDT(Q)]. Focal point extrapolations of the computational data yield a 0 K barrier for the concerted, C{sub 2v}-symmetric decomposition of o-benzyne, E{sub b}(o-C{sub 6}H{sub 4} {yields} HC {triple_bond} CH+HC {triple_bond} C-C {triple_bond} CH) = 88.0 {+-} 0.5 kcal mol{sup -1}. A barrier of this magnitude is consistent with the experimental results. A careful assessment of the

  12. Detection of Interstellar Ortho-D2H+ with SOFIA

    NASA Astrophysics Data System (ADS)

    Harju, Jorma; Sipilä, Olli; Brünken, Sandra; Schlemmer, Stephan; Caselli, Paola; Juvela, Mika; Menten, Karl M.; Stutzki, Jürgen; Asvany, Oskar; Kamiński, Tomasz; Okada, Yoko; Higgins, Ronan

    2017-05-01

    We report on the detection of the ground-state rotational line of ortho-D2H+ at 1.477 THz (203 μm) using the German REceiver for Astronomy at Terahertz frequencies (GREAT) on board the Stratospheric Observatory For Infrared Astronomy (SOFIA). The line is seen in absorption against far-infrared continuum from the protostellar binary IRAS 16293-2422 in Ophiuchus. The para-D2H+ line at 691.7 GHz was not detected with the APEX telescope toward this position. These D2H+ observations complement our previous detections of para-H2D+ and ortho-H2D+ using SOFIA and APEX. By modeling chemistry and radiative transfer in the dense core surrounding the protostars, we find that the ortho-D2H+ and para-H2D+ absorption features mainly originate in the cool (T < 18 K) outer envelope of the core. In contrast, the ortho-H2D+ emission from the core is significantly absorbed by the ambient molecular cloud. Analyses of the combined D2H+ and H2D+ data result in an age estimate of ˜5 × 105 yr for the core, with an uncertainty of ˜2 × 105 yr. The core material has probably been pre-processed for another 5 × 105 years in conditions corresponding to those in the ambient molecular cloud. The inferred timescale is more than 10 times the age of the embedded protobinary. The D2H+ and H2D+ ions have large and nearly equal total (ortho+para) fractional abundances of ˜10-9 in the outer envelope. This confirms the central role of {{{H}}}3+ in the deuterium chemistry in cool, dense gas, and adds support to the prediction of chemistry models that also {{{D}}}3+ should be abundant in these conditions.

  13. Photodissociation dynamics of the ortho- and para-xylyl radicals

    NASA Astrophysics Data System (ADS)

    Pachner, Kai; Steglich, Mathias; Hemberger, Patrick; Fischer, Ingo

    2017-08-01

    The photodissociation dynamics of the C8H9 isomers ortho- and para-xylyl are investigated in a free jet. The xylyl radicals are generated by flash pyrolysis from 2-(2-methylphenyl)- and 2-(4-methylphenyl) ethyl nitrite and are excited into the D3 state. REMPI- spectra show vibronic structure and the origin of the transition is identified at 32 291 cm-1 for the para- and at 32 132 cm-1 for the ortho-isomer. Photofragment H-atom action spectra show bands at the same energy and thus confirm H-atom loss from xylyl radicals. To gain further insight into the photodissociation dynamics, velocity map images of the hydrogen atom photofragments are recorded. Their angular distribution is isotropic and the translational energy release is in agreement with a dissociation to products in their electronic ground state. Photodissociation of para-xylyl leads to the formation of para-xylylene (C8H8), while the data for ortho-xylyl agree much better with the isomer benzocyclobutene as the dominant molecular fragment rather than ortho-xylylene. In computations we identified a new pathway for the reaction ortho-xylyl → benzocyclobutene + H with a barrier of 3.39 eV (27 340 cm-1), which becomes accessible at the employed excitation energy. It proceeds via a combination of scissoring and rotational motion of the -CH2 and -CH3 groups. However, the observed rate constants measured by delaying the excitation and ionization laser with respect to each other are significantly faster than computed ones, indicating intrinsic non-RRKM behaviour. A comparably high value of around 30% of the excess energy is released as translation of the H-atom photofragment.

  14. Dietary effects of ortho-phenylphenol and sodium ortho-phenylphenate on rat urothelium.

    PubMed

    St John, M K; Arnold, L L; Anderson, T; Cano, M; Johansson, S L; Cohen, S M

    2001-02-01

    Ortho-phenylphenol (OPP) and sodium ortho-phenylphenate (NaOPP) are pesticides used commercially in the food industry that have been shown to be carcinogenic to rat urothelium. Dietary administration of 1.25% OPP or 2.0% NaOPP caused increased incidences of urothelial hyperplasia and eventually caused tumors in male F344 rats, with NaOPP apparently having a more potent effect. In other studies, various sodium salts such as saccharin and ascorbate enhanced bladder carcinogenesis, although the acid forms of these salts did not. In studies with high dietary doses of these sodium salts, an amorphous precipitate was produced in the urine; precipitate formation was pH dependent. In previous experiments in which high doses of OPP were fed for up to 17 weeks, severe hyperplasia of the urothelium was produced, but without the formation of an urinary amorphous precipitate, calculi, or abnormal microcrystalluria. In addition, we found no evidence of OPP-DNA adduct formation in the urothelium. The present study was conducted to determine if feeding NaOPP * 4 H(2)0 to male F344 rats as 2.0% of the diet resulted in the formation of an amorphous precipitate in the urine, and if NaOPP caused an increased mineral concentration in the urine and/or kidneys. NaOPP administration produced a higher urinary pH than did OPP fed as 1.25% of the diet. Neither amorphous precipitate nor other solids were observed in the urine of the OPP or NaOPP-treated rats, and urinary calcium concentrations in the treated groups were similar to control. OPP and NaOPP had similar proliferative effects on rat urothelium after 10 weeks of treatment by light microscopy, scanning electron microscopy (SEM), and bromodeoxyuridine (BrdU) labeling indices. The results of this study indicate that formation of abnormal urinary solids is not part of the mechanism by which OPP or NaOPP exert their effects on the rat bladder epithelium.

  15. Effects of a mixture of non-ortho- and mono-ortho-polychlorinated biphenyls on reproduction in Fundulus heteroclitus (Linnaeus)

    SciTech Connect

    Black, D.E.; Gutjahr-Gobell, R.; Pruell, R.J.; Bergen, B.; McElroy, A.E.

    1998-07-01

    To assess the effects of polychlorinated biphenyls (PCBs) on reproduction, female Fundulus heteroclitus were exposed to a mixture of non-ortho- and mono-ortho-PCBs, mimicking the mixture found in fish collected from New Bedford Harbor, Massachusetts, USA, a PCB-contaminated estuary. Exposure was by intraperitoneal injection of the mixture dissolved in corn oil. Doses of 0.76, 3.8, and 19 {micro}g PCB mixture per gram of wet weight produced liver concentrations of 2.99, 12.2, and 32.8 {micro}g non-ortho- and mono-ortho-PCBs per gram of dry liver, with dioxin toxic equivalency concentrations (TEQs) of 0.0963, 0.409, and 0.720 ng/g, respectively. Female mortality was 58%, and egg production was reduced by 77% at the highest dose, compared to controls. Food consumption declined with increasing PCB concentration, suggesting that PCBs act indirectly to reduce fecundity through an energetic effect. Pituitary gonadotropin content appeared to be suppressed at the highest dose, but the ability of ovarian follicles to produce estradiol and testosterone in vitro was not impaired. Significant residue-effects linkages were found, with TEQ emerging as a potential indicator of adverse effects. Mortality was directly related, and egg production was inversely related to log{sub 10}TEQ. Multiple regression analysis indicated that egg production was directly related to pituitary gonadotropin content and food consumption.

  16. Bacterial lysates improve the protective antibody response against respiratory viruses through Toll-like receptor 4

    PubMed Central

    Coviello, Silvina; Wimmenauer, Vera; Polack, Fernando P; Irusta, Pablo M

    2014-01-01

    Respiratory viruses cause significant morbidity and mortality in infants and young children worldwide. Current strategies to modulate the immune system and prevent or treat respiratory viral infections in this age group have shown limited success. Here, we demonstrate that a lysate derived from Gram-positive and Gram-negative organisms positively modulates protective antibody responses against both respiratory syncytial virus (RSV) and influenza virus in murine models of infection. Interestingly, despite the complex mixture of Toll-like receptor (TLR) agonists present in the bacterial lysate, the modulatory effects were mostly dependent on TLR4 signaling. Our results indicate that the use of simple formulations of TLR-agonists can significantly improve the immune response against critical pediatric respiratory pathogens. PMID:25483455

  17. Chloroplast protein import inhibition by a soluble factor from wheat germ lysate.

    PubMed

    Schleiff, Enrico; Motzkus, Michael; Soll, Jürgen

    2002-09-01

    Protein import into chloroplasts occurs post-translationally in vitro. The precursor proteins are generally synthesised in a reticulocyte lysate- or wheat germ lysate-derived system and imported out of this system into chloroplast. These complex soluble protein mixtures are likely to contain factors, which influence somehow the import competence and import efficiency. Here we describe a heat-stable soluble proteinaceaous factor, which inhibits protein import into chloroplasts in vitro. The inhibitor interacts directly with the precursor protein and renders it import incompetent. This mode of action is supported by two observations: firstly, binding of the precursor to the chloroplast surface is diminished in the presence of the inhibitor. Secondly, when chloroplasts were loaded with precursor proteins under conditions, which allow only binding but not import the inhibitor was unable to abolish the subsequent translocation step.

  18. Immunogenicity of Pseudomonas aeruginosa outer membrane antigens examined by crossed immunoelectrophoresis.

    PubMed Central

    Lam, J S; Mutharia, L M; Hancock, R E; Høiby, N; Lam, K; Baek, L; Costerton, J W

    1983-01-01

    By crossed immunoelectrophoresis 36 different anode-migrating antigens were demonstrated in sonicated antigen preparations of Pseudomonas aeruginosa. We numbered these antigens to establish a reference precipitin pattern. Antigen no. 31 was identified as the lipopolysaccharide (LPS) antigen, because it was found to be responsible for the O-group specificity and because it reacted with anti-LPS monoclonal antibodies and with Limulus amoebocyte lysate. Purified outer membrane proteins F (porin), H2, and I used as antigens formed precipitins with the reference antibodies, thus establishing their antigenicity. LPS that copurified with protein F and slightly contaminated protein H2 was detectable as an extra precipitin (antigen no. 31). The use of monoclonal antibodies specific for smooth LPS and rough LPS revealed different antigenic determinants in the LPS molecule and suggested that antigen no. 5 could be the core region of the LPS which is equivalent to the rough LPS. Antibodies against these outer membrane antigens were detected in patients with chronic P. aeruginosa pneumonia and in patients with acute P. aeruginosa bacteremia. Antibodies with the same specificity were also found in rats chronically infected with P. aeruginosa 7 days postinfection. This demonstrates the surface accessibility and antigenic reactivity of outer membrane antigens. Images PMID:6194119

  19. Analysis of Reparative Activity of Platelet Lysate: Effect on Cell Monolayer Recovery In Vitro and Skin Wound Healing In Vivo.

    PubMed

    Sergeeva, N S; Shanskii, Ya D; Sviridova, I K; Karalkin, P A; Kirsanova, V A; Akhmedova, S A; Kaprin, A D

    2016-11-01

    Platelet lysate prepared from donor platelet concentrate and pooled according to a developed technique stimulates migration of multipotent mesenchymal stromal cells of the human adipose tissue and promotes healing of the monolayer defect in cultures of human fibroblasts and multipotent mesenchymal stromal cells in vitro in concentrations close those of fetal calf serum (5-10%). Lysate of platelets from platelet-rich rat blood plasma stimulated healing of the skin defect by promoting epithelialization and granulation tissue formation. The regenerative properties of platelet lysate in vivo increased with increasing its concentration.

  20. Preparation, quality criteria, and properties of human blood platelet lysate supplements for ex vivo stem cell expansion.

    PubMed

    Shih, Daniel Tzu-Bi; Burnouf, Thierry

    2015-01-25

    Most clinical applications of human multipotent mesenchymal stromal cells (MSCs) for cell therapy, tissue engineering, regenerative medicine, and treatment of immune and inflammatory diseases require a phase of isolation and ex vivo expansion allowing a clinically meaningful cell number to be reached. Conditions used for cell isolation and expansion should meet strict quality and safety requirements. This is particularly true for the growth medium used for MSC isolation and expansion. Basal growth media used for MSC expansion are supplemented with multiple nutrients and growth factors. Fetal bovine serum (FBS) has long been the gold standard medium supplement for laboratory-scale MSC culture. However, FBS has a poorly characterized composition and poses risk factors, as it may be a source of xenogenic antigens and zoonotic infections. FBS has therefore become undesirable as a growth medium supplement for isolating and expanding MSCs for human therapy protocols. In recent years, human blood materials, and most particularly lysates and releasates of platelet concentrates have emerged as efficient medium supplements for isolating and expanding MSCs from various origins. This review analyzes the advantages and limits of using human platelet materials as medium supplements for MSC isolation and expansion. We present the modes of production of allogeneic and autologous platelet concentrates, measures taken to ensure optimal pathogen safety profiles, and methods of preparing PLs for MSC expansion. We also discuss the supply of such blood preparations. Produced under optimal conditions of standardization and safety, human platelet materials can become the future 'gold standard' supplement for ex vivo production of MSCs for translational medicine and cell therapy applications.

  1. The effect of platelet lysate fibrinogen on the functionality of MSCs in immunotherapy.

    PubMed

    Copland, Ian B; Garcia, Marco A; Waller, Edmund K; Roback, John D; Galipeau, Jacques

    2013-10-01

    Human platelet lysate (PL) represents an attractive alternative to fetal bovine serum (FBS) for the ex vivo expansion of human mesenchymal stromal cells (MSCs). However, there is controversy whether MSCs propagated in unfractionated PL retain their immunosuppressive properties. Since fibrinogen can be a major component of PL, we hypothesized that the fibrinogen content in PL negatively affects the suppressor function of MSCs. Pools of outdated plateletpheresis products underwent a double freeze-thaw centrifugation and filtration to produce unfractionated platelet lysates (uPL), followed by a temperature controlled clotting procedure to produce a fibrinogen depleted platelet lysate (fdPL). Fibrinogen depletion affected neither the mitogenic properties of PL or growth factor content, however fdPL was less prone to develop precipitate over time. Functionally, fibrinogen interacted directly with MSCs, dose dependently increased IL-6, IL-8 and MCP-1 protein production, and compromised the ability of MSCs to up-regulate indoleamine dioxygenase (IDO), as well as, mitigate T-cell proliferation. Similarly uPL expanded MSCs showed a reduced capability of inducing IDO and suppressing T-cell proliferation compared to FBS expanded MSCs. Replacing uPL with fdPL largely restored the immune modulating effects of MSCs. Together these data suggest that fibrinogen negatively affects the immunomodulatory functions of MSCs and fdPL can serve as non-xenogenic mitogenic supplement for expansion of clinical grade MSCs for immune modulation.

  2. Electrochemical sensor for rapid determination of fibroblast growth factor receptor 4 in raw cancer cell lysates.

    PubMed

    Torrente-Rodríguez, Rebeca M; Ruiz-Valdepeñas Montiel, Víctor; Campuzano, Susana; Pedrero, María; Farchado, Meryem; Vargas, Eva; Manuel de Villena, F Javier; Garranzo-Asensio, María; Barderas, Rodrigo; Pingarrón, José M

    2017-01-01

    The first electrochemical immunosensor for the determination of fibroblast growth factor receptor 4 (FGFR4) biomarker is reported in this work. The biosensor involves a sandwich configuration with covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic microcarriers (HOOC-MBs) and amperometric detection at disposable carbon screen-printed electrodes (SPCEs). The biosensor exhibits a great analytical performance regarding selectivity for the target protein and a low LOD of 48.2 pg mL-1. The electrochemical platform was successfully applied for the determination of FGFR4 in different cancer cell lysates without any apparent matrix effect after a simple sample dilution and using only 2.5 μg of the raw lysate. Comparison of the results with those provided by a commercial ELISA kit shows competitive advantages by using the developed immunosensor in terms of simplicity, analysis time, and portability and cost-affordability of the required instrumentation for the accurate determination of FGFR4 in cell lysates.

  3. Electrochemical sensor for rapid determination of fibroblast growth factor receptor 4 in raw cancer cell lysates

    PubMed Central

    Torrente-Rodríguez, Rebeca M.; Ruiz-Valdepeñas Montiel, Víctor; Campuzano, Susana; Pedrero, María; Farchado, Meryem; Vargas, Eva; Manuel de Villena, F. Javier; Garranzo-Asensio, María; Barderas, Rodrigo

    2017-01-01

    The first electrochemical immunosensor for the determination of fibroblast growth factor receptor 4 (FGFR4) biomarker is reported in this work. The biosensor involves a sandwich configuration with covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic microcarriers (HOOC-MBs) and amperometric detection at disposable carbon screen-printed electrodes (SPCEs). The biosensor exhibits a great analytical performance regarding selectivity for the target protein and a low LOD of 48.2 pg mL-1. The electrochemical platform was successfully applied for the determination of FGFR4 in different cancer cell lysates without any apparent matrix effect after a simple sample dilution and using only 2.5 μg of the raw lysate. Comparison of the results with those provided by a commercial ELISA kit shows competitive advantages by using the developed immunosensor in terms of simplicity, analysis time, and portability and cost-affordability of the required instrumentation for the accurate determination of FGFR4 in cell lysates. PMID:28376106

  4. Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression.

    PubMed

    Didovyk, Andriy; Tonooka, Taishi; Tsimring, Lev; Hasty, Jeff

    2017-08-21

    Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

  5. Bacterial lysate in the prevention of acute exacerbation of COPD and in respiratory recurrent infections

    PubMed Central

    Braido, F; Tarantini, F; Ghiglione, V; Melioli, G; Canonica, G W

    2007-01-01

    Respiratory tract infections (RTIs) represent a serious problem because they are one of the most common cause of human death by infection. The search for the treatment of those diseases has therefore a great importance. In this study we provide an overview of the currently available treatments for RTIs with particular attention to chronic obstructive pulmonary diseases exacerbations and recurrent respiratory infections therapy and a description of bacterial lysate action, in particular making reference to the medical literature dealing with its clinical efficacy. Those studies are based on a very large number of clinical trials aimed to evaluate the effects of this drug in maintaining the immune system in a state of alert, and in increasing the defences against microbial infections. From this analysis it comes out that bacterial lysates have a protective effect, which induce a significant reduction of the symptoms related to respiratory infections. Those results could be very interesting also from an economic point of view, because they envisage a reduction in the number of acute exacerbations and a shorter duration of hospitalization. The use of bacterial lysate could therefore represent an important means to achieve an extension of life duration in patients affected by respiratory diseases. PMID:18229572

  6. Active immunotherapy for cancer patients using tumor lysate pulsed dendritic cell vaccine: a safety study.

    PubMed

    Ovali, E; Dikmen, T; Sonmez, M; Yilmaz, M; Unal, A; Dalbasti, T; Kuzeyli, K; Erturk, M; Omay, S B

    2007-06-01

    Cancer vaccine therapy represents a promising therapeutical option. Consistently, with these new treatment strategies, the use of dendritic cell vaccines is becoming increasingly widespread and currently in the forefront for cancer treatment. The purpose of this study was to evaluate the feasibility and safety of tumor lysate-pulsed dendritic cell (DC) vaccine in patients with advanced cancers. For this purpose, eighteen patients with relapsed or refractory cancer were vaccinated with peripheral monocyte-derived DCs generated with GM-CSF and IL-4, and pulsed consequently with 100 microg/ml of tumor lysate before maturation in culture in the presence of IL-1beta, PGE2 and TNF alpha for two days. The first two vaccinations were given intradermally every two weeks while further injections were given monthly. Tumor lysate-pulsed dendritic cell injections were well-tolerated in all patients with no more than grade 1 injection-related toxicity. Local inflammatory response was mainly erythematous which subsided in 48 hrs time. No end organ toxicity or autoimmune toxicity was identified. Clinical responses observed in our study were satisfactory for a phase I clinical study. We observed 4 (22%) objective clinical responses. These responses are significantly correlated with delayed type hypersensitivity testing (DTH) (p < 0.01). The results showed that this active immunotherapy is feasible, safe, and may be capable of eliciting immune responses against cancer.

  7. A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR.

    PubMed

    Shatzkes, Kenneth; Teferedegne, Belete; Murata, Haruhiko

    2014-04-11

    Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37 °C incubation for 1-2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at -80 °C.

  8. A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR

    PubMed Central

    Shatzkes, Kenneth; Teferedegne, Belete; Murata, Haruhiko

    2014-01-01

    Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37°C incubation for 1–2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at −80°C. PMID:24722424

  9. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

    PubMed

    Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

    2016-01-01

    The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Toxoplasma gondii Recombinant Antigens as Tools for Serodiagnosis of Human Toxoplasmosis: Current Status of Studies

    PubMed Central

    2013-01-01

    Toxoplasma gondii is a parasitic protozoan which is the cause of toxoplasmosis. Although human toxoplasmosis in healthy adults is usually asymptomatic, serious disease can occur in the case of congenital infections and immunocompromised individuals. Furthermore, despite the exact recognition of its etiology, it still presents a diagnostic problem. Diagnosis of toxoplasmosis is mainly based on the results of serological tests detecting anti-T. gondii-specific antibodies in the patient's serum sample. The specificities and sensitivities of serology tests depend mostly on the diagnostic antigen(s) used. Most of the commercial serological kits currently available are based on Toxoplasma lysate antigens (TLAs). In recent years, many studies showed that recombinant antigenic proteins of T. gondii may be an alternative source of antigens which are very useful for the serodiagnosis of toxoplasmosis. This article presents a review of current studies on the application and usefulness of different T. gondii recombinant antigens in serological tests for the diagnosis of human toxoplasmosis. PMID:23784855

  11. Using OrthoMCL to assign proteins to OrthoMCL-DB groups or to cluster proteomes into new ortholog groups

    PubMed Central

    Fischer, Steve; Brunk, Brian P.; Chen, Feng; Gao, Xin; Harb, Omar S.; Iodice, John B.; Shanmugam, Dhanasekaran; Roos, David S.; Stoeckert, Christian J.

    2011-01-01

    OrthoMCL is an algorithm for grouping proteins into ortholog groups based on their sequence similarity. OrthoMCL-DB is a public database that allows users to browse and view ortholog groups that were pre-computed using the OrthoMCL algorithm. Version 4 of this database contained 116,536 ortholog groups clustered from 1,270,853 proteins obtained from 88 eukaryotic genomes, 16 archaeal genomes and 34 bacterial genomes. Future versions of OrthoMCL-DB will include more proteomes as more genomes are sequenced. Here, we describe how you can group your proteins of interest into ortholog clusters using two different means provided by the OrthoMCL system. The OrthoMCL-DB website has a tool for uploading and grouping a set of protein sequences, typically representing a proteome. This method maps the uploaded proteins to existing groups in OrthoMCL-DB. Alternatively, if you have proteins from a set of genomes that need to be grouped, you can download, install and run the standalone OrthoMCL software. PMID:21901743

  12. Mild access to planar-chiral ortho-condensed aromatic ferrocenes via gold(i)-catalyzed cycloisomerization of ortho-alkynylaryl ferrocenes.

    PubMed

    Urbano, Antonio; Hernández-Torres, Gloria; Del Hoyo, Ana M; Martínez-Carrión, Alicia; Carmen Carreño, M

    2016-05-11

    An efficient approach to (Rp) planar-chiral tri- and tetracyclic ortho-condensed aromatic ferrocenes was developed through the enantioselective cationic Au(i)-catalyzed cycloisomerization, in the presence of bidentate phosphine ligand (R)-DTBM-Segphos, from readily available ortho-alkynylaryl ferrocenes under very mild conditions (11 examples, up to 92% yield and 93% ee).

  13. FEEDING THE MUMMICHOG (FUNDULUS HERTERCLITUS), A DIET SPIKED WITH NON-ORTHO AND MONO-ORTHO SUBSTITUTED POLYCHLORINATED BIPHENYLS:ACCUMULATION AND EFFECTS

    EPA Science Inventory

    A laboratory model was developed to predict exposure effects in the field. Accumulation of non-ortho- and mono-ortho-substituted PCBs in liver tissue and their effects on mummichogs (Fundulus heteroclitus) were investigated. An artificial diet spiked with a mixture of eight PCB c...

  14. FEEDING THE MUMMICHOG (FUNDULUS HERTERCLITUS), A DIET SPIKED WITH NON-ORTHO AND MONO-ORTHO SUBSTITUTED POLYCHLORINATED BIPHENYLS:ACCUMULATION AND EFFECTS

    EPA Science Inventory

    A laboratory model was developed to predict exposure effects in the field. Accumulation of non-ortho- and mono-ortho-substituted PCBs in liver tissue and their effects on mummichogs (Fundulus heteroclitus) were investigated. An artificial diet spiked with a mixture of eight PCB c...

  15. OrthoDisease: tracking disease gene orthologs across 100 species.

    PubMed

    Forslund, Kristoffer; Schreiber, Fabian; Thanintorn, Nattaphon; Sonnhammer, Erik L L

    2011-09-01

    Orthology is one of the most important tools available to modern biology, as it allows making inferences from easily studied model systems to much less tractable systems of interest, such as ourselves. This becomes important not least in the study of genetic diseases. We here review work on the orthology of disease-associated genes and also present an updated version of the InParanoid-based disease orthology database and web site OrthoDisease, with 14-fold increased species coverage since the previous version. Using this resource, we survey the taxonomic distribution of orthologs of human genes involved in different disease categories. The hypothesis that paralogs can mask the effect of deleterious mutations predicts that known heritable disease genes should have fewer close paralogs. We found large-scale support for this hypothesis as significantly fewer duplications were observed for disease genes in the OrthoDisease ortholog groups.

  16. Conversion of para and ortho hydrogen in the Jovian planets

    NASA Technical Reports Server (NTRS)

    Massie, S. T.; Hunten, D. M.

    1982-01-01

    A mechanism is proposed which partially equilibrates the para and ortho rotational levels of molecular hydrogen in the atmospheres of Jupiter, Saturn, and Uranus. Catalytic reactions between the free-radical surface sites of aerosol particles and hydrogen modecules yield significant equilibration near 1 bar pressure, if the efficiency of conversion per collision is between 10 to the -8th and 10 to the -10th and the effective eddy mixing coefficient is 10,000 sq cm/sec. At lower pressures the ortho-para ratio retains the value at the top of the cloud layer, except for a very small effect from conversion in the thermosphere. The influence of conversion on the specific heat and adiabatic lapse rate is also investigated. The effect is found to be generally small, though is can rise to 10% inside the aerosol layer.

  17. Flow carbonylation of sterically hindered ortho-substituted iodoarenes

    PubMed Central

    Mallia, Carl J; Walter, Gary C

    2016-01-01

    Summary The flow synthesis of ortho-substituted carboxylic acids, using carbon monoxide gas, has been studied for a number of substrates. The optimised conditions make use of a simple catalyst system compromising of triphenylphosphine as the ligand and palladium acetate as the pre-catalyst. Carbon monoxide was introduced via a reverse “tube-in-tube” flow reactor at elevated pressures to give yields of carboxylated products that are much higher than those obtained under normal batch conditions. PMID:27559403

  18. Alkali metal derivatives of an ortho-phenylene diamine.

    PubMed

    Robinson, Sarah; Davies, E Stephen; Lewis, William; Blake, Alexander J; Liddle, Stephen T

    2014-03-21

    Treatment of the ortho-phenylene diamine C6H4-1,2-{N(H)Tripp}2 (1, PDAH2, Tripp = 2,4,6-triisopropylphenyl) with two equivalents of MR (M = Li, R = Bu(n); M = Na or K, R = CH2C6H5) afforded the dimetallated alkali metal ortho-phenylene diamide dianion complexes [(PDALi2)(THF)3] (2), [{(PDANa2)(THF)2}2] (3), and [{(PDAK2)(THF)3}2] (4). In contrast, treatment of 2 with two equivalents of rubidium or cesium 2-ethylhexoxide, or treatment of 1 with two equivalents of MR (M = Rb or Cs, R = CH2C6H5) did not afford the anticipated dialkali metal ortho-phenylene diamide dianion derivatives and instead formally afforded the monometallic ortho-diiminosemiquinonate radical anion species [PDAM] (M = Rb, 5; M = Cs, 6). The structure of 2 is monomeric with one lithium coordinated to the two nitrogen centres and the other lithium η(4)-coordinated to the diazabutadiene portion of the PDA scaffold. Similar structural cores are observed for 3 and 4, except that the larger sodium and potassium ions give dimeric structures linked by multi-hapto interactions from the PDA backbone phenyl ring to an alkali metal centre. Complex 5 was not characterised in the solid state, but the structure of 6 reveals coordination of cesium ions to both PDA amide centres and multi-hapto interactions to a PDA backbone phenyl ring in the next unit to generate a one-dimensional polymer. Complexes 2-6 have been variously characterised by X-ray crystallography, multi-nuclear NMR, FTIR, and EPR spectroscopies, and CHN microanalyses.

  19. Ortho markets first FDA-approved triphasic pill.

    PubMed

    1984-06-01

    Ortho-Novum 7/7/7, the 1st triphasic oral contraceptive to be approved by the FDA (Federal Food and Drug Administration) is now being marketed in the US by the Ortho Pharmaceutical Corporation. The triphasic pill is similar to the biphasic pill as the steriod level is varied during the cycle, but the new triphasic pill more closely follows the normal ovulatory cycle cycle in regard to steriod levels. During days 1-7 of the cycle, the dosage is 0.5 mg of progestogen and 35 mcg of estrogen. Respective doses for days 8-14 are 0.75 mg amd 35 mcg and for days 15-21 they are 1.0 mg and 35 mcg. The total dosage of progestogen for the entire cycle is 15.75 mg. The new pill is associated with less breakthrough bleeding than the biphasic pill and the bleeding pattern is similar to that observed for Ortho 1-35. 2 pregnancies were observed during worldwide clinical trails but only 1 was attributed to product failure. The Pearl Formula failure rate was 0.22 or less. The incidence of side effects was low. The pill is packaged in a 28-day circular dial pack which contains 7 white, 7 light peach, and 7 dark peach pills with an additional 7 placebo pills. Some objectives have been raised concerning the packaging of the product. The pills are very similar in color and patients might find this confusing. Ortho representatives indicated that this should not be a problem if patient are given sufficient counseling.

  20. Direct Intermolecular Aniline ortho-Arylation via Benzyne Intermediates

    PubMed Central

    Truong, Thanh

    2012-01-01

    A method for direct, transition-metal-free ortho-arylation of anilines by aryl chlorides, bromides, fluorides, and triflates has been developed. This methodology provides the most direct approach to 2-arylanilines since no protecting or directing groups on nitrogen are required. The arylation is functional-group tolerant, with alkene, ether, trifluoromethyl, dimethylamino, carbonyl, chloro, and cyano functionalities tolerated. Phenylation of enantiopure binaphthyldiamine affords a product with >99% ee. PMID:23148679

  1. Biological and therapeutic effects of ortho-silicic acid and some ortho-silicic acid-releasing compounds: New perspectives for therapy

    PubMed Central

    2013-01-01

    Silicon (Si) is the most abundant element present in the Earth's crust besides oxygen. However, the exact biological roles of silicon remain unknown. Moreover, the ortho-silicic acid (H4SiO4), as a major form of bioavailable silicon for both humans and animals, has not been given adequate attention so far. Silicon has already been associated with bone mineralization, collagen synthesis, skin, hair and nails health atherosclerosis, Alzheimer disease, immune system enhancement, and with some other disorders or pharmacological effects. Beside the ortho-silicic acid and its stabilized formulations such as choline chloride-stabilized ortho-silicic acid and sodium or potassium silicates (e.g. M2SiO3; M= Na,K), the most important sources that release ortho-silicic acid as a bioavailable form of silicon are: colloidal silicic acid (hydrated silica gel), silica gel (amorphous silicon dioxide), and zeolites. Although all these compounds are characterized by substantial water insolubility, they release small, but significant, equilibrium concentration of ortho-silicic acid (H4SiO4) in contact with water and physiological fluids. Even though certain pharmacological effects of these compounds might be attributed to specific structural characteristics that result in profound adsorption and absorption properties, they all exhibit similar pharmacological profiles readily comparable to ortho-silicic acid effects. The most unusual ortho-silicic acid-releasing agents are certain types of zeolites, a class of aluminosilicates with well described ion(cation)-exchange properties. Numerous biological activities of some types of zeolites documented so far might probably be attributable to the ortho-silicic acid-releasing property. In this review, we therefore discuss biological and potential therapeutic effects of ortho-silicic acid and ortho-silicic acid -releasing silicon compounds as its major natural sources. PMID:23298332

  2. Biological and therapeutic effects of ortho-silicic acid and some ortho-silicic acid-releasing compounds: New perspectives for therapy.

    PubMed

    Jurkić, Lela Munjas; Cepanec, Ivica; Pavelić, Sandra Kraljević; Pavelić, Krešimir

    2013-01-08

    Silicon (Si) is the most abundant element present in the Earth's crust besides oxygen. However, the exact biological roles of silicon remain unknown. Moreover, the ortho-silicic acid (H4SiO4), as a major form of bioavailable silicon for both humans and animals, has not been given adequate attention so far. Silicon has already been associated with bone mineralization, collagen synthesis, skin, hair and nails health atherosclerosis, Alzheimer disease, immune system enhancement, and with some other disorders or pharmacological effects. Beside the ortho-silicic acid and its stabilized formulations such as choline chloride-stabilized ortho-silicic acid and sodium or potassium silicates (e.g. M2SiO3; M= Na,K), the most important sources that release ortho-silicic acid as a bioavailable form of silicon are: colloidal silicic acid (hydrated silica gel), silica gel (amorphous silicon dioxide), and zeolites. Although all these compounds are characterized by substantial water insolubility, they release small, but significant, equilibrium concentration of ortho-silicic acid (H4SiO4) in contact with water and physiological fluids. Even though certain pharmacological effects of these compounds might be attributed to specific structural characteristics that result in profound adsorption and absorption properties, they all exhibit similar pharmacological profiles readily comparable to ortho-silicic acid effects. The most unusual ortho-silicic acid-releasing agents are certain types of zeolites, a class of aluminosilicates with well described ion(cation)-exchange properties. Numerous biological activities of some types of zeolites documented so far might probably be attributable to the ortho-silicic acid-releasing property. In this review, we therefore discuss biological and potential therapeutic effects of ortho-silicic acid and ortho-silicic acid -releasing silicon compounds as its major natural sources.

  3. Bone-forming capacity of mesenchymal stromal cells when cultured in the presence of human platelet lysate as substitute for fetal bovine serum.

    PubMed

    Prins, Henk-Jan; Rozemuller, Henk; Vonk-Griffioen, Simone; Verweij, Vivienne G M; Dhert, Wouter J A; Slaper-Cortenbach, Ineke C M; Martens, Anton C M

    2009-12-01

    In tissue engineering, strategies are being developed to repair large bone defects by combining biomaterials and bone marrow-derived multipotent mesenchymal stromal cells (MSCs). For expansion of MSCs under good manufacturing practice conditions, human platelet lysate (PL) can serve as substitute for fetal bovine serum (FBS) in culture media. We compared the in vivo bone-forming capacity of passage 3 MSCs cultured with either PL or FBS for nine different human donors. We also tested the growth kinetics, antigen expression profile, and the multilineage differentiation capacity in vitro of these MSCs. The in vivo bone-forming capacity was determined by seeding culture-expanded MSCs onto biphasic calcium phosphate scaffolds. Hybrid constructs were implanted subcutaneously in nude mice, retrieved after 6 weeks, and analyzed using histomorphometry. PL-supplemented cultures resulted in significantly larger colonies, shorter culture time period, and higher population doublings between P1 and P3 compared to FBS-containing cultures. No differences were observed in antigen expression profiles or differentiation capacities into the osteoblastic, chondrogenic, and adipogenic lineages, qualitatively. In vivo bone formation with PL-supplemented cultures of MSCs was demonstrated in 9/9 donors versus 6/9 for FBS-supplemented cultures. These results warrant the use of PL for ex vivo expansion of human MSCs for bone tissue engineering applications.

  4. Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

    PubMed Central

    Tan, Chengbo; Wang, Zifeng; Hamauchi, Shuji; Abumiya, Takeo; Kazumata, Ken; Ito, Tsuneo; Kudo, Kohsuke; Takamoto, Shigeru; Houkin, Kiyohiro

    2016-01-01

    Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs) as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs). Platelet concentrates (PC) were harvested from healthy volunteers and made into single donor-derived PL (sPL). The PL mixtures (mPL) were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA). The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO) agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS). No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke. PMID:27840648

  5. Synthesis of β-(1→2)-Linked 6-Deoxy-l-altropyranose Oligosaccharides via Gold(I)-Catalyzed Glycosylation of an ortho-Hexynylbenzoate Donor.

    PubMed

    Shen, Zhengnan; Mobarak, Hani; Li, Wei; Widmalm, Göran; Yu, Biao

    2017-03-17

    The β-(1→2)-linked 6-deoxy-l-altropyranose di- to pentasaccharides 2-5, relevant to the O-antigen of the infectious Yersinia enterocolitica O:3, were synthesized for the first time. The challenging 1,2-cis-altropyranosyl linkage was assembled effectively via glycosylation with 2-O-benzyl-3,4-di-O-benzoyl-6-deoxy-l-altropyranosyl ortho-hexynylbenzoate (7) under the catalysis of PPh3AuNTf2. NMR and molecular modeling studies showed that the pentasaccharide (5) adopted a left-handed helical conformation.

  6. Effective biotic elicitation of Ruta graveolens L. shoot cultures by lysates from Pectobacterium atrosepticum and Bacillus sp.

    PubMed

    Orlita, A; Sidwa-Gorycka, M; Malinski, E; Czerwicka, M; Kumirska, J; Golebiowski, M; Lojkowska, E; Stepnowski, P

    2008-03-01

    Growth of Ruta graveolens shoots was induced when Bacillus sp. cell lysates were added to the culture medium. Elicitation of coumarin by this lysate was also very effective; the concentrations of isopimpinelin, xanthotoxin and bergapten increased to 610, 2120 and 1460 microg g(-1) dry wt, respectively. It also had a significant effect on the production of psoralen and rutamarin (680 and 380 microg g(-1) dry wt) and induced the biosynthesis of chalepin, which was not detected in the control sample, up to 47 microg g(-1) dry wt With lysates of the Pectobacterium atrosepticum, their effect on growth was not so significant and had no effect on the induction of coumarin accumulation. But elicitation with this lysate was much more effective for inducing the production of furoquinolone alkaloids; the concentrations of gamma-fagarine, skimmianine, dictamnine and kokusaginine rose to 99, 680, 172 and 480 microg g(-1) dry wt, respectively.

  7. Determination of the Ratio of Ortho Hydrogen and Para Hydrogen

    NASA Astrophysics Data System (ADS)

    Zhou, D.; Ihas, G. G.; Sullivan, N. S.

    2003-03-01

    The two different quantum states of hydrogen, ortho-hydrogen and para-hydrogen, possess different properties. The accurate determination of the ortho/para ratio in gaseous, liquid and solid state is important both for research needs and for applications in cryogenic engineering, such as H2 production, transport and storage. NMR can determine the ratio1 accurately, but it is cumbersome and often not practical. Cryogenic applications need a simple and reliable method. We report on the development of a thermal conductivity gauge employing a pure metal thin film that serves as both heater and thermometer for the detection of ortho-para hydrogen ratios in the gaseous state. This ratio-meter has been tested and found to have a nearly pressure-independent voltage response over a broad pressure range with a constant current. The thermal conductivity of hydrogen and nitrogen was measured and found to agree quantitatively with published data. The new development will be presented. *Thanks to Larry Phelps, Bill Malphurs, Stephen Wood, David Hernandez. # Supported by NASA Contract NAG3-2750 Ref. 1. D. Zhou, C. M. Edwards, and N. S. Sullivan, Phys. Rev. Lett. 62, 1528 (1989)

  8. ORTHO-PARA SELECTION RULES IN THE GAS-PHASE CHEMISTRY OF INTERSTELLAR AMMONIA

    SciTech Connect

    Faure, A.; Hily-Blant, P.; Le Gal, R.; Rist, C.

    2013-06-10

    The ortho-para chemistry of ammonia in the cold interstellar medium is investigated using a gas-phase chemical network. Branching ratios for the primary reaction chain involved in the formation and destruction of ortho- and para-NH{sub 3} were derived using angular momentum rules based on the conservation of the nuclear spin. We show that the 'anomalous' ortho-to-para ratio of ammonia ({approx}0.7) observed in various interstellar regions is in fact consistent with nuclear spin selection rules in a para-enriched H{sub 2} gas. This ratio is found to be independent of temperature in the range 5-30 K. We also predict an ortho-to-para ratio of {approx}2.3 for NH{sub 2}. We conclude that a low ortho-to-para ratio of H{sub 2} naturally drives the ortho-to-para ratios of nitrogen hydrides below the statistical values.

  9. [Therapeutic efficacy of dendritic cells pulsed by autologous tumor cell lysate in combination with CIK cells on advanced renal cell carcinoma].

    PubMed

    Wang, Hui; Feng, Fang; Zhu, Mingao; Wang, Roushu; Wang, Xiaoping; Wu, Ying; Zhuang, Zhixiang

    2015-01-01

    To investigate the clinical efficacy of dendritic cells (DCs) sensitization by autologous tumor cell lysate in combination with cytokine-induced killer (CIK) cells on patients with advanced renal cell carcinoma and detect their immune functions and adverse effects. The study analyzed retrospectively 82 patients with advanced renal cell carcinoma admitted in our department from January 2011 to December 2013. Peripheral blood mononuclear cells (PBMCs) were isolated from the above patients. Adherent cells were cultured to produce DCs. The DCs were pulsed with autologous tumor cell lysate (Ag) to produce Ag-DC. T lymphocytes were cultured to prepare CIK. The Ag-DC was co-cultured with CIK to produce Ag-DC-CIK vaccine, and then the phenotypes of the DCs and the secretion of IL-12 were evaluated. CIK cell proliferation was determined, too. The 41 patients received the immunotherapy of Ag-DC-CIK, and the other 41 patients received CIK cell therapy alone. After 2 cycles of treatment, the changes of T cell subtypes and cytokines released in the peripheral blood were evaluated. The therapeutic outcomes were evaluated with the help of imageology. The adverse effects were also observed. Compared with the DCs alone, DCs pulsed with autologous tumor cell lysates expressed significantly surface molecules CD11c, CD83, CD86 and HLA-DR, and the release of IL-12 significantly increased. CIK cell proliferation was significantly enhanced after co-cultured with the Ag-DC. Meanwhile, the percentages of CD3⁺CD8⁺ cells and CD3⁺CD56⁺ cells went up significantly compared with the controls. Clinical responses of the Ag-DC-CIK treatment group were much better than those of the control group. No severe adverse effects were seen in the two groups. DCs derived from PBMCs of advanced renal cell carcinoma patients harboring the autologous tumor cell antigens can differentiate into mature DCs, which facilitate the proliferation of CIK cells. Ag-DC-CIK vaccine improves the immune status of

  10. Endogenous RNase inhibitor contributes to stability of RNA in crude cell lysates: Applicability to RT-qPCR.

    PubMed

    Wang, Xiao; Teferedegne, Belete; Shatzkes, Kenneth; Tu, Wei; Murata, Haruhiko

    2016-11-15

    Crude cell lysates are increasingly used as input for direct analysis by reverse transcription quantitative PCR (RT-qPCR), particularly for high-throughput applications. We previously demonstrated that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercial cell-lysis reagents for the preparation of RT-qPCR-ready cell lysates; addition of an exogenous RNase inhibitor (RI) to the lysis buffer was found to be unnecessary to maintain RNA stability in cell lysates either freshly prepared or previously stored frozen at -80 °C. In the present study, we have demonstrated that the stability of RNA observed in our cell lysates is due to the presence of the endogenous RI. Furthermore, we have established the generalizability and applicability of this phenomenon by evaluating lysates prepared from cell lines commonly used in virology (A549, HeLa, MDCK, and Vero). Awareness of the mechanism underlying RNA stability may engender greater confidence in generating cell lysates for RT-qPCR without relying on addition of exogenous RI (a substantial cost-saving benefit) and encourage appropriate practices for handling and storage of samples.

  11. The Ratio of Ortho- to Para-H2 in Photodissociation Regions

    NASA Technical Reports Server (NTRS)

    Sternberg, Amiel; Neufeld, David A.

    1999-01-01

    We discuss the ratio of ortho- to para-H2 in photodissociation regions (PDRs). We draw attention to an apparent confusion in the literature between the ortho-to-para ratio of molecules in FUV-pumped vibrationally excited states and the total H2 ortho-to-para abundance ratio. These ratios are not the same because the process of FUV pumping of fluorescent H2 emission in PDRs occurs via optically thick absorption lines. Thus gas with an equilibrium ratio of ortho- to para-H2 equal to 3 will yield FUV-pumped vibrationally excited ortho-to-para ratios smaller than 3, because the ortho-H2 pumping rates are preferentially reduced by optical depth effects. Indeed, if the ortho and para pumping lines are on the "square root" part of the curve of growth, then the expected ratio of ortho and para vibrational line strengths is 3(sup 1/2) approximately 1.7, close to the typically observed value. Thus, contrary to what has sometimes been stated in the literature, most previous measurements of the ratio of ortho- to para-H2 in vibrationally excited states are entirely consistent with a total ortho-to-para ratio of 3, the equilibrium value for temperatures greater than 200 K. We present an analysis and several detailed models that illustrate the relationship between the total ratios of ortho- to para-H2 and the vibrationally excited ortho-to-para ratios in PDRs. Recent Infrared Space Observatory measurements of pure rotational and vibrational H2 emissions from the PDR in the star-forming region S140 provide strong observational support for our conclusions.

  12. The Ratio of Ortho- to Para-H2 in Photodissociation Regions

    NASA Technical Reports Server (NTRS)

    Sternberg, Amiel; Neufeld, David A.

    1999-01-01

    We discuss the ratio of ortho- to para-H2 in photodissociation regions (PDRs). We draw attention to an apparent confusion in the literature between the ortho-to-para ratio of molecules in FUV-pumped vibrationally excited states and the total H2 ortho-to-para abundance ratio. These ratios are not the same because the process of FUV pumping of fluorescent H2 emission in PDRs occurs via optically thick absorption lines. Thus gas with an equilibrium ratio of ortho- to para-H2 equal to 3 will yield FUV-pumped vibrationally excited ortho-to-para ratios smaller than 3, because the ortho-H2 pumping rates are preferentially reduced by optical depth effects. Indeed, if the ortho and para pumping lines are on the "square root" part of the curve of growth, then the expected ratio of ortho and para vibrational line strengths is 3(sup 1/2) approximately 1.7, close to the typically observed value. Thus, contrary to what has sometimes been stated in the literature, most previous measurements of the ratio of ortho- to para-H2 in vibrationally excited states are entirely consistent with a total ortho-to-para ratio of 3, the equilibrium value for temperatures greater than 200 K. We present an analysis and several detailed models that illustrate the relationship between the total ratios of ortho- to para-H2 and the vibrationally excited ortho-to-para ratios in PDRs. Recent Infrared Space Observatory measurements of pure rotational and vibrational H2 emissions from the PDR in the star-forming region S140 provide strong observational support for our conclusions.

  13. Carbonization Studies of Glassy Carbon Derived from Bis-Ortho-Diynylarenes (BODA) (Postprint)

    DTIC Science & Technology

    2007-02-26

    Derived from Bis-Ortho-Diynylarenes ( BODA ) (Postprint) 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Scott T. Iacono, Mark W. Perpall...have demonstrated bis-ortho-diynylarene ( BODA ) monomers undergo Bergman cyclopolymerizations to form hyper-branched, rigid naphthalene networks that...Std. 239.18 CARBONIZATION STUDIES OF GLASSY CARBON DERIVED FROM BIS-ORTHO-DIYNYLARENES ( BODA ) Scott T. Iacono1, Mark W. Perpall1, Wesley P

  14. Mutagenic activation reduces carcinogenic activity of ortho-aminoazotoluene for mouse liver.

    PubMed

    Ovchinnikova, L P; Bogdanova, L A; Kaledin, V I

    2013-03-01

    Pentachlorophenol (aromatic amine and azo stain metabolic stimulation inhibitor) reduced the hepatocarcinogenic activity of 4-aminoazobenzene and reduced that of ortho-aminoazotoluene in suckling mice. Both 4-aminoazobenzene and ortho-aminoazotoluene exhibited mutagenic activity in Ames' test in vitro on S. typhimurium TA 98 strain with activation with liver enzymes; this mutagenic activity was similarly suppressed by adding pentachlorophenol into activation medium. Induction of xenobiotic metabolism enzymes, stimulating the mutagenic activity of ortho-aminoazotoluene, suppressed its carcinogenic effect on mouse liver. Hence, ortho-aminotoluene (the initial compound), but not its mutagenic metabolites, was the direct active hepatocarcinogen for mice.

  15. Effect of the ortho alkylation of perylene bisimides on the alignment and self-assembly properties.

    PubMed

    Dasgupta, Debarshi; Kendhale, Amol M; Debije, Michael G; Ter Schiphorst, Jeroen; Shishmanova, Ivelina K; Portale, Giuseppe; Schenning, Albertus P H J

    2014-08-01

    The effect of the ortho alkylation of perylene bisimides on the alignment and self-assembly properties has been studied. It was found that the dichroic properties of perylene bisimides in a liquid crystal host can be reversed with a single synthetic step by ortho alkylation. Furthermore, a solvent-induced growth of ultralong organic n-type semiconducting fibrils from non-ortho-alkylated perylene bisimide was observed. Ortho substitution of the perylene bisimide core alters the mode of fibrillar growth, leading to isotropic crystallization.

  16. Ortho- and para-hydrogen in dense clouds, protoplanets, and planetary atmospheres

    NASA Technical Reports Server (NTRS)

    Decampli, W. M.; Cameron, A. G. W.; Bodenheimer, P.; Black, D. C.

    1978-01-01

    If ortho- and para-hydrogen achieve a thermal ratio on dynamical time scales in a molecular hydrogen cloud, then the specific heat is high enough in the temperature range 35-70 K to possibly induce hydrodynamic collapse. The ortho-para ratio in many interstellar cloud fragments is expected to meet this condition. The same may have been true for the primitive solar nebula. Detailed hydrodynamic and hydrostatic calculations are presented that show the effects of the assumed ortho-para ratio on the evolution of Jupiter during its protoplanetary phase. Some possible consequences of a thermalized ortho-para ratio in the atmospheres of the giant planets are also discussed.

  17. Quantum rotation of ortho and para-water encapsulated in a fullerene cage.

    PubMed

    Beduz, Carlo; Carravetta, Marina; Chen, Judy Y-C; Concistrè, Maria; Denning, Mark; Frunzi, Michael; Horsewill, Anthony J; Johannessen, Ole G; Lawler, Ronald; Lei, Xuegong; Levitt, Malcolm H; Li, Yongjun; Mamone, Salvatore; Murata, Yasujiro; Nagel, Urmas; Nishida, Tomoko; Ollivier, Jacques; Rols, Stéphane; Rõõm, Toomas; Sarkar, Riddhiman; Turro, Nicholas J; Yang, Yifeng

    2012-08-07

    Inelastic neutron scattering, far-infrared spectroscopy, and cryogenic nuclear magnetic resonance are used to investigate the quantized rotation and ortho-para conversion of single water molecules trapped inside closed fullerene cages. The existence of metastable ortho-water molecules is demonstrated, and the interconversion of ortho-and para-water spin isomers is tracked in real time. Our investigation reveals that the ground state of encapsulated ortho water has a lifted degeneracy, associated with symmetry-breaking of the water environment.

  18. Proteomics as a Quality Control Tool of Pharmaceutical Probiotic Bacterial Lysate Products

    PubMed Central

    Klein, Günter; Schanstra, Joost P.; Hoffmann, Janosch; Mischak, Harald; Siwy, Justyna; Zimmermann, Kurt

    2013-01-01

    Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots. PMID

  19. Influence of fine structure of lipid A on Limulus amebocyte lysate clotting and toxic activities.

    PubMed Central

    Takayama, K; Qureshi, N; Raetz, C R; Ribi, E; Peterson, J; Cantrell, J L; Pearson, F C; Wiggins, J; Johnson, A G

    1984-01-01

    We examined the relationship between the fine structure of lipid A and the toxicity of endotoxin or lipopolysaccharides as measured by the Limulus amebocyte lysate (LAL), rabbit pyrogenicity, chicken embryo lethal dose, and dermal Shwartzman reaction tests. Lipid A and lipid A-like compounds obtained from deep-rough mutants of Salmonella spp. and Escherichia coli had a wide range of structural variations. These compounds included native lipopolysaccharides, diphosphoryl and monophosphoryl lipid A's, and lipid X (a monosaccharide). The LAL test was positive for all lipids tested with lysates from Travenol Laboratories and from Associates of Cape Cod (2.9 X 10(3) to 2.6 X 10(7) endotoxin units per mg), except for O-deacylated and dephosphorylated lipid X, which were negative. The Mallinckrodt lysate gave negative tests for lipid X. In the rabbit pyrogenicity and chicken embryo lethal dose tests, only native lipopolysaccharide and diphosphoryl lipid A's were judged toxic. The Shwartzman reaction was positive for a specific purified diphosphoryl lipid A (thin-layer chromatography-3 fraction) but negative for the purified monophosphoryl lipid A (also a thin-layer chromatography-3 fraction). These results show that the LAL test is not a valid measure of all parameters of toxicity of a lipid A or lipid A-like compound and can yield false-positive results. However, these findings are not in conflict with the widespread use of the LAL assay for pyrogens in the pharmaceutical industry since a good correlation exists between LAL results and pyrogenicity when undegraded endotoxin is evaluated in parallel assays. Images PMID:6378795

  20. Ambiguities in applying traditional Limulus Amoebocyte Lysate tests to quantify endotoxin in nanoparticle formulations

    PubMed Central

    Dobrovolskaia, Marina A; Neun, Barry W; Clogston, Jeffrey D; Ding, Hui; Ljubimova, Julia; McNeil, Scott E

    2010-01-01

    Nanotechnology is finding increasing application in biology and medicine. As with other pharmaceutical formulations and medical devices intended for use in animals and human patients, contamination of nanoparticles with bacterial endotoxins should be thoroughly investigated before preclinical in vitro and in vivo characterization. Traditional methods to study endotoxin contamination include the in vitro quantitative limulus amoebocyte lysate test and the in vivo qualitative rabbit pyrogen test. Both of these tests have long history of use for traditional pharmaceuticals and medical devices and are routinely used in drug development. Here we report that nanoparticles often interfere with these traditional endotoxin detection tests and suggest approaches to detect and overcome such interferences. PMID:20528451

  1. Heat inactivation of a norovirus surrogate in cell culture lysate, abalone meat, and abalone viscera.

    PubMed

    Park, Shin Young; Bae, San-Cheong; Ha, Sang-Do

    2015-03-01

    The current study examined the effects of temperature and heat treatment duration on murine norovirus-1 (MNV-1) from both viral cell culture lysate (7-8 log10 PFU) and experimentally contaminated abalone meat and viscera (5-6 log10 PFU) as a model of human norovirus (NoV). MNV-1 titers in cell culture lysate, abalone meat, and abalone viscera were gradually reduced to 1.93-4.55, 1.79-3.00, and 2.26-3.26 log10 PFU/ml, respectively, after treatment at 70 °C for 1-10 min. Treatment at 85 °C for 1-5 min gradually reduced MNV-1 titers in abalone meat to 2.71-4.15 log10 PFU/ml. MNV-1 titers in abalone viscera were gradually reduced to 2.91-3.46 log10 PFU/ml after treatment at 85 °C for 1-3 min. No significant difference (P > 0.05) was found in MNV-1 titers in the abalone meat and viscera among treatment groups (70 °C for 5 min, 70 °C for 3 min, and 85 °C for 1 min). Complete inactivation of MNV-1 in cell culture lysate was determined at 85 °C for ≥1 min and 100 °C for ≥0.5 min. Complete inactivation of MNV-1 in abalone was determined at 100 °C for ≥0.5 min for meat, and 85 °C for 5 min and 100 °C for ≥0.5 min for viscera. At treatments at 70 °C, the Td-values (3 log reduction time) were significantly lower (P < 0.05) in the cell culture lysate (3.38) than for the abalone meat (6.07) and viscera (10.73). Td = 3 values were not significantly different (P > 0.05) between abalone meat (1.78) and abalone viscera (1.33) at treatments at 85 °C. This study suggests that 100 °C for ≥0.5 min could potentially be used to inactivate NoV in molluscan shellfishes, including viscera.

  2. Rhodium-Catalyzed Atroposelective [2 + 2 + 2] Cycloaddition of Ortho-Substituted Phenyl Diynes with Nitriles: Effect of Ortho Substituents on Regio- and Enantioselectivity.

    PubMed

    Kashima, Kenichi; Teraoka, Kota; Uekusa, Hidehiro; Shibata, Yu; Tanaka, Ken

    2016-05-06

    Axially chiral 3-(2-halophenyl)pyridines were successfully synthesized in high yields with excellent enantioselectivity by the cationic rhodium(I)/(S)-H8-BINAP complex-catalyzed atroposelective [2 + 2 + 2] cycloaddition of (o-halophenyl)diynes with nitriles. Interestingly, regio- and enantioselectivity highly depend on ortho substituents on the phenyl group of diynes. When the ortho substituents were methoxy and methoxycarbonyl groups, axially chiral 3-arylpyridines were obtained as a major product, while enantioselectivity was lowered significantly. On the other hand, when the ortho substituents were alkyl groups, regioselectivity was switched to give achiral 6-arylpyridines in high yields.

  3. The antigens - Volume VII

    SciTech Connect

    Sela, M. )

    1987-01-01

    This book contains four chapters. They are: Ir Genes: Antigen-Specific Genetic Regulation of the Immune Response; Molecular Genetics of Class II (Ia) Antigens; Antigen-Specific T Cell Clones and T Cell Factors; and Infection and Autoimmunity.

  4. Expression of the ’Bacillus anthracis’ Protective Antigen Gene by Baculovirus and Vaccinia Virus Recombinants

    DTIC Science & Technology

    1990-02-01

    ANTIIRACIS PROTI-CTIVE ANTIGEN 367 in medium containing 10% fetal bovine serum. Vaccinia nitrocellulose filters . All cell lysate samples subjected...mM glycine. and 20% methanol at 4°C for 12 to 16 h. by CaCl. precipitation. A. cali irni’, nuclear polyhedrosis Nitrocellulose filters were washed in...and monoclonal antibodies used for these pl[XTJSCRilrr(Stratagcnc. La Jolla. Calif.I. pI3LSCRPPA studies. Filters were washed and incubated with

  5. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

    PubMed

    Bosschem, Iris; Bayry, Jagadeesh; De Bruyne, Ellen; Van Deun, Kim; Smet, Annemieke; Vercauteren, Griet; Ducatelle, Richard; Haesebrouck, Freddy; Flahou, Bram

    2015-01-01

    Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

  6. Stimulation of nitric oxide, cytokine and prostaglandin production by low-molecular weight fractions of probiotic Lactobacillus casei lysate.

    PubMed

    Kmonickova, Eva; Kverka, Miloslav; Tlaskalová-Hogenová, Helena; Kostecka, Petra; Zidek, Zdenek

    2012-01-01

    Major medical indications of probiotic bacteria are conditions associated with the gastrointestinal tract. They exhibit not only the local but also systemic effects, the molecular mechanisms of which are poorly understood. We hypothesized that the action at remote sites of the body could be at least partially attributed to substances of the low molecular mass released from digested bacteria and able to cross the intestinal barrier. The aim of the study was the analysis of immunobiological properties of bacterial lysates and characterization of chemical constituents participating on this mode of action. Lactobacillus casei probiotic strain DN-114001 was employed. Lysates were prepared by passing bacteria through a French press (1500 psi) followed by lyophilisation. The fractions were prepared by the microfiltration of the crude lysate using the 3-, 10-, 30-, 50-, and 100-kDa cutoff filters (Amicon® Ultra 0.5 ml, Millipore Corp.). This procedure completely removes biologically active bacterial macromolecules such as peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS). Effects of microfiltrates on the in vitro production of nitric oxide (NO), cytokines, and prostaglandin E2 (PGE2) were investigated in rat peritoneal cells. The original crude lysate (≤10 µg/ml) activated the biosynthesis of NO, PGE2, and secretion of cytokines. The amount of the lysate needed for the preparation of microfiltered fractions exhibiting immunostimulatory effects was 10-fold higher (100 µg/ml). The molecules with the molecular mass ≤3 kDa were responsible for approximately 45% and 83% of the NO- and PGE2-enhancing activities of the crude lysate, respectively. The microfiltered fractions of the lysate also enhanced secretion of interleukin-6 and tumor necrosis factor-α but not that of interleukin-10 and interferon-γ. The Lactobacillus casei probiotic strain DN-114001 contains low molecular mass (≤3 kDa) molecules possessing immunostimulatory properties. Their

  7. Ortho-Para Mixing Hyperfine Interaction in the H2O+ Ion and Nuclear Spin Equilibration

    NASA Astrophysics Data System (ADS)

    Tanaka, Keiichi; Harada, Kensuke; Oka, Takeshi

    2013-10-01

    The ortho to para conversion of water ion, H2O+, due to the interaction between the magnetic moments of the unpaired electron and protons has been theoretically studied to calculate the spontaneous emission lifetime between the ortho- and para-levels. The electron spin-nuclear spin interaction term, Tab(Sa-Ib + Sb-Ia) mixes ortho (I = 1) and para (I = 0) levels to cause the -forbidden- ortho to para |-I| = 1 transition. The mixing term with Tab = 72.0 MHz is 4 orders of magnitude higher for H2O+ than for its neutral counterpart H2O where the magnetic field interacting with proton spins is by molecular rotation rather than the free electron. The resultant 108 increase of ortho to para conversion rate possibly makes the effect of conversion in H2O+ measurable in laboratories and possibly explains the anomalous ortho to para ratio recently reported by Herschel heterodyne instrument for the far-infrared (HIFI) observation. Results of our calculations show that the ortho - para mixings involving near-degenerate ortho and para levels are high (-10-3), but they tend to occur at high energy levels, -300 K. Because of the rapid spontaneous emission, such high levels are not populated in diffuse clouds unless the radiative temperature of the environment is very high. The low-lying 101 (para) and 111 (ortho) levels of H2O+ are mixed by -10-4 making the spontaneous emission lifetime for the para 101 - ortho 000 transition 520 years and 5200 years depending on the F value of the hyperfine structure. Thus the ortho - para conversion due to the unpaired electron is not likely to seriously affect thermalization of interstellar H2O+ unless either the radiative temperature is very high or number density of the cloud is very low.

  8. Ortho-para mixing hyperfine interaction in the H2O+ ion and nuclear spin equilibration.

    PubMed

    Tanaka, Keiichi; Harada, Kensuke; Oka, Takeshi

    2013-10-03

    The ortho to para conversion of water ion, H2O(+), due to the interaction between the magnetic moments of the unpaired electron and protons has been theoretically studied to calculate the spontaneous emission lifetime between the ortho- and para-levels. The electron spin-nuclear spin interaction term, Tab(SaΔIb + SbΔIa) mixes ortho (I = 1) and para (I = 0) levels to cause the "forbidden" ortho to para |ΔI| = 1 transition. The mixing term with Tab = 72.0 MHz is 4 orders of magnitude higher for H2O(+) than for its neutral counterpart H2O where the magnetic field interacting with proton spins is by molecular rotation rather than the free electron. The resultant 10(8) increase of ortho to para conversion rate possibly makes the effect of conversion in H2O(+) measurable in laboratories and possibly explains the anomalous ortho to para ratio recently reported by Herschel heterodyne instrument for the far-infrared (HIFI) observation. Results of our calculations show that the ortho ↔ para mixings involving near-degenerate ortho and para levels are high (∼10(-3)), but they tend to occur at high energy levels, ∼300 K. Because of the rapid spontaneous emission, such high levels are not populated in diffuse clouds unless the radiative temperature of the environment is very high. The low-lying 101 (para) and 111 (ortho) levels of H2O(+) are mixed by ∼10(-4) making the spontaneous emission lifetime for the para 101 → ortho 000 transition 520 years and 5200 years depending on the F value of the hyperfine structure. Thus the ortho ↔ para conversion due to the unpaired electron is not likely to seriously affect thermalization of interstellar H2O(+) unless either the radiative temperature is very high or number density of the cloud is very low.

  9. A compact L-band Ortho Mode Junction

    NASA Astrophysics Data System (ADS)

    Pisanu, T.; Marongiu, P.; Navarrini, A.; Valente, G.

    2010-07-01

    We describe the design construction and performance of a L-band (1300-1800 MHz) Ortho Mode Junction for the L-P dual-band receiver to be installed on the 64 m Sardinia Radio Telescope (SRT), a new radio telescope which is being built in Sardinia, Italy. The Ortho Mode Junction (OMJ) separates two orthogonal linearly polarized signals propagating in a 172 mm diameter circular waveguide and couple them into four coaxial outputs. The OMJ is part of an OMT (Ortho Mode Transducer), which includes two 1800 hybrids allowing to recombine the out-of-phase signals from the balanced OMJ outputs. The OMJ consists of four probes arranged in symmetrical configuration across the circular waveguide. A shaped tuning stub with cylindrical profile is placed a quarter wavelength away from the probes to guarantee broadband operation with low reflection coefficient across L-band. The four identical probes have a cylindrical structure, each consisting of three concentric cylinders that attach to the central pin of standard 50 Ω 7/16-type coaxial connectors. The OMJ will be cooled at 80 K inside a compact dewar together with directional couplers and Low Noise Amplifiers. The two linearly polarized signals from an input 190 mm diameter room temperature L-band feed couple into the cryogenic dewar through a vacuum window located across the waveguide. Inside the dewar, the 190 mm diameter circular waveguide is tapered down to 172 mm using a conical transition (length 85 mm) filled with a Styrodur® foam that provides mechanical support for a 0.125 mm thick Kapton vacuum barrier. A 0.6 mm air gap across the 172 mm circular waveguide provides thermal decoupling between the ambient temperature and the 80 K OMJ, which is connected to the conical transition output.

  10. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study.

    PubMed

    Fea, Antonio M; Aragno, Vittoria; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

  11. Long-term effect of platelet lysate on primary fibroblasts highlighted with a proteomic approach.

    PubMed

    Cipriani, Valentina; Ranzato, Elia; Balbo, Valeria; Mazzucco, Laura; Cavaletto, Maria; Patrone, Mauro

    2009-10-01

    The use of platelets and platelet derivatives has acquired clinical relevance as a means of accelerating wound healing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances able to regulate cellular activities. The purpose of this study was to evaluate the biological effects of platelet lysate (PL) on human primary skin fibroblasts. We studied cell viability, MAPK signalling and proteomic profile of fibroblasts exposed to a platelet lysate (PL) obtained from blood sample. Crystal violet and neutral red uptake assays showed the dose-response effects of PL on cell viability and metabolism at 3 and 6 days of exposure. Western blot demonstrated a more sustained activation of p38 than of ERK1/2. A proteomic approach was applied to identify soluble cellular components in primary fibroblasts that are differentially expressed in response to PL exposure. Protein identification was performed by mass spectrometry. The data demonstrate that human fibroblasts respond to PL exposure by modifying a number of proteins, related principally to stress response, metabolism and the cytoskeleton.

  12. Thermosensitive eyedrops containing platelet lysate for the treatment of corneal ulcers.

    PubMed

    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Ferrari, Franca; Mori, Michela; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla

    2012-04-15

    Corneal lesions cause significant pain and visual impairment and, in many cases, are unresponsive to conventional treatments. Platelet lysate (PL) is an haemoderivative rich in growth factors (GFs) that are released by platelets after freeze-thawing destruction of platelet rich plasma (PRP). The aim of the present work was to develop thermosensitive and mucoadhesive eyedrops to maintain and prolong the contact of platelet lysate (PL) with cornea ulcers. A sterile vehicle based on chondroitin sulphate sodium (CS) and hydroxypropylmethyl cellulose (HPMC) was developed. An extemporaneous loading of the vehicle with PL was performed and the obtained formulation was able to quickly thermogelify at about 32 °C and was characterized by good mucoadhesive properties. ELISA evidenced that the growth factor PDGF AB was compatible with the vehicle and stable in the formulation up to 15 days of storage at 2-8 °C. In vitro wound healing and proliferation test (performed using rabbit corneal epithelial cells (RCE)) showed that the formulation enhanced cell growth and put in evidence a synergistic effect of CS and PL in stimulating cell proliferation. The overall results indicate that PL loaded in thermosensitive and mucoadhesive eyedrops can be profitably employed to treat corneal lesions.

  13. A3R Phage and Staphylococcus aureus Lysate Do Not Induce Neutrophil Degranulation

    PubMed Central

    Borysowski, Jan; Międzybrodzki, Ryszard; Wierzbicki, Piotr; Kłosowska, Danuta; Korczak-Kowalska, Grażyna; Weber-Dąbrowska, Beata; Górski, Andrzej

    2017-01-01

    The objective of this study was to evaluate the effects of A3R phage and Staphylococcus aureus lysate obtained after phage infection on neutrophil degranulation. The exocytosis of primary and secondary granules from neutrophils was investigated in vitro in whole blood specimens by flow cytometry based on the expression of specific markers of exocytosis (CD63 for primary granules and CD66b for secondary granules). We found that both A3R and S. aureus lysate had no significant effect on the exocytosis of primary and secondary granules. These data suggest that neither A3R virions nor any products of phage-induced lysis of S. aureus are likely to induce neutrophil degranulation in patients who are treated with phage preparations. Since neutrophil granules contain some potentially toxic proteins, our results provide an important argument for the safety of phage therapy. Moreover, these data indicate that the induction of neutrophil degranulation is not likely to contribute to antibacterial effects of phages. PMID:28230780

  14. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

    PubMed Central

    Fea, Antonio M.; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M.

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications. PMID:27200376

  15. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

  16. Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

    PubMed Central

    Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

    2016-01-01

    Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

  17. In vivo effect of an immunostimulating bacterial lysate on human B lymphocytes.

    PubMed

    Lanzilli, G; Falchetti, R; Cottarelli, A; Macchi, A; Ungheri, D; Fuggetta, M P

    2006-01-01

    The aim of the present study is to investigate in humans the mechanism by which the oral vaccine Polyvalent Mechanical Bacterial Lysate (PMBL) can rapidly mobilize specific immune response and evaluate the efficacy of its immunostimulating activity in preventing recurrent infections of the upper respiratory tract (URTIs) in a group of patients with a medical history of URTI recurrence. Patients received, by sublingual route, PBML, an immunostimulating lysate obtained by mechanical lysis of the most common bacteria responsible for upper respiratory tract infections. The treatment was administered for 10 consecutive days/month for 3 consecutive months. After the end of the treatment period the patients were followed up for an additional 3 months. The frequency of IgM memory B cells and the expression of the activation marker CD25 in peripheral blood lymphocytes were measured using the flow cytometric method before the start and at days 30 and 90 of the treatment cycle. To correlate clinical results to immunological parameters, the patients were monitored at different time-points during the treatment and at the end of follow-up period. The results showed that PMBL exerts a therapeutic and preventing effect in acute and recurrent infections of the upper respiratory tract and that this effect correlated with the activation and enhancement of both IgM memory B lymphocytes (CD24+/CD27+ cells) and IL2 receptor-expressing lymphocytes (CD25+ cells) involved either in humoral or cellular immunity.

  18. Ortho stops marketing Lippes Loop; cites economic factors.

    PubMed

    1985-11-01

    Ortho Pharmaceutical Corporation has stopped marketing the Lippes Loop IUD, the only inert IUD currently available in the US. The firm cited "economic considerations" as its reason. Linda Organ, company spokeswoman, told Contraceptive Technology Update (CTU) that the number of women using IUDs has declined in the past few years and, as a result, Ortho's Lippes Loop sales dropped. Most physicians, according to Organ, currently prescribe copper-bearing IUDs. Few devices have been studied as thoroughly before marketing as the Lippes Loop, according to its developer, Dr. Jack Lippes. Lippes told CTU that the Population Council analyzed 40,000 women from 1962 to 1968 and "found no trouble with the Loop." Lippes attributes Ortho's recent decision to 2 factors: the IUD has been only "marginally profitable" and the problems of A.H. Robins with the Dalkon Shield has most likely had an effect; and the US Food and Drug Administration (FDA) published a proposed rule in August 1985 that would require any company wanting to manufacture and market IUDs like the Lippes Loop to submit a premarketing approval application to that agency. In effect, the FDA's rule would only apply to the Lippes Loop. Under the proposed rule, any company wanting to market Lippes Loops, or any nondrug IUD, would have to submit an application to the FDA with a detailed discussion and supporting clinical studies addressing the following concerns: pelvic actinomycosis; tubal infertility; duration that the IUD should remain in situ; and safety of leaving the IUD in situ when contraception is no longer indicated. According to Lillian Yin, FDA device evaluation, the clinical effectiveness and most of the safety issues regarding inert IUDs have been thoroughly covered in published data. She told CTU that "most of the information needed is straightforward, but the part that's new involves the long term use infection rate." Yin indicated that the FDA received a letter from Ortho advising the agency of the

  19. Approximate analysis of the ortho-wedgetron effect

    NASA Astrophysics Data System (ADS)

    Alekseev, G. A.; Pivovarova, A. G.

    The ortho-wedgetron was observed and named by Kirichenko and Solodovnik (1981) in a study of the effect of the entry angle of the electron beam on the starting current of a traveling wave tube. In the present work, this effect is analyzed theoretically in the case of collective interaction, taking into account the three-dimensionality of the motion of the electrons along helical trajectories in the steady-state regime. This analysis is carried out in the framework of the kinematic approach with application to an O-type resonance oscillator with an infinitely thin electron beam.

  20. A microarray method for identifying tumor antigens by screening a tumor cDNA expression library against cancer sera

    PubMed Central

    Whittemore, Kurt; Sykes, Kathryn

    2013-01-01

    The immune system responds to tumor cells. The challenge has been how to effectively use these responses to treat or protect against cancer. Toward the goal of developing a cancer vaccine, we are pursuing methodologies for the discovery and testing of useful antigens. We present an array-based approach for discovering these B cell antigens by directly screening for specific host-sera reactivity to lysates from tumor-derived cDNA expression libraries. Several cancer-specific antigens were identified, and these are currently being validated as potential candidates. PMID:23851590

  1. A microarray method for identifying tumor antigens by screening a tumor cDNA expression library against cancer sera.

    PubMed

    Whittemore, Kurt; Sykes, Kathryn

    2013-10-01

    The immune system responds to tumor cells. The challenge has been how to effectively use these responses to treat or protect against cancer. Toward the goal of developing a cancer vaccine, we are pursuing methodologies for the discovery and testing of useful antigens. We present an array-based approach for discovering these B cell antigens by directly screening for specific host-sera reactivity to lysates from tumor-derived cDNA expression libraries. Several cancer-specific antigens were identified, and these are currently being validated as potential candidates.

  2. Ortho-eugenol exhibits anti-nociceptive and anti-inflammatory activities.

    PubMed

    Fonsêca, Diogo V; Salgado, Paula R R; Aragão Neto, Humberto de C; Golzio, Adriana M F O; Caldas Filho, Marcelo R D; Melo, Cynthia G F; Leite, Fagner C; Piuvezam, Marcia R; Pordeus, Liana Clébia de Morais; Barbosa Filho, José M; Almeida, Reinaldo N

    2016-09-01

    Ortho-eugenol is a much used phenylpropanoid whose ability to reduce pain and inflammation has never been studied. Researching ortho-eugenol's antinociceptive and anti-inflammatory activity, and its possible mechanisms of action is therefore of interest. The administration of vehicle, ortho-eugenol (50, 75 and 100mg/kg i.p.), morphine (6mg/kg, i.p.) or dexamethasone (2mg/kg, s.c.) occurred 30min before the completion of pharmacological tests. Pretreatment with ortho-eugenol did not change motor coordination test results, but reduced the number of writhes and licking times in the writhing test and glutamate test, respectively. The reaction time from thermal stimulus was significantly increased in the hot plate test after administration of ortho-eugenol. Treatment with yohimbine reversed the antinociceptive effect of ortho-eugenol, suggesting involvement of the adrenergic system. In anti-inflammatory tests, ortho-eugenol inhibited acetic acid induced vascular permeability and leukocyte migration, reducing TNF-α and IL-1β by virtue of its suppression of NF-κB and p38 phosphorylated forms in the peritonitis test. From these results, ortho-eugenol antinociceptive effects mediated by the adrenergic system and anti-inflammatory activity through regulation of proinflammatory cytokines and phosphorylation of NF-kB and p38 become evident for the first time.

  3. Conventional tube and microplate Limulus amoebocyte lysate procedures for determination of gram-negative bacteria in milk.

    PubMed

    May, S A; Mikolajcik, E M; Richter, E R

    1989-05-01

    A comparison was made of the conventional tube and microplate Limulus amoebocyte lysate assay for detection of gram-negative bacterial lipopolysaccharide in milk. Raw whole milk samples were maintained frozen and portions were examined periodically on alternate days during 13-d storage to evaluate the reproducibility of both Limulus amoebocyte lysate procedures and to determine optimum reaction conditions for the microplate method. One-day-old, raw and locally purchased pasteurized milk samples, held at 7 degrees C, were analyzed during storage to establish the correlation of both procedures with aerobic and modified psychrotrophic plate counts. Vitamin- and mineral-fortified dairy-based products were examined using the microplate Limulus amoebocyte lysate test as a potential indicator of raw material or finished product bacterial quality and possible postprocessing contamination. Statistical analysis of the data collected comparing the conventional tube and the microplate Limulus amoebocyte lysate assay demonstrated no significant difference exists between the methods when either the modified psychrotrophic bacterial count or the aerobic plate count was used to determine gram-negative bacteria in pasteurized or raw milk (P less than .91). The microplate method, which uses half the lysate reagent, was a good indicator of the bacterial quality of milk and fortified dairy products, consistently detecting bacterial levels greater than 10(3) to 10(4)/ml.

  4. Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates.

    PubMed

    Guidon, P T; Perrin, D; Harrison, P

    1996-02-01

    We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.

  5. Protodeboronation of ortho- and para-phenol boronic acids and application to ortho and meta functionalization of phenols using boronic acids as blocking and directing groups.

    PubMed

    Lee, Chun-Young; Ahn, Su-Jin; Cheon, Cheol-Hong

    2013-12-06

    The first metal-free thermal protodeboronation of ortho- and para-phenol boronic acids in DMSO was developed. The protodeboronation was successfully applied to the synthesis of ortho- and meta-functionalized phenols using the boronic acid moiety as a blocking group and a directing group, respectively. Mechanistic studies suggested that this protodeboronation proceeds through the coordination of water to the boron atom followed by σ-bond metathesis.

  6. Surface-Controlled Mono/Diselective ortho C-H Bond Activation.

    PubMed

    Li, Qing; Yang, Biao; Lin, Haiping; Aghdassi, Nabi; Miao, Kangjian; Zhang, Junjie; Zhang, Haiming; Li, Youyong; Duhm, Steffen; Fan, Jian; Chi, Lifeng

    2016-03-02

    One of the most charming and challenging topics in organic chemistry is the selective C-H bond activation. The difficulty arises not only from the relatively large bond-dissociation enthalpy, but also from the poor reaction selectivity. In this work, Au(111) and Ag(111) surfaces were used to address ortho C-H functionalization and ortho-ortho couplings of phenol derivatives. More importantly, the competition between dehydrogenation and deoxygenation drove the diversity of reaction pathways of phenols on surfaces, that is, diselective ortho C-H bond activation on Au(111) surfaces and monoselective ortho C-H bond activation on Ag(111) surfaces. The mechanism of this unprecedented phenomenon was extensively explored by scanning tunneling microscopy, density function theory, and X-ray photoelectron spectroscopy. Our findings provide new pathways for surface-assisted organic synthesis via the mono/diselective C-H bond activation.

  7. Analytical characterization of four new ortho-methoxybenzylated amphetamine-type designer drugs.

    PubMed

    Westphal, Folker; Girreser, Ulrich; Waldmüller, Delia

    2016-09-01

    In a seizure of German custom authorities four N-(ortho-methoxybenzyl)amines with amphetamine partial structure were obtained as pure compounds: N-(ortho-methoxybenzyl)-3,4-dimethoxyamphetamine (3,4-DMA-NBOMe (1)), N-(ortho-methoxybenzyl)-4-ethylamphetamine (4-EA-NBOMe (2)), N-(ortho-methoxybenzyl)-4-methylmethamphetamine (4-MMA-NBOMe (3)), and N-(ortho-methoxybenzyl)-5-(2-aminopropyl)benzofuran (5-APB-NBOMe (4)). The compounds have been detected in Germany for the first time and no analytical data had been previously published. Mass spectrometric (MS), infrared (IR) spectroscopic, and nuclear magnetic resonance (NMR) spectroscopic data are presented. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Solvent-dependent intramolecular charge transfer dual fluorescence of p-dimethylaminobenzanilide bearing steric ortho,ortho-dimethyl substituents at amido aniline.

    PubMed

    Zhang, Xuan; Jiang, Yun-Bao

    2011-11-01

    Intramolecular charge transfer (ICT) dual fluorescence was observed in various organic solvents with p-dimethylaminobenzanilide (DMBA) derivatives bearing ortho-methyl (DMOMBA) and ortho,ortho-dimethyl (DMDMBA) substituents at amido aniline moiety. Ab initio calculation and absorption spectral data indicated that high steric hindrance was introduced by the ortho,ortho-dimethyl substitutions. It was found that, with DMDMBA, the CT emission initially shifted to the red with increasing solvent polarity from cyclohexane (CHX, 480 nm) to diethyl ether (DEE, 520 nm), similar to those of DMBA derivatives with the ortho-, meta- or para-methyl substitutions at amido aniline moiety. However, there is a characteristic blue-shift of the long wavelength emission between DEE and tetrahydrofuran (THF, 424 nm) then a bathochromic shift again in highly polar solvent acetonitrile (ACN, 484 nm). The unusual solvent-dependent CT emission was ascribed to two competitive CT channels. One is benzanilide (BA)-like CT, whose CT reaction occurs from amido aniline to benzoyl moiety in nonpolar solvent CHX and DEE; the other one is p-dimethylaminobenzamide (DMABA)-like, whose CT reaction occurs from dimethylamino to benzanilide moiety in highly polar solvent THF and ACN. These findings revealed the steric effect plays an important role in the ICT process, which may alter the properties of the electron donor and/or acceptor, but also change the reaction potential.

  9. Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

    PubMed

    Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

    2017-03-31

    We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

  10. Adam-Gibbs model for the supercooled dynamics in the ortho-terphenyl ortho-phenylphenol mixture

    NASA Astrophysics Data System (ADS)

    Roland, C. M.; Capaccioli, S.; Lucchesi, M.; Casalini, R.

    2004-06-01

    Dielectric measurements of the α-relaxation time were carried out on a mixture of ortho-terphenyl (OTP) with ortho-phenylphenol, over a range of temperatures at two pressures, 0.1 and 28.8 MPa. These are the same conditions for which heat capacity, thermal expansivity, and compressibility measurements were reported by Takahara et al. [S. Takahara, M. Ishikawa, O. Yamamuro, and T. Matsuo, J. Phys. Chem. B 103, 3288 (1999)] for the same mixture. From the combined dynamic and thermodynamic data, we determine that density and temperature govern to an equivalent degree the variation of the relaxation times with temperature. Over the measured range, the dependence of the relaxation times on configurational entropy is in accord with the Adam-Gibbs model, and this dependence is invariant to pressure. Consistent with the implied connection between relaxation and thermodynamic properties, the kinetic and thermodynamic fragilities are found to have the same pressure independence. In comparing the relaxation properties of the mixture to those of neat OTP, density effects are stronger in the former, perhaps suggestive of less efficient packing.

  11. Adam-Gibbs model for the supercooled dynamics in the ortho-terphenyl ortho-phenylphenol mixture.

    PubMed

    Roland, C M; Capaccioli, S; Lucchesi, M; Casalini, R

    2004-06-08

    Dielectric measurements of the alpha-relaxation time were carried out on a mixture of ortho-terphenyl (OTP) with ortho-phenylphenol, over a range of temperatures at two pressures, 0.1 and 28.8 MPa. These are the same conditions for which heat capacity, thermal expansivity, and compressibility measurements were reported by Takahara et al. [S. Takahara, M. Ishikawa, O. Yamamuro, and T. Matsuo, J. Phys. Chem. B 103, 3288 (1999)] for the same mixture. From the combined dynamic and thermodynamic data, we determine that density and temperature govern to an equivalent degree the variation of the relaxation times with temperature. Over the measured range, the dependence of the relaxation times on configurational entropy is in accord with the Adam-Gibbs model, and this dependence is invariant to pressure. Consistent with the implied connection between relaxation and thermodynamic properties, the kinetic and thermodynamic fragilities are found to have the same pressure independence. In comparing the relaxation properties of the mixture to those of neat OTP, density effects are stronger in the former, perhaps suggestive of less efficient packing.

  12. Preparation of pooled human platelet lysate (pHPL) as an efficient supplement for animal serum-free human stem cell cultures.

    PubMed

    Schallmoser, Katharina; Strunk, Dirk

    2009-10-30

    Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo(1,2) and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin(3-5) or lysed by freeze/thaw cycles(6-14) before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.(1,15-17) Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors(18,19) and a current lack of standardized protocols for platelet preparation.(15,16) This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

  13. ELISA-Based Crossmatching Allowing the Detection of Emerging Donor-Specific Anti-HLA Antibodies through the Use of Stored Donors' Cell Lysates

    PubMed Central

    Schlaf, G.; Stöhr, K.; Rothhoff, A.; Altermann, W.

    2015-01-01

    About forty years ago the complement-dependent crossmatch assay (CDC-CM) was developed as standard procedure in order to select recipients without donor-specific antibodies directed against human leukocyte antigens of their given donors since the negative outcome of pretransplant crossmatching represents one of the most important requirements for a successful kidney graft survival. However, as a functional assay the CDC-CM strongly depends on the availability of donors' isolated lymphocytes and in particular on their vitality highly limiting its applicability for recipients treated with special drugs and therapeutic antibodies or suffering from underlying autoimmune diseases. In the great majority of these cases ELISA-based crossmatching has been demonstrated to be an adequate alternative procedure nevertheless leading to valid results. With these case reports we show for the first time that ELISA-based crossmatching is suitable to demonstrate the upcoming donor-specific anti-HLA antibodies as a consequence of allografting using deep-frozen deceased donor's material such as blood or spleen detergent lysate. Thus, this ELISA-based procedure first provides the option to routinely perform crossmatching using stored material of deceased donors in order to substitute or at least to complement virtual crossmatching, that is, the comparison of the recipients' anti-HLA antibody specificities with the donors' historically identified HLA types. PMID:26634169

  14. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications

    PubMed Central

    Fortunato, Tiago M.; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A.; Pula, Giordano

    2016-01-01

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. PMID:27141997

  15. Routine limulus amoebocyte lysate (LAL) test for endotoxin determination in milk using a Toxinometer ET-201.

    PubMed

    Mottar, J; De Block, J; Merchiers, M; Vantomme, K; Moermans, R

    1993-05-01

    A rapid method of performing the Limulus amoebocyte lysate (LAL) test in milk is proposed using the Toxinometer ET-201. This instrument measured the increase in turbidity due to the interaction between the endotoxins of the Gram-negative bacteria and the LAL reagent, monitored the ratio Rt of the sequential to the initial transmission at 12 s intervals and quantified endotoxins by determination of the reaction time Tr required to obtain a 5% decrease in Rt. There was a good correlation between the toxinometrically determined endotoxin concentrations and the number of Gram-negative bacteria (SD, 0.18 log(plate count units)), and the repeatability (CV, 6-10%) was high. The assay may be useful for screening raw materials for UHT milk production, as the endotoxin content of the raw material is related to the rest proteinase activity in the UHT milk.

  16. The impact of non-endotoxin LAL-reactive materials on Limulus amebocyte lysate analyses.

    PubMed

    Cooper, J F; Weary, M E; Jordan, F T

    1997-01-01

    Limulus amebocyte lysate (LAL) is activated by bacterial endotoxins and certain glucans (beta-D-glucan, LAL-RM). The potential for conflicting inter-laboratory results for LAL tests exists because commercial LAL reagents are highly variable in response to LAL-reactive glucans. The nature of beta-D-glucan activation of LAL and means for rendering LAL non-responsive to glucan are reviewed to provide a background for resolving conflicting data. Kinetic LAL methods are particularly useful for screening materials potentially contaminated with glucan. The presence of beta-D-glucan in parenterals is uncommon and is likely limited to products exposed to microbial or cellulosic materials. A scheme is suggested for identifying LAL-reactive glucans and for LAL release-testing without glucan interference.

  17. Cell-Free Production of Membrane Proteins in Escherichia coli Lysates for Functional and Structural Studies.

    PubMed

    Rues, Ralf-Bernhardt; Henrich, Erik; Boland, Coilin; Caffrey, Martin; Bernhard, Frank

    2016-01-01

    The complexity of membrane protein synthesis is largely reduced in cell-free systems and it results into high success rates of target expression. Protocols for the preparation of bacterial lysates have been optimized in order to ensure reliable efficiencies in membrane protein production that are even sufficient for structural applications. The open accessibility of the semisynthetic cell-free expression reactions allows to adjust membrane protein solubilization conditions according to the optimal folding requirements of individual targets. Two basic strategies will be exemplified. The post-translational solubilization of membrane proteins in detergent micelles is most straightforward for crystallization approaches. The co-translational integration of membrane proteins into preformed nanodiscs will enable their functional characterization in a variety of natural lipid environments.

  18. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

    PubMed

    Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

    2016-05-04

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

  19. Methods for the Measurement of a Bacterial Enzyme Activity in Cell Lysates and Extracts

    PubMed Central

    Mendz, George; Hazell, Stuart

    1998-01-01

    The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of Helicobacter pylori by three diffirent methods. Nuclear magnetic resonance spectroscopy, radioactive tracer analysis, and spectrophotometry were employed in conjunction to identify the properties of the enzyme activity and to validate the results obtained with each assay. NMR spectroscopy was the most direct method to provide proof of ACTase activity; radioactive tracer analysis was the most sensitive technique and a microtitre-based colorimetric assay was the most cost-and time-efficient for large scale analyses. Freeze-thawing was adopted as the preferred method for cell lysis in studying enzyme activity in situ. This study showed the benefits of employing several different complementary methods to investigate bacterial enzyme activity. PMID:12734591

  20. Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate

    PubMed Central

    Sokolova, Ekaterina; Spruijt, Evan; Hansen, Maike M. K.; Dubuc, Emilien; Groen, Joost; Chokkalingam, Venkatachalam; Piruska, Aigars; Heus, Hans A.; Huck, Wilhelm T. S.

    2013-01-01

    Liquid–liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation. PMID:23818642

  1. Binding Rate Screen - a high-throughput assay in soluble lysate for prioritizing protein expression constructs.

    PubMed

    Tian-Yu, Jiamin; Licht, Stuart; Pardee, Gwynn; Bhat, Arun; Cao, Ying; Gao, Wei; Sangalang, Emma; Zaror, Isabel

    2010-04-15

    Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts, as most methods require purified proteins and/or are labor-intensive. Here, we present a novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening expression constructs in crude soluble lysate. This functional screen utilizes the frequently employed hexahistidine (His(6)) tag as a reporter, and measures its binding rate to an affinity matrix as a metric to reflect aggregation, concentration, and purifiability of the target protein. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein as judged by analytical size-exclusion chromatography. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, which is at least 10-100 times faster than any other technologies currently available.

  2. Detecting kinase activities from single cell lysate using concentration-enhanced mobility shift assay.

    PubMed

    Cheow, Lih Feng; Sarkar, Aniruddh; Kolitz, Sarah; Lauffenburger, Douglas; Han, Jongyoon

    2014-08-05

    Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates.

  3. Natural assembly of platelet lysate-loaded nanocarriers into enriched 3D hydrogels for cartilage regeneration.

    PubMed

    Santo, Vítor E; Popa, Elena G; Mano, João F; Gomes, Manuela E; Reis, Rui L

    2015-06-01

    The role of Platelet Lysates (PLs) as a source of growth factors (GFs) and as main element of three-dimensional (3D) hydrogels has been previously described. However, the resulting hydrogels usually suffer from high degree of contraction, limiting their usefulness. This work describes the development of a stable biomimetic 3D hydrogel structure based on PLs, through the spontaneous assembling of a high concentration of chitosan-chondroitin sulfate nanoparticles (CH/CS NPs) with PLs loaded by adsorption. The interactions between the NPs and the lysates resemble the ones observed in the extracellular matrix (ECM) native environment between glycosaminoglycans and ECM proteins. In vitro release studies were carried out focusing on the quantification of PDGF-BB and TGF-β1 GFs. Human adipose derived stem cells (hASCs) were entrapped in these 3D hydrogels and cultured in vitro under chondrogenic stimulus, in order to assess their potential use for cartilage regeneration. Histological, immunohistological and gene expression analysis demonstrated that the PL-assembled constructs entrapping hASCs exhibited results similar to the positive control (hASCS cultured in pellets), concerning the levels of collagen II expression and immunolocalization of collagen type I and II and aggrecan. Moreover, the deposition of new cartilage ECM was detected by alcian blue and safranin-O positive stainings. This work demonstrates the potential of PLs to act simultaneously as a source/carrier of GFs and as a 3D structure of support, through the application of a "bottom-up" approach involving the assembly of NPs, resulting in an enriched construct for cartilage regeneration applications.

  4. Virus production and lysate recycling in different sub-basins of the northern Baltic Sea.

    PubMed

    Holmfeldt, Karin; Titelman, Josefin; Riemann, Lasse

    2010-10-01

    In the Gulf of Bothnia, northern Baltic Sea, a large freshwater inflow creates north-southerly gradients in physico-chemical and biological factors across the two sub-basins, the Bothnian Bay (BB) and the Bothnian Sea. In particular, the sub-basins differ in nutrient limitation (nitrogen vs. phosphorus; P). Since viruses are rich in P, and virus production is commonly connected with bacterial abundance and growth, we hypothesized that the role of viral lysis differs between the sub-basins. Thus, we examined virus production and the potential importance of lysate recycling in surface waters along a transect in the Gulf of Bothnia. Surprisingly, virus production and total P were negatively correlated. In the BB, virus production rates were double those elsewhere in the system, although bacterial abundance and production were the lowest. In the BB, virus-mediated cell lysates could account for 70-180% and 100-250% of the bacterial carbon and P demand, respectively, while only 4-15% and 8-21% at the other stations. Low concentrations of dissolved DNA (D-DNA) with a high proportion of encapsulated DNA (viruses) in the BB suggested rapid turnover and high uptake of free DNA. The correlation of D-DNA and total P indicates that D-DNA is a particularly important nutrient source in the P-limited BB. Our study demonstrates large and counterintuitive differences in virus-mediated recycling of carbon and nutrients in two basins of the Gulf of Bothnia, which differ in microbial community composition and nutrient limitation.

  5. Transcriptome Analysis of Escherichia coli O157:H7 Exposed to Lysates of Lettuce Leaves ▿

    PubMed Central

    Kyle, Jennifer L.; Parker, Craig T.; Goudeau, Danielle; Brandl, Maria T.

    2010-01-01

    Harvesting and processing of leafy greens inherently cause plant tissue damage, creating niches on leaves that human pathogens can exploit. We previously demonstrated that Escherichia coli O157:H7 (EcO157) multiplies more rapidly on shredded leaves than on intact leaves (M. T. Brandl, Appl. Environ. Microbiol. 74:5285-5289, 2008). To investigate how EcO157 cells adapt to physicochemical conditions in injured lettuce tissue, we used microarray-based whole-genome transcriptional profiling to characterize gene expression patterns in EcO157 after 15- and 30-min exposures to romaine lettuce lysates. Multiple carbohydrate transport systems that have a role in the utilization of substrates known to be prevalent in plant cells were activated in EcO157. This indicates the availability to the human pathogen of a variety of carbohydrates released from injured plant cells that may promote its extensive growth in leaf lysates and, thus, in wounded leaf tissue. In addition, microarray analysis revealed the upregulation of numerous genes associated with EcO157 attachment and virulence, with oxidative stress and antimicrobial resistance (including the OxyR and Mar regulons), with detoxification of noxious compounds, and with DNA repair. Upregulation of oxidative stress and antimicrobial resistance genes in EcO157 was confirmed on shredded lettuce by quantitative reverse transcription-PCR. We further demonstrate that this adaptation to stress conditions imparts the pathogen with increased resistance to hydrogen peroxide and calcium hypochlorite. This enhanced resistance to chlorinated sanitizers combined with increased expression of virulence determinants and multiplication at sites of injury on the leaves may help explain the association of processed leafy greens with outbreaks of EcO157. PMID:20061451

  6. Suppression of sulfoconjugation reduces the protective effect of ortho-aminoazotoluene on hepatocarcinogenesis induced by diethylnitrosamine in mice.

    PubMed

    Kaledin, V I; Il'nitskaya, S I; Popova, N A; Bogdanova, L A

    2014-07-01

    The effects of ortho-aminoazotoluene on carcinogenic activity of diethylnitrosamine were studied in CBA and ICR mice. Injection of ortho-aminoazotoluene before and after diethylnitrosamine led to a significant reduction of its anticarcinogenic effect, judging from significantly lower level of liver tumors. Pentachlorophenol, inhibitor of sulfotransferase (catalyzing the terminal stage of ortho-aminoazotoluene metabolic activity), stimulated its carcinogenic effect on mouse liver. On the other hand, pentachlorophenol reduced the protective effect of ortho-aminoazotoluene on diethylnitrosamine-induced hepatocarcinogenesis in mice. Presumably, the carcinogenic and anticarcinogenic effects of ortho-aminoazotoluene were realized by its initial form or intermediate (non-sulfated) metabolites.

  7. Synthesis, structure, and reactivity of N-benzoyl iminophosphoranes ortho lithiated at the benzoyl group.

    PubMed

    Aguilar, David; Fernández, Ignacio; Cuesta, Luciano; Yañez-Rodríguez, Víctor; Soler, Tatiana; Navarro, Rafael; Urriolabeitia, Esteban P; López Ortiz, Fernando

    2010-10-01

    Ortho lithiation of N-benzamido-P,P,P-triaryliminophosphoranes through deprotonation with alkyllithium bases was achieved with ortho-C═O and ortho-P═N chemoselectivity. However, the synthetic scope of these processes was rather limited. Ortho-lithiated N-benzamido-P,P,P-triphenyliminophosphorane 8 was efficiently prepared via lithium/halogen exchange of the corresponding ortho-brominated precursor with s-BuLi in THF at -90 °C. The reaction of 8 with a variety of electrophiles provides an easy and mild method for the regioselective synthesis of ortho-modified iminophosphoranes via C-C (alkylation and hydroxyalkylation) and C-X (X = I, Si, P, Sn, and Hg) bond-forming reactions. NMR characterization of 8 in THF solution showed that 8 exists as an equilibrium mixture of one monomer and two dimers. The Li atoms of these species become members of five-membered rings through chelation by the ortho-metalated carbon and the carbonyl oxygen. The dimers differ in the relative orientation of the two chelates with respect to the plane defined by the C(2)Li(2) core. The equilibrium between all species is established by splitting the dimers into monomers and subsequent recombination with formation of a different dimer.

  8. Measurement of ortho-positronium properties in liquid scintillators

    SciTech Connect

    Perasso, S.; Franco, D.; Tonazzo, A.; Consolati, G.; Hans, S.; Yeh, M.; Jollet, C.; Meregaglia, A.

    2013-08-08

    Pulse shape discrimination in liquid scintillator detectors is a well-established technique for the discrimination of heavy particles from light particles. Nonetheless, it is not efficient in the separation of electrons and positrons, as they give rise to indistinguishable scintillator responses. This inefficiency can be overtaken through the exploitation of the formation of ortho-Positronium (o-Ps), which alters the time profile of light pulses induced by positrons. We characterized the o-Ps properties in the most commonly used liquid scintillators, i.e. PC, PXE, LAB, OIL and PC + PPO. In addition, we studied the effects of scintillator doping on the o-Ps properties for dopants currently used in neutrino experiments, Gd and Nd. Further measurements for Li-loaded and Tl-loaded liquid scintillators are foreseen. We found that the o-Ps properties are suitable for enhancing the electron-positron discrimination.

  9. Measurement of ortho-positronium properties in liquid scintillators

    NASA Astrophysics Data System (ADS)

    Perasso, S.; Consolati, G.; Franco, D.; Hans, S.; Jollet, C.; Meregaglia, A.; Tonazzo, A.; Yeh, M.

    2013-08-01

    Pulse shape discrimination in liquid scintillator detectors is a well-established technique for the discrimination of heavy particles from light particles. Nonetheless, it is not efficient in the separation of electrons and positrons, as they give rise to indistinguishable scintillator responses. This inefficiency can be overtaken through the exploitation of the formation of ortho-Positronium (o-Ps), which alters the time profile of light pulses induced by positrons. We characterized the o-Ps properties in the most commonly used liquid scintillators, i.e. PC, PXE, LAB, OIL and PC + PPO. In addition, we studied the effects of scintillator doping on the o-Ps properties for dopants currently used in neutrino experiments, Gd and Nd. Further measurements for Li-loaded and Tl-loaded liquid scintillators are foreseen. We found that the o-Ps properties are suitable for enhancing the electron-positron discrimination.

  10. Quantum rotation of ortho and para-water encapsulated in a fullerene cage

    PubMed Central

    Beduz, Carlo; Carravetta, Marina; Chen, Judy Y.-C.; Concistrè, Maria; Denning, Mark; Frunzi, Michael; Horsewill, Anthony J.; Johannessen, Ole G.; Lawler, Ronald; Lei, Xuegong; Levitt, Malcolm H.; Li, Yongjun; Mamone, Salvatore; Murata, Yasujiro; Nagel, Urmas; Nishida, Tomoko; Ollivier, Jacques; Rols, Stéphane; Rõõm, Toomas; Sarkar, Riddhiman; Turro, Nicholas J.; Yang, Yifeng

    2012-01-01

    Inelastic neutron scattering, far-infrared spectroscopy, and cryogenic nuclear magnetic resonance are used to investigate the quantized rotation and ortho–para conversion of single water molecules trapped inside closed fullerene cages. The existence of metastable ortho-water molecules is demonstrated, and the interconversion of ortho-and para-water spin isomers is tracked in real time. Our investigation reveals that the ground state of encapsulated ortho water has a lifted degeneracy, associated with symmetry-breaking of the water environment. PMID:22837402

  11. Ruthenium(II)-Catalyzed Regioselective Ortho Amidation of Imidazo Heterocycles with Isocyanates.

    PubMed

    Shakoor, S M Abdul; Kumari, Santosh; Khullar, Sadhika; Mandal, Sanjay K; Kumar, Anil; Sakhuja, Rajeev

    2016-12-16

    Direct ortho amidation at the phenyl ring of 2-phenylimidazo heterocycles with aryl isocyanates has been achieved via a chelation-assisted cationic ruthenium(II) complex catalyzed mechanism. The methodology provides a straightforward, high-yielding regioselective approach toward the synthesis of an array of ortho-amidated phenylimidazo heterocycles without prior activation of C(sp(2))-H. This also reports the first method for coupling of aryl isocyanates with the imidazo[1,2-a]pyridine system via a pentacyclometalated intermediate. The methodology is found to be easily scalable and could be applied toward the selective ortho amidation of 2-heteroarylimidazo[1,2-a]pyridine frameworks.

  12. Measurement of the formaldehyde ortho to para ratio in three molecular clouds

    NASA Technical Reports Server (NTRS)

    Kahane, C.; Lucas, R.; Frerking, M. A.; Langer, W. D.; Encrenaz, P.

    1984-01-01

    Observations of ortho and para H2CO in two types of clouds, a warm cloud (Orion A) and two cold clouds (L183 and TMC1), are presented. The ortho to para ratio in Orion deduced from the H2(C-13)O data is about three, while that for TMC1 is about one and that for L183 is 1-2. The former value is in agreement with the value calculated from chemical models of ortho and para H2CO production. The values for the cold clouds are consistent with thermal equilibrium at a temperature slightly smaller than 10 K.

  13. Silver-Catalyzed Cyclization of ortho-Carbonylarylacetylenols for the Synthesis of Dihydronaphthofurans.

    PubMed

    Akkachairin, Bhornrawin; Tummatorn, Jumreang; Supantanapong, Nantamon; Nimnual, Phongprapan; Thongsornkleeb, Charnsak; Ruchirawat, Somsak

    2017-03-13

    ortho-Carbonylarylacetylenols have been employed for the synthesis of dihydronaphthofurans via AgTFA-catalyzed annulation reaction. A broad range of substrates both ortho-keto- and ortho-formylarylacetylenols could be employed in this transformation providing the desired products in good yields. However, the reaction pathways of these two substrates are different. The reaction of the ketone precursors could directly lead to the desired products in a single operation while the reaction of the aldehyde precursors required a one-pot two-step approach, without isolation of the bicyclic acetal intermediates. In addition, this method was also successfully used for the synthesis of dihydronaphthopyrans in very good yields.

  14. Measurement of the formaldehyde ortho to para ratio in three molecular clouds

    NASA Technical Reports Server (NTRS)

    Kahane, C.; Lucas, R.; Frerking, M. A.; Langer, W. D.; Encrenaz, P.

    1984-01-01

    Observations of ortho and para H2CO in two types of clouds, a warm cloud (Orion A) and two cold clouds (L183 and TMC1), are presented. The ortho to para ratio in Orion deduced from the H2(C-13)O data is about three, while that for TMC1 is about one and that for L183 is 1-2. The former value is in agreement with the value calculated from chemical models of ortho and para H2CO production. The values for the cold clouds are consistent with thermal equilibrium at a temperature slightly smaller than 10 K.

  15. OrthoParaMap: Distinguishing orthologs from paralogs by integrating comparative genome data and gene phylogenies

    PubMed Central

    Cannon, Steven B; Young, Nevin D

    2003-01-01

    Background In eukaryotic genomes, most genes are members of gene families. When comparing genes from two species, therefore, most genes in one species will be homologous to multiple genes in the second. This often makes it difficult to distinguish orthologs (separated through speciation) from paralogs (separated by other types of gene duplication). Combining phylogenetic relationships and genomic position in both genomes helps to distinguish between these scenarios. This kind of comparison can also help to describe how gene families have evolved within a single genome that has undergone polyploidy or other large-scale duplications, as in the case of Arabidopsis thaliana – and probably most plant genomes. Results We describe a suite of programs called OrthoParaMap (OPM) that makes genomic comparisons, identifies syntenic regions, determines whether sets of genes in a gene family are related through speciation or internal chromosomal duplications, maps this information onto phylogenetic trees, and infers internal nodes within the phylogenetic tree that may represent local – as opposed to speciation or segmental – duplication. We describe the application of the software using three examples: the melanoma-associated antigen (MAGE) gene family on the X chromosomes of mouse and human; the 20S proteasome subunit gene family in Arabidopsis, and the major latex protein gene family in Arabidopsis. Conclusion OPM combines comparative genomic positional information and phylogenetic reconstructions to identify which gene duplications are likely to have arisen through internal genomic duplications (such as polyploidy), through speciation, or through local duplications (such as unequal crossing-over). The software is freely available at . PMID:12952558

  16. Platelet lysate mucohadesive formulation to treat oral mucositis in graft versus host disease patients: a new therapeutic approach.

    PubMed

    Del Fante, Claudia; Perotti, Cesare; Bonferoni, Maria Cristina; Rossi, Silvia; Sandri, Giuseppina; Ferrari, Franca; Scudeller, Luigia; Caramella, Carla Marcella

    2011-09-01

    Optimal treatment of oral mucositis (OM) due to graft versus host disease (GvHD) is currently not available. Platelet-derived growth factors (PDGFs) have high capability for tissue healing and may play a role in repairing the mucosal barrier. The aim of the present work was to develop a mucoadhesive formulation to administer platelet lysate to oral cavity prolonging contact time of platelet lysate with oral mucosa. The mucoadhesive formulation was characterized for in vitro properties (PDGF-AB concentration, mucoadhesive properties, cytotoxicity, fibroblast proliferation, wound healing). Moreover, a preliminary clinical study on seven GvHD patients with OM refractory to other therapies was conducted, to evaluate feasibility, safety, and efficacy. GVPL (mucoadhesive gel vehicle mixed with platelet lysate)showed good mucoadhesive properties; additionally, it was characterized by good biocompatibility in vitro on fibroblasts and it was able to enhance fibroblast proliferation and wound healing, maintaining the efficacy for up to 14 days following storage at 2-8°C. In vivo, clinical response was good-to-complete in five, fair in one, none in the remaining one. The in vitro results indicate that GVPL has optimal mucoadhesive and healing enhancer properties, maintained over time (up to 14 days); preliminary clinical results suggest that oral application of platelet lysate-loaded mucoadhesive formulation is feasible, safe, well tolerated, and effective. A larger controlled randomized study is needed.

  17. Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant.

    PubMed

    Camussone, Cecilia M; Veaute, Carolina M; Pujato, Nazarena; Morein, Bror; Marcipar, Iván S; Calvinho, Luis F

    2014-02-01

    Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 whole-cell vaccine adjuvanted with ISCOM Matrix to increase specific antibodies production in blood and milk, improving opsonic capacity, compared with the same vaccine formulated with Al(OH)3. However, there is no information about the use of ISCOM Matrix for the formulation of bacterial lysates. The aim of this study was to characterize the innate and humoral immune responses induced by a S. aureus CP5 whole-cell or lysate vaccine, formulated with ISCOM Matrix after immunization of pregnant heifers. Both immunogens stimulated strong humoral immune responses in blood and milk, raising antibodies that increased opsonic capacity. Lysate formulation generated a higher and longer lasting antibody titer and stimulated a higher expression of regulatory and pro-inflammatory cytokines compared with the whole-cell vaccine. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Assessment of bone healing ability of calcium phosphate cements loaded with platelet lysate in rat calvarial defects.

    PubMed

    Babo, Pedro S; Carvalho, Pedro P; Santo, Vítor E; Faria, Susana; Gomes, Manuela E; Reis, Rui L

    2016-11-01

    Injectable calcium phosphate cements have been used as a valid alternative to autologous bone grafts for bone augmentation with the additional advantage of enabling minimally invasive implantation procedures and for perfectly fitting the tissue defect. Nevertheless, they have low biodegradability and lack adequate biochemical signaling to promote bone healing and remodeling. In previous in vitro studies, we observed that the incorporation of platelet lysate directly into the cement paste or loaded in hyaluronic acid microspheres allowed to modulate the cement degradation and the in vitro expression of osteogenic markers in seeded human adipose derived stem cells. The present study aimed at investigating the possible effect of this system in new bone formation when implanted in calvarial bilateral defects in rats. Different formulations were assessed, namely plain calcium phosphate cements, calcium phosphate cements loaded with human platelet lysate, hybrid injectable formulations composed of the calcium phosphate cement incorporating hyaluronin acid non-loaded microparticles (20% hyaluronin acid) or with particles loaded with platelet lysate. The degradability and new bone regrowth were evaluated in terms of mineral volume in the defect, measured by micro-computed tomography and histomorphometric analysis upon 4, 8 and 12 weeks of implantation. We observed that the incorporation of hyaluronin acid microspheres induced an overly rapid cement degradation, impairing the osteoconductive properties of the cement composites. Moreover, the incorporation of platelet lysate induced higher bone healing than the materials without platelet lysate, up to four weeks after surgery. Nevertheless, this effect was not found to be significant when compared to the one observed in the sham-treated group.

  19. Intranasal coadministration of the Cry1Ac protoxin with amoebal lysates increases protection against Naegleria fowleri meningoencephalitis.

    PubMed

    Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A; López-Revilla, Rubén; Reséndiz-Albor, Aldo A; Moreno-Fierros, Leticia

    2004-08-01

    Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 x 10(4) live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines.

  20. Intranasal Coadministration of the Cry1Ac Protoxin with Amoebal Lysates Increases Protection against Naegleria fowleri Meningoencephalitis

    PubMed Central

    Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A.; López-Revilla, Rubén; Reséndiz-Albor, Aldo A.; Moreno-Fierros, Leticia

    2004-01-01

    Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 × 104 live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines. PMID:15271892

  1. Adjuvant treatment with the bacterial lysate (OM-85) improves management of atopic dermatitis: A randomized study

    PubMed Central

    Bodemer, Christine; Guillet, Gerard; Cambazard, Frederic; Boralevi, Franck; Ballarini, Stefania; Milliet, Christian; Bertuccio, Paola; La Vecchia, Carlo; Bach, Jean-François; de Prost, Yves

    2017-01-01

    Background Environmental factors play a major role on atopic dermatitis (AD) which shows a constant rise in prevalence in western countries over the last decades. The Hygiene Hypothesis suggesting an inverse relationship between incidence of infections and the increase in atopic diseases in these countries, is one of the working hypothesis proposed to explain this trend. Objective This study tested the efficacy and safety of oral administration of the bacterial lysate OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), in the treatment of established AD in children. Methods Children aged 6 months to 7 years, with confirmed AD diagnosis, were randomized in a double-blind, placebo-controlled trial to receive, in addition to conventional treatment with emollients and topical corticosteroids, 3.5mg of the bacterial extract OM-85 or placebo daily for 9 months. The primary end-point was the difference between groups in the occurrence of new flares (NF) during the study period, evaluated by Hazard Ratio (HR) derived from conditional Cox proportional hazard regression models accounting for repeated events. Results Among the 179 randomized children, 170 were analysed, 88 in the OM-85 and 82 in the placebo group. As expected most children in both treatment groups experienced at least 1 NF during the study period (75 (85%) patients in the OM-85 group and 72 (88%) in the placebo group). Patients treated with OM-85 as adjuvant therapy had significantly fewer and delayed NFs (HR of repeated flares = 0.80; 95% confidence interval (CI): 0.67–0.96), also when potential confounding factors, as family history of atopy and corticosteroids use, were taken into account (HR = 0.82; 95% CI: 0.69–0.98). No major side effect was reported, with comparable and good tolerability for OM-85 and placebo. Conclusions Results show an adjuvant therapeutic effect of a well standardized bacterial lysate OM-85 on established AD. PMID:28333952

  2. Ruthenium-catalyzed meta/ortho-selective C-H alkylation of azoarenes using alkyl bromides.

    PubMed

    Li, Gang; Ma, Xingxing; Jia, Chunqi; Han, Qingqing; Wang, Ya; Wang, Junjie; Yu, Liuyang; Yang, Suling

    2017-01-19

    meta/ortho-Selective CAr-H (di)alkylation reactions of azoarenes have been achieved via [Ru(p-cymene)Cl2]2 catalyzed ortho-metalation using various types of alkyl bromides. Particularly, dual meta-alkylation of azoarene and reduction offer an attractive strategy for the synthesis of meta-alkylanilines, which are difficult to access via traditional aniline functionalization methods.

  3. Dihydrobiphenylenes through ruthenium-catalyzed [2+2+2] cycloadditions of ortho-alkenylarylacetylenes with alkynes.

    PubMed

    García-Rubín, Silvia; González-Rodríguez, Carlos; García-Yebra, Cristina; Varela, Jesús A; Esteruelas, Miguel A; Saá, Carlos

    2014-02-10

    A new synthetic route to dihydrobiphenylenes has been developed. The process involves a mild Ru(II) -catalyzed [2+2+2] dimerization of ortho-alkenylarylacetylenes or its more versatile variant, the Ru-catalyzed [2+2+2] cycloaddition of ortho-ethynylstyrenes with alkynes. Mechanistic aspects of this [2+2+2] cycloaddition are discussed. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Novel pH-Sensitive Cationic Lipids with Linear Ortho Ester Linkers for Gene Delivery

    PubMed Central

    Chen, Haigang; Zhang, Huizhen; Thor, Der; Rahimian, Roshanak; Guo, Xin

    2012-01-01

    In an effort to develop pH-sensitive lipoplexes for efficient gene delivery, we report three novel cationic lipids containing a linear ortho ester linker that conjugates either the headgroup (Type I) or one hydrocarbon chain (Type II) with the rest of the lipid molecule. The cationic lipids carry either an iodide or a chloride counterion. Compared to our previously reported cyclic ortho ester linker, the linear ortho ester linker facilitated the construction of cationic liposomes and lipoplexes with different helper lipids. The chloride counterion not only facilitated the hydration of the lipid films during liposome construction, but also enhanced the hydrolysis of the ortho ester linker in the lipoplexes. After incubation at endosomal pH 5.5, the Type I lipoplexes aggregated and destabilized the endosome-mimicking model liposomes, but not the Type II lipoplexes. The helper lipids (DOPE or cholesterol) of the lipoplexes enhanced the pH-sensitivity of the Type I lipoplexes. In CV-1 cells (monkey kidney fibroblast), the Type I ortho ester-based lipoplexes, especially those with the chloride counterion, significantly improved the gene transfection efficiency, in some cases by more than 100 fold, compared to their pH-insensitive counterparts consisting of DOTAP. The gene transfection efficiency of the ortho ester-based lipoplexes was well correlated with their rate of aggregation and membrane destabilization in response to the endosomal pH 5.5. PMID:22480493

  5. Novel pH-sensitive cationic lipids with linear ortho ester linkers for gene delivery.

    PubMed

    Chen, Haigang; Zhang, Huizhen; Thor, Der; Rahimian, Roshanak; Guo, Xin

    2012-06-01

    In an effort to develop pH-sensitive lipoplexes for efficient gene delivery, we report three novel cationic lipids containing a linear ortho ester linker that conjugates either the headgroup (Type I) or one hydrocarbon chain (Type II) with the rest of the lipid molecule. The cationic lipids carry either an iodide or a chloride counterion. Compared to our previously reported cyclic ortho ester linker, the linear ortho ester linker facilitated the construction of cationic liposomes and lipoplexes with different helper lipids. The chloride counterion not only facilitated the hydration of the lipid films during liposome construction, but also enhanced the hydrolysis of the ortho ester linker in the lipoplexes. After incubation at endosomal pH 5.5, the Type I lipoplexes aggregated and destabilized the endosome-mimicking model liposomes, but not the Type II lipoplexes. The helper lipids (DOPE or cholesterol) of the lipoplexes enhanced the pH-sensitivity of the Type I lipoplexes. In CV-1 cells (monkey kidney fibroblast), the Type I ortho ester-based lipoplexes, especially those with the chloride counterion, significantly improved the gene transfection efficiency, in some cases by more than 100 fold, compared to their pH-insensitive counterparts consisting of DOTAP. The gene transfection efficiency of the ortho ester-based lipoplexes was well correlated with their rate of aggregation and membrane destabilization in response to the endosomal pH 5.5.

  6. Facile synthesis of acid-labile polymers with pendent ortho esters.

    PubMed

    Cheng, Jing; Ji, Ran; Gao, Shi-Juan; Du, Fu-Sheng; Li, Zi-Chen

    2012-01-09

    This work presents a facile approach for preparation of acid-labile and biocompatible polymers with pendent cyclic ortho esters, which is based on the efficient and mild reactions between cyclic ketene acetal (CKA) and hydroxyl groups. Three CKAs, 2-ethylidene-1,3-dioxane (EDO), 2-ethylidene-1,3-dioxolane (EDL), and 2-ethylidene-4- methyl-1,3-dioxolane (EMD) were prepared from the corresponding cyclic vinyl acetals by catalytic isomerization of the double bond. The reaction of CKAs with different alcohols and diols was examined using trace of p-toluenesulfonic acid as a catalyst. For the monohydroxyl alcohols, cyclic ortho esters were formed by simple addition of the hydroxyl group toward CKAs with ethanol showing a much greater reactivity than iso-propanol. When 1,2- or 1,3-diols were used to react with the CKAs, we observed the isomerized cyclic ortho esters besides the simple addition products. Biocompatible polyols, that is, poly(2-hydroxyethyl acrylate) (PHEA) and poly(vinyl alcohol) (PVA) were then modified with CKAs, and the degree of substitution of the pendent ortho esters can be easily tuned by changing feed ratio. Both the small molecule ortho esters and the CKA-modified polymers demonstrate the pH-dependent hydrolysis profiles, which depend also on the chemical structure of the ortho esters as well as the polymer hydrophobicity.

  7. Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay.

    PubMed

    Kucki, Melanie; Cavelius, Christian; Kraegeloh, Annette

    2014-04-01

    Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here, we tested the traditional Limulus amebocyte lysate gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of, or covered by, the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement controls revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein-particle interactions are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination.

  8. Platelet lysate induces in vitro wound healing of human keratinocytes associated with a strong proinflammatory response.

    PubMed

    El Backly, Rania; Ulivi, Valentina; Tonachini, Laura; Cancedda, Ranieri; Descalzi, Fiorella; Mastrogiacomo, Maddalena

    2011-07-01

    Platelet lysates (PL), which are derived from platelets, are cocktails of growth factors and cytokines that can promote tissue regeneration. Until today, most studies have focused on growth factor content of platelets rather than on their potential as a reservoir of mediators and cytokines. Taking advantage of an in vitro scratch assay performed under both normal and inflammatory conditions, in the present work, we report that at physiologic concentrations, PL enhanced wound closure rates of NCTC 2544 human keratinocytes. This effect was clearly detectable 6 h after wounding. Moreover, PL induced a strong cell actin cytoskeletal re-organization that persisted up to 24 h. The accelerated wound closure promoted by PL, in either presence or absence of serum, was associated with a high expression of the inflammatory cytokine interleukin-8. Further, after 24 h PL treatment, confluent keratinocytes also expressed low amounts of interleukin-8 and of the antimicrobial peptide neutrophil gelatinase-associated lipocalin, which dramatically increased under inflammatory conditions. These effects were associated with activation of the inflammatory pathways, p38 mitogen-activated protein kinase, and NF-κB. Our findings support the concept that platelet-derived preparations could accelerate regeneration of difficult-to-heal wounds by triggering an inflammatory cascade and having an antimicrobial role.

  9. Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

    PubMed

    Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2014-03-01

    Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products.

  10. Culture of human cell lines by a pathogen-inactivated human platelet lysate.

    PubMed

    Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

    2016-08-01

    Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

  11. Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate.

    PubMed

    Oliveira, Sara M; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L; Mano, João F

    2015-04-01

    Great efforts have been made to introduce growth factors (GFs) onto 2D/3D constructs in order to control cell behavior. Platelet lysate (PL) presents itself as a cost-effective source of multiple GFs and other proteins. The instruction given by a construct-PL combination will depend on how its instructive cues are presented to the cells. The content, stability and conformation of the GFs affect their instruction. Strategies for a controlled incorporation of PL are needed. Herein, PL was incorporated into nanocoatings by layer-by-layer assembling with polysaccharides presenting different sulfation degrees (SD) and charges. Heparin and several marine polysaccharides were tested to evaluate their PL and GF incorporation capability. The consequent effects of those multilayers on human adipose derived stem cells (hASCs) were assessed in short-term cultures. Both nature of the polysaccharide and SD were important properties that influenced the adsorption of PL, vascular endothelial growth factor (VEGF), fibroblast growth factor b (FGFb) and platelet derived growth factor (PDGF). The sulfated polysaccharides-PL multilayers showed to be efficient in the promotion of morphological changes, serum-free adhesion and proliferation of high passage hASCs (P > 5). These biomimetic multilayers promise to be versatile platforms to fabricate instructive devices allowing a tunable incorporation of PL.

  12. Immunomodulative efficacy of bone marrow-derived mesenchymal stem cells cultured in human platelet lysate.

    PubMed

    Flemming, Antoinette; Schallmoser, Katharina; Strunk, Dirk; Stolk, Meaghan; Volk, Hans-Dieter; Seifert, Martina

    2011-12-01

    Human mesenchymal stem cells (hMSCs) are considered to be a promising tool for novel cell-based therapies. Clinical applications in solid organ transplantation were hampered by the dependence on animal serum for hMSCs clinical scale expansion until substitution with human platelet lysate (HPL) became a promising alternative. Therefore we focused on a direct comparison of immunomodulatory properties of hMSCs cultured in HPL or fetal calf serum (FCS). Phenotypic characterization, detection of cytokine secretion and effects on alloantigen- and mitogen-induced lymphocyte proliferation as well as degranulation of cytomegalovirus-specific cytotoxic T cells were applied in potency assays. We demonstrated that HPL-cultured MSCs have comparable immunomodulatory capacities to their FCS-cultured counterparts. The observed immunomodulatory properties include a beneficial inhibitory effect on immune cell proliferation and an unaffected viral T cell immunity. Thus, culturing hMSCs in HPL generates an efficient and safe expansion combined with intriguing immunomodulatory properties making these cells an attractive cell therapeutic tool.

  13. Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.

    PubMed

    Cholewa, Dominik; Stiehl, Thomas; Schellenberg, Anne; Bokermann, Gudrun; Joussen, Sylvia; Koch, Carmen; Walenda, Thomas; Pallua, Norbert; Marciniak-Czochra, Anna; Suschek, Christoph V; Wagner, Wolfgang

    2011-01-01

    The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm(2). The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.

  14. Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

    PubMed

    Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

    2016-01-13

    The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds.

  15. GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

    PubMed

    Carducci, Augusto; Scafetta, Gaia; Siciliano, Camilla; Carnevale, Roberto; Rosa, Paolo; Coccia, Andrea; Mangino, Giorgio; Bordin, Antonella; Vingolo, Enzo Maria; Pierelli, Luca; Lendaro, Eugenio; Ragona, Giuseppe; Frati, Giacomo; De Falco, Elena

    2016-01-01

    Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

  16. Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors.

    PubMed

    Ott, Christine A; Baljinnyam, Bolormaa; Zakharov, Alexey V; Jadhav, Ajit; Simeonov, Anton; Zhuang, Zhihao

    2017-09-15

    The deubiquitinases, or DUBs, are associated with various human diseases, including neurological disorders, cancer, and viral infection, making them excellent candidates for pharmacological intervention. Drug discovery campaigns against DUBs require enzymatic deubiquitination assays amenable for high-throughput screening (HTS). Although several DUB substrates and assays have been developed in recent years, they are largely limited to recombinantly purified DUBs. Many DUBs are large multidomain proteins that are difficult to obtain recombinantly in sufficient quantities for HTS. Therefore, an assay that obviates the need of recombinant protein generation and also recapitulates a physiologically relevant environment is highly desirable. Such an assay will open doors for drug discovery against many therapeutically relevant, but currently inaccessible, DUBs. Here, we report a cell lysate DUB assay based on AlphaLISA technology for high throughput screening. This assay platform uses a biotin-tagged ubiquitin probe and a HA-tagged DUB expressed in human cells. The assay was validated and adapted to a 1536-well format, which enabled a screening against UCHL1 as proof of principle using a library of 15 000 compounds. We expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes.

  17. A spectrophotometric assay for routine measurement of mammalian target of rapamycin activity in cell lysates.

    PubMed

    Dekter, Hinke E; Romijn, Fred P H T M; Temmink, Wouter P M; van Pelt, Johannes; de Fijter, Johan W; Smit, Nico P M

    2010-08-01

    The mammalian target of rapamycin (mTOR) is an important mediator in the PI3K/AKT signaling pathway. mTOR is the target of immunosuppressive drugs, such as rapamycin and everolimus, that are used in transplant patients but also for the treatment of various cancers. We have developed a method for mTOR activity measurement in cell lysates that measures the phosphorylation of p70 S6 kinase by an enzyme linked immunosorbent assay (ELISA) protocol. Using an optimized lysis composition, activity could be measured in the peripheral blood mononuclear cells (PBMCs) isolated from blood. For the PBMCs, intra- and interassay variations of 7 and 10%, respectively, were found using one lot number of the kit. With different lot numbers, the interassay variation increased up to 21%. Activity remained constant for PBMC pool samples on storage for a period of more than 7 months. Activity could also be measured in CD3+ T-cells isolated from blood. In vitro experiments revealed maximum mTOR inhibition of 30% in PBMCs and 44% in T-cells. The in vitro inhibition in PBMCs could also be demonstrated by Western blotting. The mTOR activity measurements may be used to show in vivo inhibition in renal allograft patients during everolimus treatment and to study mTOR activity in various (tumor) cell types.

  18. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    NASA Astrophysics Data System (ADS)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  19. Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips.

    PubMed

    Witek, Malgorzata A; Llopis, Shawn D; Wheatley, Abigail; McCarley, Robin L; Soper, Steven A

    2006-06-06

    We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of approximately 7.6 +/- 1.6 microg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 +/- 5%. The immobilization and purification assay using this PPC microchip could be performed within approximately 25 min as follows: (i) DNA immobilization approximately 6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption approximately 6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for 'reactivation' of the PC surface with UV light.

  20. Limulus amebocyte lysate assay for endotoxins by an adsorption method with polycation-immobilized cellulose beads.

    PubMed

    Sakata, Masayo; Inoue, Tomofumi; Todokoro, Masami; Kunitake, Masashi

    2010-01-01

    To assay lipopolysaccharides (LPSs) in solutions containing Limulus amebocyte lysate (LAL)-inhibiting or LAL-enhancing compounds, we developed a selective endotoxin (LPS) assay using poly(epsilon-lysine)-immobilized cellulose beads (PL-Cellufine) and LAL. The PL-Cellufine can adsorb LPSs in a solution containing certain compounds (NaCl, proteins and amino acids) at an ionic strength of mu = 0.05-0.4 at neutral pH. The LPSs adsorbed on the PL-Cellufine were separated from the compounds by centrifugation and then the PL-Cellufine was suspended in LPS-free water. The LPS activities of the suspension are directly assayed by a turbidimetric time assay with the LAL reagent. The accuracy of the adsorption method was high compared with those of common solution methods. As for the common method, the apparent recovery of LPS from the compounds was 40-95%. This suggests that these compounds inhibit the LAL procedure. By contrast, the adsorption method showed good LPS recovery (88-120%) in all cases, without being inhibited or enhanced by the compounds.

  1. Comparison of limulus amebocyte lysates and correlation with the United States Pharmacopeial pyrogen test.

    PubMed Central

    Wachtel, R E; Tsuji, K

    1977-01-01

    Six limulus amebocyte lysate (LAL) preparations obtained from five different suppliers were evaluated for sensitivity, dependability, cost, convenience of use, and correlation with the United States Pharmacopeial (USP) rabbit pyrogen test method. Endotoxins from various gram-negative microorganisms were used for the evaluation. Major differences among the LAL preparations lie in the area of sensitivity. Differences, up to 100-fold, exist in the sensitivity of the various LAL preparations to the same endotoxin. The LAL tests in general were 3 to 300 times more sensitive than was the USP rabbit pyrogen test method. The LAL and the USP rabbit pyrogen test data correlated well when the endotoxin in a relatively pure and undegraded form was examined. However, large discrepancies in correlation were found when partially degraded endotoxins were compared. One LAL preparation responded to both intact and degraded endotoxin, whereas others responded only to intact endotoxin; the latter closely correlated with the febrile response of the rabbit. Therefore, proper selection of an LAL preparation is important for its application in clinical, pharmaceutical, public health, and environmental areas. PMID:327936

  2. Black cobra (Naja naja karachiensis) lysates exhibit broad-spectrum antimicrobial activities.

    PubMed

    Sagheer, Mehwish; Siddiqui, Ruqaiyyah; Iqbal, Junaid; Khan, Naveed Ahmed

    2014-04-01

    It is hypothesized that animals living in polluted environments possess antimicrobials to counter pathogenic microbes. The fact that snakes feed on germ-infested rodents suggests that they encounter pathogenic microbes and likely possess antimicrobials. The venom is used only to paralyze the rodent, but the ability of snakes to counter potential infections in the gut due to disease-ridden rodents requires robust action of the immune system against a broad range of pathogens. To test this hypothesis, crude lysates of different organs of Naja naja karachiensis (black cobra) were tested for antimicrobial properties. The antimicrobial activities of extracts were tested against selected bacterial pathogens (neuropathogenic Escherichia coli K1, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Streptococcus pneumonia), protist (Acanthamoeba castellanii), and filamentous fungus (Fusarium solani). The findings revealed that plasma and various organ extracts of N. n. karachiensis exhibited antimicrobial activity against E. coli K1, MRSA, P. aeruginosa, S. pneumoniae, A. castellanii, and F. solani in a concentration-dependent manner. The results of this study are promising for the development of new antimicrobials.

  3. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

    PubMed

    Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

    2016-09-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.

  4. In vitro effects of an immunostimulating bacterial lysate on human lymphocyte function.

    PubMed

    Lanzilli, G; Falchetti, R; Tricarico, M; Ungheri, D; Fuggetta, M P

    2005-01-01

    MLBL is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by mechanical lysis of different strains of Gram-positive and Gram-negative bacteria that can cause acute and chronic infections of the respiratory tract. Previous studies suggested a stimulating effect of MLBL both on humoral and cellular immune responses. In the present study, the in vitro effects of MLBL on human lymphocyte effector functions and its mechanisms of action were evaluated. The results show that the most remarkable effects of MLBL on the immune system are: i) activation of the IL-2 receptor (IL-2Ralpha) on different lymphocyte subsets (B, CD4+ T and CD8+ T cells) involved both in humoral and cellular immune responses; ii) induction of cytokine synthesis (IL-2, IL-10, IL-12, IFNgamma) in the immune competent cells that induce and regulate immune responses; iii) generation of CD4+ and CD8+ effector T cells. Overall, these results suggest that the therapeutic effect of MLBL on acute and recurrent infections of the respiratory tract is related to its ability to activate the responses of different subsets of immune competent cells both for humoral and cellular immunity. Moreover, these effects can be induced either by direct immune cell activation or through the generation and activation of immune effector cells.

  5. Electrochemical monitoring of an important biomarker and target protein: VEGFR2 in cell lysates

    PubMed Central

    Wei, Tianxiang; Tu, Wenwen; Zhao, Bo; Lan, Yaqian; Bao, Jianchun; Dai, Zhihui

    2014-01-01

    Vascular endothelial growth factor receptor 2 (VEGFR2) is a potential cell-type biomarker in clinical diagnoses. Besides, it's the target protein of many tyrosine kinase inhibitors and its expression significantly associates with clinical performance of these inhibitors. VEGFR2 detection provides an early warning for diseases and a basis for therapy and drug screening. Some methods have been developed for VEGFR2 determination. However, they are usually performed indirectly and complexly. Herein, an electrochemical biosensing platform for VEGFR2 analysis has been first proposed. It can detect the total concentrations of the VEGFR2 protein in cells lysates directly and can be used to monitor the changes of VEGFR2 expression levels induced by treatments of different inhibitors. Moreover, the inhibitor-VEGFR2 interactions are illuminated through theoretical simulation. The simulation results agree well with the experimental data, indicating the veracity of the proposed method. The electrochemical detection methodology for VEGFR2 would be promising in clinical diagnosis and drug screening. PMID:24496270

  6. Proteomic analysis using 2-D liquid separations of intact proteins from whole-cell lysates.

    PubMed

    Zhu, Kan; Yan, Fang; O'Neil, Kimberly A; Hamler, Rick; Lubman, David M; Lin, Linda; Barder, Timothy J

    2004-02-01

    This unit describes procedures for 2-D liquid separations of proteins from whole-cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI-based fractionation using a batch-phase electrophoretic method, while chromatofocusing uses a column-based chromatographic method to generate the pH gradient. Using either method, a second-dimension fractionation is provided in the liquid phase using nonporous silica-based reversed-phase HPLC (NPS-RP-HPLC) to generate a 2-D liquid map of the protein content of the cell. The eluate of the 2-D liquid fractionation is directly coupled to a mass spectrometer for on-line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.

  7. Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

    PubMed Central

    Buhrman, Jason S.; Cook, Laura C.; Rayahin, Jamie E.; Federle, Michael J.; Gemeinhart, Richard A.

    2013-01-01

    Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the Gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of approximately 0.5 to 1 mg/L of E. coli culture. PMID:23926061

  8. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts

    PubMed Central

    Budiyanto, Arief; Soebono, Hardyanto

    2016-01-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm-2) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined. PMID:27401663

  9. Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.

    PubMed Central

    Zhang, G H; Baek, L; Buchardt, O; Koch, C

    1994-01-01

    The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL. PMID:8077400

  10. Use of magnesium to increase sensitivity of Limulus amoebocyte lysate for detection of endotoxin.

    PubMed Central

    Tsuji, K; Steindler, K A

    1983-01-01

    Increased sensitivity of the Limulus amoebocyte lysate (LAL) assay for the detection of endotoxin was attained by the reconstitution of commercially available LAL reagent with a magnesium-containing solution. As little as 2 to 6 pg (0.002 to 0.006 ng) of Escherichia coli O127:B8 endotoxin per ml was detected, an increase in sensitivity of 10 to 30 times. The optimum magnesium concentration range for the LAL reagent used and the optimum pH range were approximately 50 to 65 mM and pH 6.0 to 8.0, respectively. Reconstitution of five commercially available brands of LAL with a solution containing magnesium resulted in greater assay sensitivity than the identical LAL reconstituted with pyrogen-free water. Use of LAL reconstituted with a solution containing magnesium is crucial for the assay of some parenteral products, wherein increased sensitivity is essential to meet the requirement for the maximum valid dilution criteria. The mode of action of magnesium for enhanced sensitivity of LAL has been postulated. Images PMID:6859851

  11. Chaperonin GroEL a Brucella immunodominant antigen identified using Nanobody and MALDI-TOF-MS technologies.

    PubMed

    Abbady, A Q; Al-Daoude, A; Al-Mariri, A; Zarkawi, M; Muyldermans, S

    2012-05-15

    The deployment of today's antibodies that are able to distinguish Brucella from the closely similar pathogens, such as Yersinia, is still considered a great challenge since both pathogens share identical LPS (lipopolysaccharide) O-ring epitopes. In addition, because of the great impact of Brucella on health and economy in many countries including Syria, much effort is going to the development of next generation vaccines, mainly on the identification of new immunogenic proteins of this pathogen. In this context, Brucella-specific nanobodies (Nbs), camel genetic engineered heavy-chain antibody fragments, could be of great value. Previously, a large Nb library was constructed from a camel immunized with heat-killed Brucella. Phage display panning of this 'immune' library with Brucella total lysate resulted in a remarkable fast enrichment for a Nb referred to as NbBruc02. In the present work, we investigated the main characteristics of this Nb that can efficiently distinguish under well-defined conditions the Brucella from other bacteria including Yersinia. NbBruc02 showed a strong and specific interaction with its antigen within the crude lysate as tested by a surface plasmon resonance (SPR) biosensor and it was also able to pull down its cognate antigen from such lysate by immuno-capturing. Using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), NbBruc02 specific antigen was identified as chaperonin GroEL, also known as heat shock protein of 60 kDa (HSP-60), which represents a Brucella immunodominant antigen responsible of maintaining proteins folding during stress conditions. Interestingly, the antigen recognition by NbBruc02 was found to be affected by the state of GroEL folding. Thus, the Nb technology applied in the field of infectious diseases, e.g. brucellosis, yields two outcomes: (1) it generates specific binders that can be used for diagnosis, and perhaps treatment, and (2) it identifies the immunogenic candidate

  12. Cost-effectiveness analysis, prevention of atopic dermatitis by oral application of bacterial lysate in newborns/small children.

    PubMed

    Kiencke, Peter; Viehmann, Kristina; Rychlik, Reinhard

    2013-12-01

    The aim of this analysis was to determine the cost-effectiveness compared to placebo of prophylactic treatment with sterile bacterial lysate (Escherichia coli and Enterococcus faecalis) (verum) of newborns/small children with heredity for atopy [atopic dermatitis (AD)]. Infants were followed from the age of 5 weeks until 3 years of age. During this time, the number of children with AD who were treated with verum or placebo was observed at eight visits. Cost-effectiveness analyses were performed at different time points. A randomized, double-blind placebo-controlled clinical trial performed in Germany included 606 newborns. After randomization, n = 303 patients were classified in the placebo group and n = 303 in the verum group. A total of 119 participants left the study, so data from n = 250 patients of the placebo group and n = 237 patients of the verum group were available for analysis. At the beginning of the study, newborns were treated prophylactically with bacterial lysate or placebo for 26 weeks. After this, children were observed until the age of 3 years. A systematic literature research was done to evaluate treatment costs of atopic eczema in newborn/small children. Finally, 17 publications were included and checked for searched treatment costs of AD. A study was then initiated to evaluate the direct costs to statutory health insurance. Based on the described clinical trial, a decision tree model was developed. Using the evaluated direct costs and prevalence according to the clinical trial, the developed model can be used in cost-effectiveness analyses. The focus of the analyses was on the subgroup "single heredity for atopy" in clinical trials. Cost-effectiveness analysis showed an advantage for bacterial lysate after 3 years. To further support this result a model extension was executed; the model was expanded from 3 to 6 years. Cost-effectiveness of bacterial lysate was also proven after 6 years. Prophylactic treatment with bacterial lysate of infants

  13. Microbial Transformation of Bioactive Compounds and Production of ortho-Dihydroxyisoflavones and Glycitein from Natural Fermented Soybean Paste

    PubMed Central

    Roh, Changhyun

    2014-01-01

    Recently, there has been a great deal of remarkable interest in finding bioactive compounds from nutritional foods to replace synthetic compounds. In particular, ortho-dihydroxyisoflavones and glycitein are of growing scientific interest owing to their attractive biological properties. In this study, 7,8-ortho-dihydroxyisoflavone, 6,7-ortho-dihydroxyisoflavone, 3',4'-ortho-dihydroxyisoflavone and 7,4'-dihydroxy-6-methoxyisoflavone were characterized using microorganism screened from soybean Doenjang. Three ortho-dihydroxyisoflavones and glycitein were structurally elucidated by 1H-NMR and GC-MS analysis. Furthermore, bacterial strains from soybean Doenjang with the capacity of biotransformation were screened. The bacterial strain, identified as Bacillus subtilis Roh-1, was shown to convert daidzein into ortho-dihydroxyisoflavones and glycitein. Thus, this study has, for the first time, demonstrated that a bacterial strain had a substrate specificity for multiple modifications of the bioactive compounds. PMID:25513748

  14. Microbial transformation of bioactive compounds and production of ortho-dihydroxyisoflavones and glycitein from natural fermented soybean paste.

    PubMed

    Roh, Changhyun

    2014-12-12

    Recently, there has been a great deal of remarkable interest in finding bioactive compounds from nutritional foods to replace synthetic compounds. In particular, ortho-dihydroxyisoflavones and glycitein are of growing scientific interest owing to their attractive biological properties. In this study, 7,8-ortho-dihydroxyisoflavone, 6,7-ortho-dihydroxyisoflavone, 3',4'-ortho-dihydroxyisoflavone and 7,4'-dihydroxy-6-methoxyisoflavone were characterized using microorganism screened from soybean Doenjang. Three ortho-dihydroxyisoflavones and glycitein were structurally elucidated by 1H-NMR and GC-MS analysis. Furthermore, bacterial strains from soybean Doenjang with the capacity of biotransformation were screened. The bacterial strain, identified as Bacillus subtilis Roh-1, was shown to convert daidzein into ortho-dihydroxyisoflavones and glycitein. Thus, this study has, for the first time, demonstrated that a bacterial strain had a substrate specificity for multiple modifications of the bioactive compounds.

  15. Changes in the community structure of free-living heterotrophic bacteria in the open tropical Pacific Ocean in response to microalgal lysate-derived dissolved organic matter.

    PubMed

    Tada, Yuya; Suzuki, Koji

    2016-07-01

    Dissolved organic matter derived from phytoplankton (DOMP) can affect the bacterial biomass and community structure in aquatic ecosystems. Here, we examined the community response of free-living heterotrophic bacteria, with respect to cellular nucleic acid levels, to the DOMP lysates derived from three phytoplankton strains in the open tropical Pacific. The free amino acid (FAA) composition of each DOMP lysate differed among the microalgal strains. Terminal restriction fragment-length polymorphism analyses with 16S rRNA genes revealed that the community shifts of high nucleic acid (HNA) and low nucleic acid (LNA) bacteria varied significantly with the different DOMP lysate treatments. Furthermore, the FAA composition in DOMP lysates significantly affected the bacterial community shifts in HNA and LNA. Similarity percentage analysis using 16S rRNA gene deep-sequencing revealed that the DOMP lysates from the pelagophyte Pelagomonas calceolata caused relatively large community shifts with Alcaligenes predominating in the HNA fraction. In contrast, the DOMP lysate from the diatom Thalassiosira oceanica induced a community shift in the LNA fraction with a predominance of uncultured Actinobacteria Thus, the data indicate that the DOMP lysates from different microalgae constitute a primary factor altering the dominant bacterial groups in the open ocean. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Antigen provoking gamma interferon production in response to Mycobacterium bovis BCG and functional difference in T-cell responses to this antigen between viable and killed BCG-immunized mice.

    PubMed Central

    Kawamura, I; Yang, J; Takaesu, Y; Fujita, M; Nomoto, K; Mitsuyama, M

    1994-01-01

    It has been shown that gamma interferon (IFN-gamma)-producing CD4+ T cells, which are generated only by immunization with viable bacteria, exert a significant role in protective immunity against mycobacteria in mice. In this study, we have tried to determine the antigen recognized by the T cells in search of a possible protective antigen. T cells from viable Mycobacterium bovis BCG-immunized mice were stimulated with several antigens, and IFN-gamma production was measured. Purified protein derivative and viable and killed BCG lysates caused significant IFN-gamma production, and almost the same level of IFN-gamma activity was detected in both groups stimulated with viable and killed BCG lysates. However, heat shock protein (HSP) 65 and HSP 70 were not a major antigen for IFN-gamma production. The antigen provoking IFN-gamma production is localized mainly in the membrane fraction of BCG cells, and the approximate molecular size was 18 kDa. On the other hand, T cells from killed BCG-immunized mice never responded to this antigen for IFN-gamma production, whereas they could mount a delayed-type hypersensitivity response. These results showed that the antigen provoking IFN-gamma production was present in killed as well as viable BCG. In addition to the antigen presentation by antigen-presenting cells, some kinds of differentiation factor (such as monokines) that are produced only by stimulation with viable cells seemed to be necessary for the development of IFN-gamma-producing T cells. Images PMID:7927701

  17. A bioinspired and biocompatible ortho-sulfiliminyl phenol synthesis

    NASA Astrophysics Data System (ADS)

    Xiong, Feng; Lu, Liang; Sun, Tian-Yu; Wu, Qian; Yan, Dingyuan; Chen, Ying; Zhang, Xinhao; Wei, Wei; Lu, Yi; Sun, Wei-Yin; Li, Jie Jack; Zhao, Jing

    2017-06-01

    Synthetic methods inspired by Nature often offer unique advantages including mild conditions and biocompatibility with aqueous media. Inspired by an ergothioneine biosynthesis protein EgtB, a mononuclear non-haem iron enzyme capable of catalysing the C-S bond formation and sulfoxidation, herein, we discovered a mild and metal-free C-H sulfenylation/intramolecular rearrangement cascade reaction employing an internally oxidizing O-N bond as a directing group. Our strategy accommodates a variety of oxyamines with good site selectivity and intrinsic oxidative properties. Combining an O-N bond with an X-S bond generates a C-S bond and an S=N bond rapidly. The newly discovered cascade reaction showed excellent chemoselectivity and a wide substrate scope for both oxyamines and sulfenylation reagents. We demonstrated the biocompatibility of the C-S bond coupling reaction by applying a coumarin-based fluorogenic probe in bacterial lysates. Finally, the C-S bond coupling reaction enabled the first fluorogenic formation of phospholipids, which self-assembled to fluorescent vesicles in situ.

  18. Two ortho-rhom-bic polymorphs of hydro-morphone.

    PubMed

    Mazurek, Jaroslaw; Hoffmann, Marcel; Fernandez Casares, Ana; Cox, D Phillip; Minardi, Mathew D; Sasine, Josh

    2016-05-01

    Conditions to obtain two polymorphic forms by crystallization from solution were determined for the analgesic drug hydro-morphone [C17H19NO3; systematic name: (4R,4aR,7aR,12bS)-9-hy-droxy-3-methyl-1,2,4,4a,5,6,7a,13-octa-hydro-4,12-methano-benzofuro[3,2-e]iso-quinolin-7-one]. These two crystalline forms, designated as I and II, belong to the P212121 ortho-rhom-bic space group. In both polymorphs, the hydro-morphone mol-ecules adopt very similar conformations with some small differences observed only in the N-methyl amine part of the mol-ecule. The crystal structures of both polymorphs feature chains of mol-ecules connected by hydrogen bonds; however, in form I this inter-action occurs between the hydroxyl group and the tertiary amine N atom whereas in form II the hydroxyl group acts as a donor of a hydrogen bond to the O atom from the cyclic ether part.

  19. ortho-Phenylene oligomers with terminal push-pull substitution.

    PubMed

    He, Jian; Mathew, Sanyo M; Cornett, Sarah D; Grundy, Stephan C; Hartley, C Scott

    2012-05-07

    ortho-Phenylenes are an emerging class of helical oligomers and polymers. We have synthesized a series of push-pull-substituted o-phenylene oligomers (dimethylamino/nitro) up to the octamer. Conformational analysis of the hexamer using a combination of low-temperature NMR spectroscopy and ab initio predictions of (1)H NMR chemical shifts indicates that, like other o-phenylenes, they exist as compact helices in solution. However, the substituents are found to have a significant effect on their conformational behavior: the nitro-functionalized terminus is 3-fold more likely to twist out of the helix. Protonation of the dimethylamino group favors the helical conformer. UV/vis spectroscopy indicates that the direct charge-transfer interaction between the push-pull substituents attenuates quickly compared to other conjugated systems, with no significant charge-transfer band for oligomers longer than the trimer. On protonation of the dimethylamino group, significant bathochromic shifts with increasing oligomer length are observed: the effective conjugation length is 9 repeat units, more than twice that of the parent oligomer. This behavior may be rationalized through examination of the frontier molecular orbitals of these compounds, which exhibit greater delocalization after protonation, as shown by DFT calculations.

  20. Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives.

    PubMed

    Feng, Pengju; Ngai, Ming-Yu

    2016-01-19

    Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 °C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

  1. The carcinogenicity of the biocide ortho-phenylphenol.

    PubMed

    Appel, K E

    2000-04-01

    The biocides ortho-phenylphenol and its sodium salt (OPP and SOPP) are widely used as fungicides and antibacterial agents for commercial and consumer purposes. The carcinogenicity of OPP/SOPP toward the urinary bladder was demonstrated when rats were chronically fed concentrations of 0.5%-4% in their diet. Other species tested so far did not develop tumours. Understanding the mechanisms underlying OPP/SOPP-induced bladder carcinogenesis is critical to determine whether risks observed at high doses in rats are of relevance to humans exposed at much lower levels. This overview details experimental studies of carcinogenicity, genotoxicity as well as metabolism/toxicokinetics and other mechanistic studies which bear on cancer hazard and risk evaluation of exposure to humans. Based on the presently available knowledge, it is concluded that reactive quinoid metabolites exhibiting redox cycling activities are the crucial factors. At certain concentration levels, these metabolites are able to produce cytotoxic events with concomitant enhanced cell proliferation of the target tissue. Further important risk factors are probably promutagenic lesions induced by oxidative stress and a higher urinary pH. Supposed that these mechanisms are the basis for the tumourigenicity observed, then suitable low doses of OPP/SOPP will practically pose no cancer risk.

  2. Cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

    PubMed

    Nakagawa, Y; Moldéus, P; Moore, G A

    1992-01-22

    The effects of ortho-phenylphenol (OPP) and its metabolites, phenyl-hydroquinol (PHQ) and phenyl-benzoquinone (PBQ), on isolated rat hepatocytes were investigated. Addition of OPP (0.5-1.0 mM) to cells caused a dose-dependent cell death accompanied by the depletion of intracellular levels of ATP, glutathione (GSH) and protein thiols. GSH loss correlated with the formation of oxidized GSH. In addition, PHQ and especially PBQ (both at 0.5 mM) resulted in acute cell death with rapid depletion of ATP, GSH and protein thiols, and further low doses of PBQ (10-50 microM) elicited serious impairment of mitochondrial functions related to oxidative phosphorylation and Ca fluxes in isolated liver mitochondria. These results indicate that mitochondria are a target for these compounds and that OPP is itself toxic to hepatocytes even when metabolism is inhibited. The loss of cellular GSH and protein thiols accompanied by the impairment of mitochondrial function may be the main mechanisms of cytotoxicity induced by OPP and its metabolites.

  3. Orthos, an alarm system for the ALICE DAQ operations

    NASA Astrophysics Data System (ADS)

    Chapeland, Sylvain; Carena, Franco; Carena, Wisla; Chibante Barroso, Vasco; Costa, Filippo; Denes, Ervin; Divia, Roberto; Fuchs, Ulrich; Grigore, Alexandru; Simonetti, Giuseppe; Soos, Csaba; Telesca, Adriana; Vande Vyvre, Pierre; von Haller, Barthelemy

    2012-12-01

    ALICE (A Large Ion Collider Experiment) is the heavy-ion detector studying the physics of strongly interacting matter and the quark-gluon plasma at the CERN LHC (Large Hadron Collider). The DAQ (Data Acquisition System) facilities handle the data flow from the detectors electronics up to the mass storage. The DAQ system is based on a large farm of commodity hardware consisting of more than 600 devices (Linux PCs, storage, network switches), and controls hundreds of distributed hardware and software components interacting together. This paper presents Orthos, the alarm system used to detect, log, report, and follow-up abnormal situations on the DAQ machines at the experimental area. The main objective of this package is to integrate alarm detection and notification mechanisms with a full-featured issues tracker, in order to prioritize, assign, and fix system failures optimally. This tool relies on a database repository with a logic engine, SQL interfaces to inject or query metrics, and dynamic web pages for user interaction. We describe the system architecture, the technologies used for the implementation, and the integration with existing monitoring tools.

  4. Measurement of ortho-positronium properties in liquid scintillators

    NASA Astrophysics Data System (ADS)

    Perasso, S.; Consolati, G.; Franco, D.; Jollet, C.; Meregaglia, A.; Tonazzo, A.; Yeh, M.

    2014-03-01

    Pulse shape discrimination is a well-established technique for background rejection in liquid scintillator detectors. It is particularly effective in separating heavy particles from light particles, but not in distinguishing electrons from positrons. This inefficiency can be overtaken by exploiting the formation of ortho-positronium (o-Ps), which alters the time profile of light pulses induced by positrons. We characterized the o-Ps properties in the most commonly used liquid scintillators, i.e. PC, PXE, LAB, OIL and PC + PPO. In addition, we studied the effects of scintillator doping on the o-Ps properties for dopants used in neutrino-less double beta decay experiments (Nd and Te) and in anti-neutrino and neutron detection (Gd and Li respectively). We found that the o-Ps properties are similar in all the tested scintillators, with a lifetime around 3 ns and a formation probability of about 50%. This result indicates that an o-Ps-enhanced pulse shape discrimination can be applied in liquid scintillator detectors for neutrino and anti-neutrino detection and for neutrino-less double beta decay search.

  5. Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product.

    PubMed Central

    Tsuji, K; Steindler, K A; Harrison, S J

    1980-01-01

    A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test. PMID:6448582

  6. Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units.

    PubMed

    Sellberg, Felix; Berglund, Erik; Ronaghi, Martin; Strandberg, Gabriel; Löf, Helena; Sommar, Pehr; Lubenow, Norbert; Knutson, Folke; Berglund, David

    2016-12-01

    Platelet lysate is a readily available source of growth factors, and other mediators, which has been used in a variety of clinical applications. However, the product remains poorly standardized and the present investigation evaluates the composition of platelet lysate obtained from either fresh or stored pathogen-inactivated platelet units. Platelet pooled units (n = 10) were obtained from healthy blood donors and tested according to standard procedures. All units were pathogen inactivated using amotosalen hydrochloride and UVA exposure. Platelet lysate was subsequently produced at two separate time-points, either from fresh platelet units or after 5 days of storage, by repeated freeze-thaw cycles. The following mediators were determined at each time-point: EGF, FGF-2, VEGF, IGF-1, PDGF-AB/BB, BMP-2, PF4, TGF-β isoform 1, IL-1β, IL-2, IL-6, IL-10, IL-12p70, 1L-17A, TNF-α, and IFN-γ. The concentration of growth factors and cytokines was affected by time in storage. Notably, TGF-β, PDGF-AB/BB, and PF4 showed an increase of 27.2% (p < 0.0001), 29.5% (p = 0.04) and 8.2% (p = 0.0004), respectively. A decrease was seen in the levels of IGF-1 and FGF-2 with 22% (p = 0.041) and 11% (p = 0.01), respectively. Cytokines were present only in very low concentrations and all other growth factors remained stable with time in storage. The composition of mediators in platelet lysate obtained from pathogen-inactivated platelet units differs when produced from fresh and stored platelet units, respectively. This underscores the need for further standardization and optimization of this important product, which potentially may influence the clinical effects. Copyright © 2016. Published by Elsevier Ltd.

  7. Equine platelet lysate as an alternative to fetal bovine serum in equine mesenchymal stromal cell culture - too much of a good thing?

    PubMed

    Russell, K A; Koch, T G

    2016-03-01

    Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Randomised dose escalation study. Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1 × 10(12) platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB-MSC cultures was determined after 4 days using a resazurin semiquantitative assay. Cord blood-MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Expansion medium enriched with PL can support short-term equine CB-MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB-MSC at higher PL concentrations, an in vivo dose study is indicated to investigate if combinational therapies of CB-MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB-MSC function. © 2015 EVJ Ltd.

  8. Copper-catalyzed direct amination of ortho-functionalized haloarenes with sodium azide as the amino source.

    PubMed

    Zhao, Haibo; Fu, Hua; Qiao, Renzhong

    2010-05-21

    A simple copper-catalyzed direct amination of ortho-functionalized haloarenes (2-halobenzoic acid, 2-halobenzamide, and N-(2-bromophenyl)acetamide derivatives) has been developed with use of NaN(3) as the amino source in ethanol, and the corresponding ortho-functionalized aromatic amines were synthesized in good to excellent yields. The protocol undergoes one-pot Ullmann-type coupling of ortho-functionalized haloarenes with NaN(3) to lead to ortho-functionalized azidoarenes, followed by reduction with ethanol.

  9. Gilch Synthesis of Poly(ortho-Phenylene Vinylenes): A Powerful Access to High-Molecular-Weight Blue-Emitting Polymers.

    PubMed

    Rittscher, Valentina; Kuch, Serena; Rehahn, Matthias

    2016-05-01

    For the first time, the successful Gilch synthesis of poly(ortho-phenylene vinylenes) (ortho-PPVs) is reported. The molar mass of the constitutionally homogeneous ortho-PPVs reaches values as high as Mw ≈ 300 000 Da. The ortho-connectivity of the repeating units forces the chains to assume closely packed conformations even in good solvents. Significant perturbation of the π-electron systems and considerable shortening of the conjugation lengths are the consequences. UV-vis absorption and photoluminescence maxima consequently are shifted clearly toward shorter wavelengths compared to, e.g., classic para-PPVs.

  10. An S(N)Ar approach to sterically hindered ortho-alkoxybenzaldehydes for the synthesis of olefin metathesis catalysts.

    PubMed

    Engle, Keary M; Luo, Shao-Xiong; Grubbs, Robert H

    2015-04-17

    A three-step procedure has been developed for preparing ortho-alkoxybenzaldehydes from ortho-fluorobenzaldehydes that tolerates the use of sterically hindered sodium alkoxide nucleophiles. The protocol is modular and operationally convenient. The ortho-alkoxybenzaldehyde products can be converted in one additional step to ortho-alkoxystyrenes by a Wittig reaction. These styrenes are precursors to the chelating benzylidene moiety in a proposed series of novel ruthenium complexes for use in olefin metathesis. Chelation with three representative styrenes has been demonstrated.

  11. Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering.

    PubMed

    Zaky, S H; Ottonello, A; Strada, P; Cancedda, R; Mastrogiacomo, M

    2008-12-01

    The heterogeneous population of non-haematopoietic cells residing in the bone marrow (bone marrow stromal cells, BMSCs) and the different fractions and components obtained from platelet-rich plasma provide an invaluable source of autologous cells and growth factors for bone and other connective tissue reconstruction. In this study, we investigated the effect of an allogenic platelet lysate on human BMSCs proliferation and differentiation. Cell proliferation and number of performed cell doublings were enhanced in cultures supplemented with the platelet-derived growth factors (platelet lysate, PL), either with or without the concomitant addition of fetal bovine serum (FBS), compared to cultures performed in the presence of FBS and FGF2. Both in vitro and in vivo osteogenic differentiation were unaltered in cells maintained in medium supplemented with PL and not FBS (Only PL) and in cells maintained in medium containing FBS and FGF2. Interestingly, the in vitro cartilage formation was more effective in the pellet of BMSCs expanded in the Only PL medium. In particular, a chondrogenic differentiation was observed in pellets of some in vitro-expanded BMSCs in the Only PL medium, whereas pellets from parallel cell cultures in medium containing FBS did not respond to the chondrogenic induction. We conclude that the platelet lysate from human source is an effective and even more beneficial substitute for fetal bovine serum to support the in vitro expansion of human BMSCs for subsequent tissue-engineering applications.

  12. Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates.

    PubMed

    Ochiai, Masaki; Yamamoto, Akihiko; Naito, Seishiro; Maeyama, Jun-Ichi; Masumi, Atsuko; Hamaguchi, Isao; Horiuchi, Yoshinobu; Yamaguchi, Kazunari

    2010-11-01

    Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1-3)-β-D-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.

  13. Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.

    PubMed

    Schoch, Reto B; Ronaghi, Mostafa; Santiago, Juan G

    2009-08-07

    We present a technique which enables the separation of small RNAs-such as microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs)-from >or=66 nucleotide RNAs and other biomolecules contained in a cell lysate. In particular, the method achieves separation of small RNAs from precursor miRNAs (pre-miRNAs) in less than 3 min. We use on-chip isotachophoresis (ITP) for the simultaneous extraction, isolation, preconcentration and quantitation of small RNAs (approximately 22 nucleotides) and employ the high-efficiency sieving matrix Pluronic F-127; a thermo-responsive triblock copolymer which allows convenient microchannel loading at low temperature. We present the isolation of small RNAs from the lysate of 293A human kidney cells, and quantitate the number of short RNA molecules per cell to be 2.9x10(7). We estimate this quantity is an aggregate of roughly 500 types of short RNA molecules per 293A cell. Currently, the minimal cell number for small RNA extraction and detection with our method is approximately 900 (from a 5 microL sample volume), and we believe that small RNA analysis from tens of cells is realizable. Techniques for rapid and sensitive extraction and isolation of small RNAs from cell lysate are much-needed to further uncover their full range and functionality, including RNA interference studies.

  14. Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability.

    PubMed

    Crespo-Diaz, Ruben; Behfar, Atta; Butler, Greg W; Padley, Douglas J; Sarr, Michael G; Bartunek, Jozef; Dietz, Allan B; Terzic, Andre

    2011-01-01

    With favorable regenerative and immunotolerant profiles, patient-derived human mesenchymal stem cells (hMSCs) are increasingly considered in cell therapy. Derived from bone marrow (BM) and standardized with culture in fetal bovine serum (FBS), translation of hMSC-based approaches is impeded by protracted expansion times, risk of xenogenic response, and exposure to zoonoses. Here, human platelet lysate adherent to good manufacturing practices (GMP-hPL) provided a nonzoonotic adjuvant that enhanced the capacity of BM-hMSC to proliferate. The nurturing benefit of GMP-hPL was generalized to hMSC from adipose tissue evaluated as an alternative to bone marrow. Long-term culture in GMP-hPL maintained the multipotency of hMSC, while protecting against clonal chromosomal instability detected in the FBS milieu. Proteomic dissection identified TGF-β, VEGF, PDGF, FGF, and EGF as highly ranked effectors of hPL activity, revealing a paradigm of healing that underlies platelet lysate adjuvancy. Thus, GMP-adherent human platelet lysate accelerates hMSC proliferation with no chromosomal aberrancy, through an innate repair paradigm.

  15. Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates.

    PubMed

    Feng, Yiran; Yang, Xiaolan; Wang, Deqiang; Hu, Xiaolei; Chong, Huimin; Liao, Juan; Zhan, Chang-Guo; Liao, Fei

    2017-06-01

    With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs. With 0.4 μg crude polyclonal antibody for immobilization, Vs was consistent for ECAP in cell lysates bearing fourfold differences in its apparent specific activities when its abundance was greater than 0.9%. The ratio of Vs of the mutant R168K to that of ECAP was 1.5 ± 0.1 (n = 2), consistent with that of their specific activities after affinity purification. Unfortunately, the prediction of Vs with polyclonal antibodies that saturated microplate wells was ineffective to Pseudomonas aeruginosa arylsulfatase bearing less than 2% specific activity of ECAP. Therefore, with microplate-immobilized polyclonal antibodies to adsorb enzyme/mutants from cell lysates, high-throughput prediction of Vs was practical to recognize weakly-positive mutants of starting enzymes bearing fairly-high activities.

  16. Antiestrogenic potentials of ortho-PCB congeners by single or complex exposure.

    PubMed

    Oh, Seung Min; Ryu, Byung Taek; Lee, Sang Ki; Chung, Kyu Hyuck

    2007-02-01

    Di-ortho PCB congeners 52, 138, 153 and 180, and the mono-ortho coplanar congener 118 have been detected as a complex mixture in human tissue in Korea. This study examined the antiestrogenic effects of samples exposed to single or combination treatment of the ortho-PCB congeners. In order to determined the combined toxicity, a sample mixture (M1, M2, M3, M4, and M5) was designed based on the ortho-PCB congeners found in Korean human tissue. With the exception of PCB 52, the ortho-PCB congeners (PCB 118, 138, 153, and 180) showed weak antiestrogenic activity. The antiestrogenic activity of di-ortho PCB congeners (PCB 138, 153, and 180) was induced by the depletion of endogenous E2 as well as through the ER-dependent pathway, whereas the antiestrogenic activity of mono-ortho PCB 118 was only induced through the depletion of endogenous E2. When the MCF7-BUS cells were treated with mixtures containing the no effective concentration (10(-6) M) of the PCB congeners, M3 (PCB 118 + PCB 138 + PCB 180) and M4 (PCB 118 + PCB 138) had an antiestrogenic effect but the other mixtures (M1; PCB 52 + PCB 118 + PCB 138 + PCB 180, M2; PCB 118 + PCB 138 + PCB 153 + PCB 180, M5; PCB 118 + PCB 180) did not. Although the mechanism for the interaction between the PCB congeners is not completely understood, it was presumed that exposure to a mixture of the PCB congeners might have synergistic effects on their antiestrogenicity through the ER-independent pathway.

  17. A novel mucosal vaccine targeting Peyer's patch M cells induces protective antigen-specific IgA responses.

    PubMed

    Shima, Hideaki; Watanabe, Takashi; Fukuda, Shinji; Fukuoka, Shin-Ichi; Ohara, Osamu; Ohno, Hiroshi

    2014-11-01

    Mucosal vaccines can induce mucosal immunity, including antigen-specific secretory IgA production, to protect from invasion by pathogens and to neutralize toxins at mucosal surfaces. We established an effective antigen-delivering fusion protein, anti-GP2-SA, as a mucosal vaccine. The anti-GP2-SA consists of streptavidin (SA) fused to the antigen-binding fragment region from a mAb against glycoprotein 2 (GP2), an antigen-uptake receptor specifically expressed on M cells. Anti-GP2-SA targets antigen-sampling M cells in the follicle-associated epithelium covering Peyer's patches. Immunofluorescence showed that anti-GP2-SA specifically bound to M cells. Orally administered biotinylated ovalbumin peptide (bOVA) conjugated with anti-GP2-SA more efficiently induced OVA-specific fecal IgA secretion compared with bOVA alone or bOVA conjugated with SA. Furthermore, mice immunized by oral administration of the biotinylated Salmonella enterica serovar Typhimurium (S. Typhimurium) lysate conjugated with anti-GP2-SA were significantly better protected from subsequent infection by virulent S. Typhimurium than mice treated with the bacterial lysate alone or conjugated with SA. These results suggest that anti-GP2-SA-based M-cell-targeting vaccines are a novel strategy for inducing efficient mucosal immunity.

  18. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    PubMed

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations.

  19. Development and validation of a production process of platelet lysate for autologous use.

    PubMed

    Plöderl, Karin; Strasser, Cornelia; Hennerbichler, Simone; Peterbauer-Scherb, Anja; Gabriel, Christian

    2011-01-01

    Growth factors (GF) contained in platelets are a potential source to improve wound healing by the stimulation and acceleration of soft tissue and bone healing. This resulted in the idea that autologous platelet-rich plasma or platelet lysate (PL) containing high levels of GF might improve healing processes. Today platelet products are already applied in bone and maxillofacial surgery. In recent years, cosmetic surgery and facial rejuvenation procedures are growing steadily. New methods including platelet products aiming to induce non-surgical reduction of wrinkles upon topical injection and to minimize surgical risks in general are developed. Several point-of-care devices are already available on the market. However, the amount of PL obtained by these kits is far too high for certain applications in cosmetic surgery and they offer no possibility of storing the remaining material in a sterile manner. Therefore we developed a procedure for the sterile production of smaller amounts of PL in a closed system that can also be split into several products for repeated administration. The closed system was determined to be a bag system designed for an autologous blood donation of 100 ml whole blood. We set a special focus on the validation of the production procedure, mainly regarding sterility and platelet recovery. For validation 22 healthy volunteers were asked for a blood donation, which was centrifuged twice to obtain concentrated platelets (CP). A freeze-thaw cycle caused lysis of the CP to get approximately 8.48 ± 1.36 ml PL. We yielded satisfying results of 100% sterility and a platelet recovery of 36.92% ± 18.71%. We therefore conclude that the PL obtained is ready for studies comparing it with traditional treatments.

  20. Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

    PubMed

    Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

    2012-01-01

    The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

  1. Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications.

    PubMed

    Govindasamy, Vijayendran; Ronald, Veronica Sainik; Abdullah, Aimi Naim Binti; Ganesan Nathan, Kavitha R; Aziz, Zeti Adura Che Abdul; Abdullah, Mariam; Zain, Rosnah Binti; Kasim, Noor Hayaty Abu; Musa, Sabri; Bhonde, Ramesh R

    2011-11-01

    BACKGROUND AIMS. Dental pulp stromal cells (DPSC) are considered to be a promising source of stem cells in the field of regenerative therapy. However, the usage of DPSC in transplantation requires large-scale expansion to cater for the need for clinical quantity without compromising current good manufacturing practice (cGMP). Existing protocols for cell culturing make use of fetal bovine serum (FBS) as a nutritional supplement. Unfortunately, FBS is an undesirable additive to cells because it carries the risk of transmitting viral and prion diseases. Therefore, the present study was undertaken to examine the efficacy of human platelet lysate (HPL) as a substitute for FBS in a large-scale set-up. METHODS. We expanded the DPSC in Dulbecco's modified Eagle's medium-knock-out (DMEM-KO) with either 10% FBS or 10% HPL, and studied the characteristics of DPSC at pre- (T25 culture flask) and post- (5-STACK chamber) large-scale expansion in terms of their identity, quality, functionality, molecular signatures and cytogenetic stability. RESULTS. In both pre- and post-large-scale expansion, DPSC expanded in HPL showed extensive proliferation of cells (c. 2-fold) compared with FBS; the purity, immune phenotype, colony-forming unit potential and differentiation were comparable. Furthermore, to understand the gene expression profiling, the transcriptomes and cytogenetics of DPSC expanded under HPL and FBS were compared, revealing similar expression profiles. CONCLUSIONS. We present a highly economized expansion of DPSC in HPL, yielding double the amount of cells while retaining their basic characteristics during a shorter time period under cGMP conditions, making it suitable for therapeutic applications.

  2. Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways.

    PubMed

    Dastan, Maryam; Najafzadeh, Nowruz; Abedelahi, Ali; Sarvi, Mohammadreza; Niapour, Ali

    2016-12-01

    Minoxidil and human platelet lysate (HPL) are commonly used to treat patients with hair loss. However, the roles of HPL versus minoxidil in hair follicle biology largely remain unknown. Here, we hypothesized that bulge and dermal papilla (DP) cells may express specific genes, including Kras, Erk, Akt, Shh and β-catenin after exposure to minoxidil or HPL. The mouse hair follicles were isolated on day 10 after depilation and bulge or DP regions were dissected. The bulge and DP cells were cultured for 14days in DMEM/F12 medium. Then, the cells were treated with 100μM minoxidil and 10% HPL for 10 days. Nuclear morphology was identified using DAPi staining. Reverse transcriptase and real-time polymerase chain reaction (PCR) analysis were also performed to examine the expression of Kras, Erk, Akt, Shh and β-catenin mRNA levels in the treated bulge and DP regions after organ culture. Here, we found that minoxidil influences bulge and DP cell survival (P<0.05). Apoptosis in DP cells was also meaningfully decreased by HPL treatment (P=0.014). In addition, Kras, Akt, Erk, Shh and β-catenin mRNA levels were changed in response to minoxidil treatment in both bulge and DP cells. HPL mediated Erk upregulation in both bulge and DP cells (P<0.05), but Kras and Akt mRNA levels were not considerably different in the HPL-treated cells. β-catenin mRNA level was also significantly increased in the bulge region by HPL. We also found that Shh mRNA level was considerably higher in HPL-treated bulge cells than in minoxidil-treated bulge cells. In contrast, the expression of β-cateinin and Shh in the DP cells was not meaningfully increased after treatment with HPL. Our results suggest that minoxidil and HPL can promote hair growth by activating the main anagen inducing signaling pathways.

  3. Corynebacterium cutis Lysate Treatment Can Increase the Efficacies of PPR Vaccine.

    PubMed

    Dik, Burak; Dik, Irmak; Bahcivan, Emre; Avci, Oguzhan

    2016-10-01

    This study aimed to evaluate the effects of Peste des petits ruminants (PPR) vaccine on cytokine and antibody levels in sheep when administered alone or in combination with Corynebacterium cutis lysate (CCL). The PPR vaccine group received a single subcutaneous axillary injection of the PPR vaccine (1 mL containing tissue culture infectious dose (TCID)50 attenuated live PPRV, n = 6) and the combination treatment (1 mL CCL and 1 mL PPR vaccine, n = 6) groups received a single subcutaneous axillary injection of both CCL and PPR vaccine. Blood samples were collected from sheep before the treatment and at different points after treatment (1, 3, 7, 14, 21, and 28 days). Plasma and serum samples were evaluated for antibody percentage, levels of cytokines IL-6, IL-10, IFN-γ, IL-4, IL-12, and IL-18, oxidative stress marker Thiobarbituric acid reactive substances, and hematological and biochemical parameters. Maximum protective antibody levels reach 3-4 weeks after vaccine administration. The combination treatment resulted in significant changes in IFN-γ, IL-4, IL-12, and IL-18 cytokine levels. These changes were not evident when only the PPR vaccine was administered and antibody percentage against PPRV was short term in PPR vaccine group. In conclusion, combined usage of the PPR vaccine with CCL resulted in a heightened cytokine response, leading to improved antibody level against PPR virus. Repeated CCL treatments can lead to earlier vaccine potency, provide protective efficacy for a longer time, and increase passive immunity.

  4. Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

    PubMed

    Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

    2017-10-01

    The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy.

  5. Epitopes shared by unrelated antigens of Borrelia burgdorferi.

    PubMed Central

    Anda, P; Backenson, P B; Coleman, J L; Benach, J L

    1994-01-01

    An immunoglobulin M kappa-chain murine monoclonal antibody (CAB) reacted in a Western blot (immunoblot) with approximately 30 polypeptides from a whole-cell lysate of several American and European Borrelia burgdorferi strains. The reactive antigen with the highest M(r) was measured at 93 kDa (p93) and had an NH2-terminal sequence identical to the one previously reported for this antigen. The lowest reactive antigen had an M(r) of 16,000. All antigens recognized by CAB had isoelectric points within a narrow acidic range, between 5.4 and 6.2. Thus, the objective of this study was to determine whether the broad reactivity of CAB could be due to degradation of the antigen with the highest M(r), since such spontaneous degradation of p93 has already been reported, and to determine whether CAB could recognize shared epitopes in different antigens. Treatment of B. burgdorferi with protease inhibitors did not result in changes in CAB reactivity, indicating that if such degradation existed, it was most likely not due to the action of endogenous proteases. Likewise, protease treatment of intact organisms and recovery of the antigens in the insoluble fraction of a Triton X-114 partition indicated that they were internal and thus less likely to be degraded by experimental procedures. Amino-terminal sequences of other reactive polypeptides showed one approximately 72-kDa polypeptide to be identical to the DnaK homolog of B. burgdorferi. Two other antigens at approximately 49 and 47 kDa were blocked to Edman degradation. Finally, one sequenced polypeptide with a molecular mass of approximately 38.5 kDa had a strong identity with glyceraldehyde-3-phosphate dehydrogenase of other bacteria and vertebrates. Thus, while it cannot be ruled out that some of the CAB reactivity may be due to fragmentation of p93, there is strong evidence to indicate the presence of a shared epitope in at least three, possibly five, unrelated antigens of B. burgdorferi. A linear epitope within amino acid

  6. AntigenMap 3D: an online antigenic cartography resource.

    PubMed

    Barnett, J Lamar; Yang, Jialiang; Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2012-05-01

    Antigenic cartography is a useful technique to visualize and minimize errors in immunological data by projecting antigens to 2D or 3D cartography. However, a 2D cartography may not be sufficient to capture the antigenic relationship from high-dimensional immunological data. AntigenMap 3D presents an online, interactive, and robust 3D antigenic cartography construction and visualization resource. AntigenMap 3D can be applied to identify antigenic variants and vaccine strain candidates for pathogens with rapid antigenic variations, such as influenza A virus. http://sysbio.cvm.msstate.edu/AntigenMap3D

  7. Heat-shocked tumor cell lysate-pulsed dendritic cells induce effective anti-tumor immune response in vivo

    PubMed Central

    Qiu, Jian; Li, Guo-Wei; Sui, Yan-Fang; Song, Hong-Ping; Si, Shao-Yan; Ge, Wei

    2006-01-01

    AIM: To study whether heat-shocked tumor cells could enhance the effect of tumor cell lysate-pulsed dendritic cells (DCs) in evoking anti-tumor immune response in vivo. METHODS: Mouse undifferentiated colon cancer cells (CT-26) were heated at 42 °C for 1 h and then frozen-thawed. The bone marrow-derived DCs pulsed with heat-shocked CT-26 cell lysate (HSCT-26 DCs) were recruited to immunize syngeneic naïve BALB/c mice. The cytotoxic activity of tumor specific cytotoxic T lymphocytes (CTLs) in mouse spleen was evaluated by IFN-enzyme-linked immunospot (ELISpot) and LDH release assay. The immunoprophylactic effects induced by HSCT-26 DCs in mouse colon cancer model were compared to those induced by single CT-26 cell lysate-pulsed DCs (CT-26 DCs) on tumor volume, peritoneal metastasis and survival time of the mice. RESULTS: Heat-treated CT-26 cells showed a higher hsp70 protein expression. Heat-shocked CT-26 cell lysate pulsing elevated the co-stimulatory and MHC-II molecule expression of bone marrow-derived DCs as well as interleukin-12 p70 secretion. The IFN-γ secreting CTLs induced by HSCT-26 DCs were significantly more than those induced by CT-26 DCs (P = 0.002). The former CTLs’ specific cytotoxic activity was higher than the latter CTLs’ at a serial E/T ratio of 10:1, 20:1, and 40:1. Mouse colon cancer model showed that the tumor volume of HSCT-26 DC vaccination group was smaller than that of CT-26 DC vaccination group on tumor volume though there was no statistical difference between them (24 mm3 vs 8 mm3, P = 0.480). The median survival time of mice immunized with HSCT-26 DCs was longer than that of those immunized with CT-26 DCs (57 d vs 43 d, P = 0.0384). CONCLUSION: Heat-shocked tumor cell lysate-pulsed DCs can evoke anti-tumor immune response in vivo effectively and serve as a novel DC-based tumor vaccine. PMID:16489653

  8. Heat-shocked tumor cell lysate-pulsed dendritic cells induce effective anti-tumor immune response in vivo.

    PubMed

    Qiu, Jian; Li, Guo-Wei; Sui, Yan-Fang; Song, Hong-Ping; Si, Shao-Yan; Ge, Wei

    2006-01-21

    To study whether heat-shocked tumor cells could enhance the effect of tumor cell lysate-pulsed dendritic cells (DCs) in evoking anti-tumor immune response in vivo. Mouse undifferentiated colon cancer cells (CT-26) were heated at 42 degrees Celsius for 1 h and then frozen-thawed. The bone marrow-derived DCs pulsed with heat-shocked CT-26 cell lysate (HSCT-26 DCs) were recruited to immunize syngeneic naive BALB/c mice. The cytotoxic activity of tumor specific cytotoxic T lymphocytes (CTLs) in mouse spleen was evaluated by IFN-enzyme-linked immunospot (ELISpot) and LDH release assay. The immunoprophylactic effects induced by HSCT-26 DCs in mouse colon cancer model were compared to those induced by single CT-26 cell lysate-pulsed DCs (CT-26 DCs) on tumor volume, peritoneal metastasis and survival time of the mice. Heat-treated CT-26 cells showed a higher hsp70 protein expression. Heat-shocked CT-26 cell lysate pulsing elevated the co-stimulatory and MHC-II molecule expression of bone marrow-derived DCs as well as interleukin-12 p70 secretion. The IFN-gamma secreting CTLs induced by HSCT-26 DCs were significantly more than those induced by CT-26 DCs (P=0.002). The former CTLs' specific cytotoxic activity was higher than the latter CTLs' at a serial E/T ratio of 10:1, 20:1, and 40:1. Mouse colon cancer model showed that the tumor volume of HSCT-26 DC vaccination group was smaller than that of CT-26 DC vaccination group on tumor volume though there was no statistical difference between them (24 mm3 vs 8 mm3, P=0.480). The median survival time of mice immunized with HSCT-26 DCs was longer than that of those immunized with CT-26 DCs (57 d vs 43 d, P=0.0384). Heat-shocked tumor cell lysate-pulsed DCs can evoke anti-tumor immune response in vivo effectively and serve as a novel DC-based tumor vaccine.

  9. Urinary ortho-tyrosine excretion in diabetes mellitus and renal failure: evidence for hydroxyl radical production.

    PubMed

    Molnár, Gergõ A; Wagner, Zoltán; Markó, Lajos; Kó Szegi, Tamás; Mohás, Márton; Kocsis, Béla; Matus, Zoltán; Wagner, László; Tamaskó, Mónika; Mazák, István; Laczy, Boglárka; Nagy, Judit; Wittmann, István

    2005-11-01

    Phenylalanine is converted to para- and ortho-tyrosine by hydroxyl free radical, or to para-tyrosine by the phenylalanine hydroxylase enzyme. The aim of this study was to measure para- and ortho-tyrosine in the urine and plasma of patients with chronic renal disease and/or diabetes, to obtain information on the renal handling of the different tyrosine isomers and, furthermore, to measure urinary levels of 8-epi-prostaglandin-F(2alpha), a marker of lipid peroxidation. In our cross-sectional study we measured para-, ortho-tyrosine, and phenylalanine levels, using high performance liquid chromatography and 8-epi-prostaglandin-F(2alpha) with enzyme-linked immunosorbent assay (ELISA). We compared 4 groups: (1) controls (CONTR, N = 14), (2) patients with chronic kidney disease (CKD, N = 12), (3) patients with type 2 diabetes mellitus (DIAB, N = 17), (4) patients with chronic kidney disease and type 2 diabetes (DIAB-CKD, N = 19). We found a decreased plasma para-tyrosine level and decreased urinary para-tyrosine excretion in CKD patients, while the fractional excretion of para-tyrosine was similar in all 4 groups, approximately 1%. There was no difference in the plasma ortho-tyrosine levels between the groups. However, urinary ortho-tyrosine excretion was higher in all 3 groups of patients than in the CONTR group, and higher in DIAB and in DIAB-CKD patients than in CKD patients. The fractional excretion of ortho-tyrosine was significantly higher in DIAB and in DIAB-CKD patients than in the CONTR group. The fractional excretion of ortho-tyrosine exceeded 100% in the 2 diabetic groups. Urinary 8-epi-prostaglandin-F(2alpha)/creatinine ratio did not correlate with urinary ortho-tyrosine excretion. The difference between para-tyrosine levels of the groups is probably due to renal impairment, while there is indirect evidence for an increased tubular secretion or production of ortho-tyrosine in the kidney in diabetic patients with or without CKD.

  10. Ortho-para conversion of endohedral water in the fullerene C60 at cryogenic temperatures

    NASA Astrophysics Data System (ADS)

    Shugai, Anna; Nagel, U.; Rõõm, T.; Mamone, S.; Concistrè, M.; Meier, B.; Krachmalnicoff, A.; Whitby, R. J.; Levitt, M. H.; Lei, Xuegong; Li, Yongjun; Turro, N. J.

    2015-03-01

    Water displays the phenomenon of spin isomerism in which the two proton spins either couple to form a triplet (ortho water, I = 1) or a singlet nuclear spin state (para water, I = 0). Here we study the interconversion of para and ortho water. The exact mechanism of this process is still not fully understood. In order to minimize interactions between molecules we use a sample where a single H2O is trapped in the C60 molecular cage (H2O@C60)andH2O@C60iscrystallized.H2O@C60 has long-lived ortho state and ortho-para conversion kinetics is non-exponential at LHeT. We studied mixtures of H2O@C60, D2O@C60 and C60 using IR absorption, NMR and dielectric measurements. We saw the speeding up of the interconversion with the growth of H2O@C60 concentration in C60 or when D2O@C60 was added. At some temperatures the kinetics is exponential. Models are discussed in order to explain the T and concentration dependence of ortho-para interconversion kinetics. This work was supported by institutional research funding IUT23-3 of the Estonian Ministry of Education and Research.

  11. Reevaluating the carcinogenicity of ortho-toluidine: a new conclusion and its implications.

    PubMed

    Sellers, C; Markowitz, S

    1992-12-01

    The aromatic amine ortho-toluidine has been recognized by IARC as an animal carcinogen for the past decade. Three recent epidemiological studies of worker populations have now implicated this chemical as a human bladder carcinogen. In a study by E. Ward, A. Carpenter, S. Markowitz, D. Roberts, and W. Halperin ((1991), J. Natl. Cancer Inst. 83, 501-506), workers definitely exposed to ortho-toluidine for at least 10 years experienced a Standardized Incidence Ratio (SIR) of 27.2 (90% CI = 11.8-53.7). The other major exposure was to aniline, which significant epidemiological studies have failed to confirm as a human carcinogen. In retrospect, studies by G. F. Rubino, G. Scansetti, G. Piolatto ((1982) Environ. Res. 27, 241-254) and M. J. Stasik ((1988) Int. Arch. Occup. Environ. Health 60, 21-24) also support the hypothesis that ortho-toluidine is a human bladder carcinogen. Animal studies of both ortho-toluidine and its possible confounders in these epidemiological investigations further confirm this hypothesis. When evaluated in a suitably comprehensive way, according to the traditional standards for assessing causality outlined by A. B. Hill ((1977) A Short Textbook of Medical Statistics, pp. 288-294, Lippincott, Philadelphia) the evidence that ortho-toluidine causes human bladder cancer has become much more conclusive. In this case, animal tests have proven a good predictor of human carcinogenicity.

  12. Hydrogen-atom attack on phenol and toluene is ortho-directed.

    PubMed

    Krechkivska, Olha; Wilcox, Callan M; Troy, Tyler P; Nauta, Klaas; Chan, Bun; Jacob, Rebecca; Reid, Scott A; Radom, Leo; Schmidt, Timothy W; Kable, Scott H

    2016-03-28

    The reaction of H + phenol and H/D + toluene has been studied in a supersonic expansion after electric discharge. The (1 + 1') resonance-enhanced multiphoton ionization (REMPI) spectra of the reaction products, at m/z = parent + 1, or parent + 2 amu, were measured by scanning the first (resonance) laser. The resulting spectra are highly structured. Ionization energies were measured by scanning the second (ionization) laser, while the first laser was tuned to a specific transition. Theoretical calculations, benchmarked to the well-studied H + benzene → cyclohexadienyl radical reaction, were performed. The spectrum arising from the reaction of H + phenol is attributed solely to the ortho-hydroxy-cyclohexadienyl radical, which was found in two conformers (syn and anti). Similarly, the reaction of H/D + toluene formed solely the ortho isomer. The preference for the ortho isomer at 100-200 K in the molecular beam is attributed to kinetic, not thermodynamic effects, caused by an entrance channel barrier that is ∼5 kJ mol(-1) lower for ortho than for other isomers. Based on these results, we predict that the reaction of H + phenol and H + toluene should still favour the ortho isomer under elevated temperature conditions in the early stages of combustion (200-400 °C).

  13. The ortho/para ratio of water vapor in Comet Halley

    NASA Technical Reports Server (NTRS)

    Mumma, Michael J.; Larson, Harold P.; Weaver, Harold A.

    1986-01-01

    The ortho/para ratio of H2O is shown to be an invariant in the cometary coma. The dependence of ortho-para ratio on temperature in thermal equilibrium is given, and the nuclear-spin-temperature is defined. Its relation to the physical temperature of the cometary ices is discussed, and the prospects for using the observed ortho/para ratio to infer properties of the cometary nucleus are explored. The ortho/para ratio in Halley's comet is derived from high resolution infrared spectra of near 2.7 microns wavelength. On UT December 24.1, 1985 it was 2.73 + or - 0.17, and on UT March 22.7, 1986 it was 3.23 + or - 0.37. The nuclear-spin-temperature was 35 K (+9 K, -5 K) pre-perihelion, and less than 40 K post-perihelion, at the 67% confidence limit. Both numbers are consistent with modeled values of the equilibrium temperature of the cometary nucleus at aphelion (47 K). However, at the 95% confidence limit they are also fully consistent with temperatures less than 50 K, corresponding to an ortho/para ratio of about 3.0.

  14. The formaldehyde ortho/para ratio as a probe of dark cloud chemistry and evolution

    NASA Technical Reports Server (NTRS)

    Dickens, J. E.; Irvine, W. M.

    1999-01-01

    We present measurements of the H2CO ortho/para ratio toward four star-forming cores, L723, L1228, L1527, and L43, and one quiescent core, L1498. Combining these data with earlier results by Minh et al., three quiescent cores are found to have ortho/para ratios near 3, the ratio of statistical weights expected for gas-phase formation processes. In contrast, ortho/para ratios are 1.5-2.1 in five star-forming cores, suggesting thermalization at a kinetic temperature of 10 K. We attribute modification of the ortho/para ratio in the latter cores to formation and/or equilibration of H2CO on grains with sub-sequent release back into the gas phase due to the increased energy inputs from the forming star and outflow. We see accompanying enhancements in the H2CO abundance relative to H, to support this idea. The results suggest that the formaldehyde ortho/para ratio can differentiate between quiescent cores and those in which low-mass star formation has occurred.

  15. Adaptation of the ORTHO-15 test to Polish women and men.

    PubMed

    Brytek-Matera, Anna; Krupa, Magdalena; Poggiogalle, Eleonora; Donini, Lorenzo Maria

    2014-03-01

    There is a lack of Polish tools to measure behaviour related to orthorexia nervosa. The purpose of the present study was to validate the Polish version of the ORTHO-15 test. 341 women and 59 men (N = 400) were recruited, whose age ranged from 18 to 35 years. Mean age was 23.09 years (SD = 3.14) in women and 24.02 years (SD = 3.87) in men. The ORTHO-15 test and the EAT-26 test were used in the present study. Factor analysis (exploratory and confirmatory analysis) was used in the present study. Exploratory factor analysis performed on the initial 15 items from a random split half of the study group suggested a nine-item two-factor structure. Confirmatory factor analysis performed on the second randomly selected half of the study group supported this two-factor structure of the ORTHO-15 test. The Polish version of the ORTHO-15 test demonstrated an internal consistency (Cronbach's alpha) equal to 0.644. The Polish version of the ORTHO-15 test is a reliable and valuable instrument to assess obsessive attitudes related to healthy and proper nutrition in Polish female and male population.

  16. Influence of Molecular Oxygen on Ortho-Para Conversion of Water Molecules

    NASA Astrophysics Data System (ADS)

    Valiev, R. R.; Minaev, B. F.

    2017-07-01

    The mechanism of influence of molecular oxygen on the probability of ortho-para conversion of water molecules and its relation to water magnetization are considered within the framework of the concept of paramagnetic spin catalysis. Matrix elements of the hyperfine ortho-para interaction via the Fermi contact mechanism are calculated, as well as the Maliken spin densities on water protons in H2O and O2 collisional complexes. The mechanism of penetration of the electron spin density into the water molecule due to partial spin transfer from paramagnetic oxygen is considered. The probability of ortho-para conversion of the water molecules is estimated by the quantum chemistry methods. The results obtained show that effective ortho-para conversion of the water molecules is possible during the existence of water-oxygen dimers. An external magnetic field affects the ortho-para conversion rate given that the wave functions of nuclear spin sublevels of the water protons are mixed in the complex with oxygen.

  17. The formaldehyde ortho/para ratio as a probe of dark cloud chemistry and evolution

    NASA Technical Reports Server (NTRS)

    Dickens, J. E.; Irvine, W. M.

    1999-01-01

    We present measurements of the H2CO ortho/para ratio toward four star-forming cores, L723, L1228, L1527, and L43, and one quiescent core, L1498. Combining these data with earlier results by Minh et al., three quiescent cores are found to have ortho/para ratios near 3, the ratio of statistical weights expected for gas-phase formation processes. In contrast, ortho/para ratios are 1.5-2.1 in five star-forming cores, suggesting thermalization at a kinetic temperature of 10 K. We attribute modification of the ortho/para ratio in the latter cores to formation and/or equilibration of H2CO on grains with sub-sequent release back into the gas phase due to the increased energy inputs from the forming star and outflow. We see accompanying enhancements in the H2CO abundance relative to H, to support this idea. The results suggest that the formaldehyde ortho/para ratio can differentiate between quiescent cores and those in which low-mass star formation has occurred.

  18. The Planck-LFI flight model ortho-mode transducers

    NASA Astrophysics Data System (ADS)

    D'Arcangelo, O.; Simonetto, A.; Figini, L.; Pagana, E.; Villa, F.; Pecora, M.; Battaglia, P.; Bersanelli, M.; Butler, R. C.; Garavaglia, S.; Guzzi, P.; Mandolesi, N.; Sozzi, C.

    2009-12-01

    The Low Frequency Instrument (LFI) of the ESA Planck CMB mission is an array of 22 ultra sensitive pseudocorrelation radiometers working at 30, 44, and 70 GHz. LFI has been calibrated and delivered for integration with the satellite to the European Space Agency on November 2006. The aim of Planck is to measure the anisotropy and polarization of the Cosmic Background Radiation with a sensitivity and angular resolution never reached before over the full sky. LFI is intrinsically sensitive to polarization thanks to the use of Ortho-Mode Transducers (OMT) located between the feedhorns and the pseudo-correlation radiometers. The OMTs are microwave passive components that divide the incoming radiation into two linear orthogonal components. A set of 11 OMTs (2 at 30 GHz, 3 at 44 GHz, and 6 at 70 GHz) were produced and tested. This work describes the design, development and performance of the eleven Flight Model OMTs of LFI. The final design was reached after several years of development. At first, Elegant Bread Board OMTs were produced to investigate the manufacturing technology and design requirements. Then, a set of 3 Qualification Model (QM) OMTs were designed, manufactured and tested in order to freeze the design and the manufacturing technology for the flight units. Finally, the Flight Models were produced and tested. It is shown that all the OMT units have been accepted for flight and the electromagnetic performance is at least marginally compliant with the requirements. Mechanically, the units passed all the thermoelastic qualification tests after a reworking necessary after the QM campaign.

  19. Ortho-phthalaldehyde exposure levels among endoscope disinfection workers.

    PubMed

    Miyajima, Keiko; Yoshida, Jin; Kumagai, Shinji

    2010-01-01

    Recently, the use of ortho-phthalaldehyde (OPA) has been increasing as an alternative to glutaraldehyde for endoscope disinfection. To better understand OPA exposure and its health effects among disinfection workers, we conducted environmental monitoring and administered a questionnaire in 17 endoscope disinfection rooms. There were 9 manual disinfection rooms using immersion vats for scope disinfection and 8 automatic rooms using automatic washers. OPA exposure concentration during the disinfection process of scope was significantly higher in the manual group (median: 1.43ppb, range: not detected (ND-5.37ppb) than in the automatic group (median: 0.35 ppb, range: ND-0.69 ppb). Similarly, during charging and discharging the antiseptic solution, OPA levels were significantly higher in the manual group (median: 2.58 ppb, range: 0.92-10.0 ppb) than in the automatic group (median: 0.46ppb, range: ND-1.35 ppb). Time-weighted averages of OPA exposure concentration during work shifts were 0.33 to 1.15 ppb (median 0.66 ppb) in the manual group and 0.13 to 1.28 ppb (median 0.33 ppb) in the automatic group, which suggests that manual workers are exposed to OPA at higher levels. Among 80 female disinfection workers who used only antiseptic solutions containing OPA, the incidence of disinfection-related complaints were 10% skin, 9% eye, and 16% respiratory symptoms. These findings suggest that it is desirable to introduce automatic washers to decrease OPA exposure levels among disinfection workers.

  20. Ortho Image and DTM Generation with Intelligent Methods

    NASA Astrophysics Data System (ADS)

    Bagheri, H.; Sadeghian, S.

    2013-10-01

    Nowadays the artificial intelligent algorithms has considered in GIS and remote sensing. Genetic algorithm and artificial neural network are two intelligent methods that are used for optimizing of image processing programs such as edge extraction and etc. these algorithms are very useful for solving of complex program. In this paper, the ability and application of genetic algorithm and artificial neural network in geospatial production process like geometric modelling of satellite images for ortho photo generation and height interpolation in raster Digital Terrain Model production process is discussed. In first, the geometric potential of Ikonos-2 and Worldview-2 with rational functions, 2D & 3D polynomials were tested. Also comprehensive experiments have been carried out to evaluate the viability of the genetic algorithm for optimization of rational function, 2D & 3D polynomials. Considering the quality of Ground Control Points, the accuracy (RMSE) with genetic algorithm and 3D polynomials method for Ikonos-2 Geo image was 0.508 pixel sizes and the accuracy (RMSE) with GA algorithm and rational function method for Worldview-2 image was 0.930 pixel sizes. For more another optimization artificial intelligent methods, neural networks were used. With the use of perceptron network in Worldview-2 image, a result of 0.84 pixel sizes with 4 neurons in middle layer was gained. The final conclusion was that with artificial intelligent algorithms it is possible to optimize the existing models and have better results than usual ones. Finally the artificial intelligence methods, like genetic algorithms as well as neural networks, were examined on sample data for optimizing interpolation and for generating Digital Terrain Models. The results then were compared with existing conventional methods and it appeared that these methods have a high capacity in heights interpolation and that using these networks for interpolating and optimizing the weighting methods based on inverse

  1. Comparison of corneal epitheliotrophic capacities among human platelet lysates and other blood derivatives

    PubMed Central

    Huang, Chien-Jung; Sun, Yi-Chen; Christopher, Karen; Pai, Amy Shih-I; Lu, Chia-Ju; Hu, Fung-Rong; Lin, Szu-Yuan; Chen, Wei-Li

    2017-01-01

    Purpose To evaluate the corneal epitheliotropic abilities of two commercialized human platelet lysates (HPLs) and to compare the results with other blood derivatives, including human peripheral serum (HPS) and bovine fetal serum (FBS). Methods In vitro, human corneal epithelial cells were incubated in various concentrations (0%, 3%, 5% and 10%) of blood derivatives. Two commercialized HPLs, including UltraGRO TM (Helios, Atlanta, GA) and PLTMax (Mill Creek, Rochester, MI), were tested and compared with HPS and FBS. Scratch-induced directional wounding assay was performed to evaluate cellular migration. MTS assay was used to evaluate cellular proliferation. Cellular differentiation was examined by scanning electron microscopy, inverted microscopy and transepithelial electrical resistance. Sprague-Dawley rats were used to evaluate the effects of the blood derivatives on corneal epithelial wound healing in vivo. Different blood derivatives were applied topically every 2 hours for 2 days after corneal epithelial debridement. The concentrations of epidermal growth factor (EGF), transforming growth factor -β1 (TGF-β1), fibronectin, platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, and hyaluronic acid in different blood derivatives were evaluated by enzyme-linked immunosorbent assay (ELISA). Results In vitro experiments demonstrated statistically comparable epitheliotropic characteristics in cellular proliferation, migration, and differentiation for the two commercialized HPLs compared to FBS and HPS. Cells cultured without any serum were used as control group. The epitheliotropic capacities were statistically higher in the two commercialized HPLs compared to the control group (p<0.05). Among the different concentrations of blood derivatives, the preparations with 3% yielded better outcomes compared to 5% and 10%. In rats, HPLs also caused improved but not statistically significant wound healing compared to HPS. All the blood derivatives had better wound healing

  2. Detection of peptidoglycan and endotoxin in dialysate, using silkworm larvae plasma and limulus amebocyte lysate methods.

    PubMed

    Tsuchida, K; Takemoto, Y; Yamagami, S; Edney, H; Niwa, M; Tsuchiya, M; Kishimoto, T; Shaldon, S

    1997-01-01

    Silkworm larvae plasma (SLP) reagent is activated by peptidoglycan (PG), a fragment of both the gram-positive and gram-negative bacterial cell wall, as well as beta-glucan (BG), a component of fungi. It is possible to measure contamination of gram-positive bacteria quantitatively by combining the conventional limulus amebocyte lysate (LAL) and PG measurement methods. Therefore, a more highly accurate analysis of dialysate can be made using both SLP and LAL methods to detect endotoxin (ET) and/or PG contamination. We studied the effects of contaminated dialysate on human peripheral blood mononuclear cells (PBMC) by producing various cytokines in vitro. Muramyl dipeptide (MDP) was used as the biologically active minimum constituent of PG. A total of 54 dialysate samples were obtained under sterile conditions from 4 sites: (1) reverse osmosis water unit; (2) proportioning unit; (3) multiple dialysate preparation console, and (4) personal dialysate preparation console, at 9 dialysis facilities. To detect bacterial contamination, the samples were measured with LAL(C), LAL(G) and SLP methods. PBMC were collected from 10 healthy controls and from 10 hemodialysis patients and cultured for 24 h with ET, MDP, ET + MDP and contaminated dialysate. IL-1 receptor antagonist (IL-1Ra), IL-1 beta and TNF-alpha in the culture medium supernatants were measured using the ELISA method. PG was not detected in dialysate from sites 1 or 2. However, dialysate from the inlet of the dialyzer at the bedside monitor of the central supply and personal console showed 4.1 +/- 6.1 ng/ml for site 3 (in 7 of 18 samples) and 3.3 +/- 4.6 ng/ml for site 4 (in 3 of 18 samples). Contamination by PG alone and complex contamination by PG and ET were also detected. Furthermore, IL-1Ra, IL-1 beta and TNF-alpha production by PBMC increased in accordance with the concentrations of MDP. Cytokine production was enhanced 5-10 times more where MDP and ET coexisted than where either MDP or ET existed alone, showing

  3. Transcutaneous antigen delivery system

    PubMed Central

    Lee, Mi-Young; Shin, Meong-Cheol; Yang, Victor C.

    2013-01-01

    Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24] PMID:23351379

  4. [ICO-166 monoclonal antibodies against the CD45RA antigen].

    PubMed

    Frolova, E A; Baryshnikov, A Iu; Novikov, V V; Syrkin, A B

    1993-07-01

    Monoclonal antibodies (MCA) ICO-166 against CD45RA antigen were generated and characterized. In the indirect IFA, MCA ICO-166 reacted with 54.1 +/- 1.9% lymphocytes of human peripheral blood and 15.2 +/- 2.3% monocytes but not with granulocytes or thrombocytes. The method of double labelling of cells demonstrated that MCA ICO-166 detected all B-lymphocytes, all NK-cells and 31% of mature T-lymphocytes but only 55% of CD8 suppressor cells and only 21% of CDA helper cells carried this antigen on the surface. Experiments were carried out to block binding of FITC-labeled MCA ALB11 against CD45RA antigen with human lymphocytes by pretreatment of cells with different concentrations of MCA ICO-166. Treatment of cells with MCA ALB11 blocked binding of MCA ALB11-FITC by 85% on the average. MCA ICO-166 blocked binding of MCA ALB11-FITC by 66% on the average. When different dilutions of MCA ICO-166 were used, the dose-dependent effect of blocking of MCA ALB11-FITC binding was observed. MCA ICO-166 immunoprecipitated a protein band of molecular weight 220 kDa from lysates of mononuclear cells of the human peripheral blood.

  5. Ortho effect in electron ionization mass spectrometry of N-acylanilines bearing a proximal halo substituent.

    PubMed

    Jariwala, Freneil B; Figus, Margaret; Attygalle, Athula B

    2008-08-01

    Electron ionization (EI) mass spectra are not very helpful for characterizing ortho, meta, and para isomers of underivatized haloanilines since their spectra are virtually identical. In contrast, when the amino group of chloro-, bromo-, or iodoanilines is transformed to an N-formyl, N-acetyl, or N-benzoyl derivative, the spectra of the derivatives reveal a highly dramatic loss of a halogen radical, instead of an HX elimination usually expected from an "ortho effect." For example, the spectra of N-formyl, N-acetyl, and N-benzoyl derivatives of ortho isomers of chloro-, bromo-, and iodoanilines show a very prominent peak at m/z 120, 134, and 196, respectively, for the loss of the corresponding halogen atom.

  6. Evolution of tRNA Repertoires in Bacillus Inferred with OrthoAlign.

    PubMed

    Tremblay-Savard, Olivier; Benzaid, Billel; Lang, B Franz; El-Mabrouk, Nadia

    2015-06-01

    OrthoAlign, an algorithm for the gene order alignment problem (alignment of orthologs), accounting for most genome-wide evolutionary events such as duplications, losses, rearrangements, and substitutions, was presented. OrthoAlign was used in a phylogenetic framework to infer the evolution of transfer RNA repertoires of 50 fully sequenced bacteria in the Bacillus genus. A prevalence of gene duplications and losses over rearrangement events was observed. The average rate of duplications inferred in Bacillus was 24 times lower than the one reported in Escherichia coli, whereas the average rates of losses and inversions were both 12 times lower. These rates were extremely low, suggesting a strong selective pressure acting on tRNA gene repertoires in Bacillus. An exhaustive analysis of the type, location, distribution, and length of evolutionary events was provided, together with ancestral configurations. OrthoAlign can be downloaded at: http://www.iro.umontreal.ca/~mabrouk/.

  7. The ortho-substituent effect on the Ag-catalysed decarboxylation of benzoic acids.

    PubMed

    Grainger, Rachel; Cornella, Josep; Blakemore, David C; Larrosa, Igor; Campanera, Josep M

    2014-12-08

    A combined experimental and computational investigation on the Ag-catalysed decarboxylation of benzoic acids is reported herein. The present study demonstrates that a substituent at the ortho position exerts dual effects in the decarboxylation event. On one hand, ortho-substituted benzoic acids are inherently destabilised starting materials compared to their meta- and para-substituted counterparts. On the other hand, the presence of an ortho-electron-withdrawing group results in an additional stabilisation of the transition state. The combination of both effects results in an overall reduction of the activation energy barrier associated with the decarboxylation event. Furthermore, the Fujita-Nishioka linear free energy relationship model indicates that steric bulk of the substituent can also exert a negative effect by destabilising the transition state of decarboxylation.

  8. Poly(ortho-phenylenediamine-co-aniline) based copolymer with improved capacitance

    NASA Astrophysics Data System (ADS)

    Olmedo-Martínez, Jorge L.; Farías-Mancilla, Bárbara I.; Vega-Rios, Alejandro; Zaragoza-Contreras, E. Armando

    2017-10-01

    A poly(ortho-phenylenediamine-co-aniline) copolymer is synthesized via the oxidative route, using a 1:1 M ratio of aniline to ortho-phenylenediamine (oPDA) and ammonium persulfate as the oxidizing agent. Infrared spectroscopy indicates that the copolymer contains the functional groups typically present in polyaniline and poly(ortho-phenylenediamine); whereas UV-vis-NIR spectroscopy shows that the copolymer adopts a phenazine-type structure. Cyclic voltammetry evidences the copolymer synthesis, as a redox peak at -65 mV, different from those exhibited by polyaniline (160 mV and 600 mV) or poly(o-phenylenediamine) (-240 mV) is observed. Finally, electrochemical impedance spectroscopy and the charge/discharge test provide support to propose the copolymer application in electrodes for supercapacitors.

  9. Electrical detection of ortho-para conversion in fullerene-encapsulated water

    NASA Astrophysics Data System (ADS)

    Meier, Benno; Mamone, Salvatore; Concistrè, Maria; Alonso-Valdesueiro, Javier; Krachmalnicoff, Andrea; Whitby, Richard J.; Levitt, Malcolm H.

    2015-08-01

    Water exists in two spin isomers, ortho and para, that have different nuclear spin states. In bulk water, rapid proton exchange and hindered molecular rotation obscure the direct observation of two spin isomers. The supramolecular endofullerene H2O@C60 provides freely rotating, isolated water molecules even at cryogenic temperatures. Here we show that the bulk dielectric constant of this substance depends on the ortho/para ratio, and changes slowly in time after a sudden temperature jump, due to nuclear spin conversion. The attribution of the effect to ortho-para conversion is validated by comparison with nuclear magnetic resonance and quantum theory. The change in dielectric constant is consistent with an electric dipole moment of 0.51+/-0.05 Debye for an encapsulated water molecule, indicating the partial shielding of the water dipole by the encapsulating cage. The dependence of bulk dielectric constant on nuclear spin isomer composition appears to be a previously unreported physical phenomenon.

  10. Environmental occurrence and potential toxicity of planar, mono-, and di-ortho polychlorinated biphenyls in the biota

    SciTech Connect

    Hong, C.S.; Xiao, J.; Bush, B.

    1995-12-31

    Coplanar PCBs without ortho-chlorine substituents and semicoplanar PCBs with one ortho-chlorination are stereochemically similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and, because of this similarity, exert biochemical activity and toxicity to biota comparable to that of TCDD. Four non-ortho-, eight mono-ortho-, and two di-ortho-chlorinated congeners have been determined in insect larvae, fresh water and salt water mussels, fish, mallard duck, seals, and in human milk and adipose tissue. The PCB congeners are separated from the remainder of PCBs by activated carbon chromatography or HPLC on porous graphitic carbon followed by gas chromatography with electron capture detection. PCB toxic equivalency factors (TEFs) (1) recommended by WHO for 3 non-ortho, 8 mono-ortho and 2 di-ortho PCBs and a TEF for congener 81 (3,4,4{prime},5-tetrachlorobiphenyl) suggested by Harris et al. from AHH induction in rainbow trout (2) were used for calculation of the contribution to dioxin-like toxicity to each life form. In all the biota examined, PCB congener number 126 was the major contributor to PCB toxic equivalents (TEQs), followed by congener numbers 118, 114, 156, and 105. The ability to separate out planar PCBs from the majority of PCBs has allowed the use of TCDD toxicity equivalence to compare the relative dioxin-like potency of PCB residues in various species from 12 different locations.

  11. 78 FR 51733 - Draft Report on Carcinogens Monographs for ortho-Toluidine and Pentachlorophenol and By-Products...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-21

    ... HUMAN SERVICES National Institutes of Health Draft Report on Carcinogens Monographs for ortho-Toluidine... Carcinogens (RoC) Monographs for ortho-Toluidine and Pentachlorophenol and By-products of its Synthesis... on Carcinogens (ORoC), Division of the National Toxicology Program (DNTP), National Institute of...

  12. 78 FR 67371 - Draft Report on Carcinogens Monographs for ortho-Toluidine and Pentachlorophenol and By-products...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-12

    ... HUMAN SERVICES National Institutes of Health Draft Report on Carcinogens Monographs for ortho-Toluidine... Report on Carcinogens (RoC) Monographs for ortho-Toluidine and Pentachlorophenol and By-products of its... the Report on Carcinogens (ORoC), Division of the National Toxicology Program (DNTP), National...

  13. Toxicological significance of non-, mono- and di-ortho-substituted polychlorinated biphenyls in oysters from Galveston and Tampa bays

    SciTech Connect

    Sericano, J.L.; Safe, S.H.; Wade, T.L.; Brooks, J.M. )

    1994-11-01

    Concentrations of non-ortho (77, 126, and 169), mono-ortho (105 and 118) and di-ortho (128 and 138)-substituted PCB congeners were measured in oysters from Galveston and Tampa bays, and reported toxic equivalent factors were used to assess their toxicity. Most of the relative toxicity encountered in the oysters analyzed during this study was due to the presence of planar non-ortho-PCBs, particularly congener 126. In contrast, the contribution of di-ortho-substituted PCB congeners to the total relative toxicity of the samples was negligible. On average, the contribution of each of these non-, mono-, and di-ortho-substituted PCB congeners to the total toxicity encountered in oysters from Galveston and Tampa bays were 126 > 118 [ge] 169 [ge] 105 > 77 [much gt] 138 > 128 and 126 > 118 > 169 [ge] 77 > 105 [much gt] 138 > 128, respectively. Based on the reported lower clearance rates of non-ortho- and mono-ortho-substituted PCB congeners compared to other congeners within the same chlorination level, contaminated oysters that are depurated in clean environments will lower their total PCB concentrations, but their original toxicity may not be proportionally reduced.

  14. A convenient synthesis of anthranilic acids by Pd-catalyzed direct intermolecular ortho-C-H amidation of benzoic acids.

    PubMed

    Ng, Ka-Ho; Ng, Fo-Ning; Yu, Wing-Yiu

    2012-12-11

    An efficient method for synthesis of anthranilic acids by Pd-catalyzed ortho-C-H amidation of benzoic acids is disclosed. The amidation is proposed to proceed by carboxylate-assisted ortho-C-H palladation to form an arylpalladium(II) complex, followed by nitrene insertion to the Pd-C bond.

  15. 17O NMR studies of ortho-substituent effects in substituted phenyl tosylates.

    PubMed

    Nummert, Vilve; Mäemets, Vahur; Piirsalu, Mare; Koppel, Ilmar A

    2012-10-01

    (17)O NMR spectra for 35 ortho-, para-, and meta-substituted phenyl tosylates (phenyl 4-methylbenzenesulfonates), 4-CH(3)-C(6)H(4) SO(2)OC(6)H(4)-X, at natural abundance in acetonitrile at 50 °C were recorded. The (17)O NMR chemical shifts, δ((17)O), of the sulfonyl (SO(2)) and the single-bonded phenoxy (OPh) oxygens for para and meta derivatives correlated well with dual substituent parameter treatment using the Taft inductive, σ(I), and resonance, σº(R), constants. The influence of ortho substituents on the sulfonyl oxygen and the single-bonded phenoxy oxygen chemical shifts, δ((17)O), was found to be nicely described by the Charton equation: δ((17)O)(ortho) = δ((17)O)(H) + ρ(I)σ(I) + ρ(R)σ°(R) + δE(s)(B) when the data treatment was performed separately for electron-donating +R substituents and electron-attracting -R substituents. Electron-attracting meta and para substituents in the phenyl moiety caused deshielding while the electron-donating meta, para and ortho +R substituents produce shielding effects on the sulfonyl (SO(2)) and single-bonded phenoxy (OPh) oxygens. The influence of ortho inductive and resonance effects in the case of +R substituents was found to be approximately twice higher than the corresponding influence from the para position. Due to the steric effect of ortho substituents a decrease in shielding of the oxygens at the sulfonyl group (δE(s)(B) > 0, E(s)(B) < 0) was detected.

  16. Characterization of Slosh Damping for Ortho-Grid and Iso-Grid Internal Tank Structures

    NASA Technical Reports Server (NTRS)

    Westra, Douglas G.; Sansone, Marco D.; Eberhart, Chad J.; West, Jeffrey S.

    2016-01-01

    Grid stiffened tank structures such as Ortho-Grid and Iso-Grid are widely used in cryogenic tanks for providing stiffening to the tank while reducing mass, compared to tank walls of constant cross-section. If the structure is internal to the tank, it will positively affect the fluid dynamic behavior of the liquid propellant, in regard to fluid slosh damping. As NASA and commercial companies endeavor to explore the solar system, vehicles will by necessity become more mass efficient, and design margin will be reduced where possible. Therefore, if the damping characteristics of the Ortho-Grid and Iso-Grid structure is understood, their positive damping effect can be taken into account in the systems design process. Historically, damping by internal structures has been characterized by rules of thumb and for Ortho-Grid, empirical design tools intended for slosh baffles of much larger cross-section have been used. There is little or no information available to characterize the slosh behavior of Iso-Grid internal structure. Therefore, to take advantage of these structures for their positive damping effects, there is much need for obtaining additional data and tools to characterize them. Recently, the NASA Marshall Space Flight Center conducted both sub-scale testing and computational fluid dynamics (CFD) simulations of slosh damping for Ortho-Grid and Iso-Grid tanks for cylindrical tanks containing water. Enhanced grid meshing techniques were applied to the geometrically detailed and complex Ortho-Grid and Iso-Grid structures. The Loci-STREAM CFD program with the Volume of Fluid Method module for tracking and locating the water-air fluid interface was used to conduct the simulations. The CFD simulations were validated with the test data and new empirical models for predicting damping and frequency of Ortho-Grid and Iso-Grid structures were generated.

  17. Serological studies of actionomyces israelii by crossed immunoelectrophoresis: standard antigen-antibody system for A. israelii.

    PubMed

    Holmberg, K; Nord, C E; Wadström, T

    1975-08-01

    Standard preparations of crude cytoplasmic and whole cell-associated antigen mixtures of Actinomyces israelii were analyzed by crossed immunoelectrophoresis (CIE), with a standard polyvalent antiserum comprising purified and concentrated immunoglobulin G antibodies to formolized whole cells of A. israelii serotypes 1 and 2. The standard antigens provided four antigen-antibody systems for A. israelii. The immunoprecipitation patterns of the system were compared, and the immunochemical characteristics of individual precipitates were analyzed. Each system contained specific precipitates, but also one or two precipitates which were immunochemically identical to precipitates of the other systems. The standard system for A. israelii based on cytoplasmic antigens was best reproducible and revealed the highest number of immunoprecipitates. These precipitates possessed immunochemical properties which made them suitable for CIE studies. The cytoplasmic antigen mixture of A. israelii was, therefore, adopted as the most suitable for further development of a crossed immunoelectrophoretic system for A. israelii. In subsequent assays the cytoplasmic antigen mixture was raised in rabbit against cell lysates of A. israelii, serotypes 1 and 2. A standard antigen-antibody system for A. israelii was obtained which revealed an immunoprecipitation pattern of 10 distinguishable precipitates. The resolving power and separation by CIE of this standard system for A. israelii was compared with that of crossed immunoelectrofocusing. The results suggest that these methods supplement each other. Crossed immunoelectrofocusing appeared to be a useful tool for separation of specific components of the protein-antigen complex of A. israelii for analytic serology. The CIE in conjunction with a standard reference antigen-antibody system for A. israelii based on cytoplasmic antigens offers great potentialities in diagnostic A. israelii serology.

  18. Trypanosoma vivax GM6 Antigen: A Candidate Antigen for Diagnosis of African Animal Trypanosomosis in Cattle

    PubMed Central

    Pillay, Davita; Izotte, Julien; Fikru, Regassa; Büscher, Philipe; Mucache, Hermogenes; Neves, Luis; Boulangé, Alain; Seck, Momar Talla; Bouyer, Jérémy; Napier, Grant B.; Chevtzoff, Cyrille; Coustou, Virginie; Baltz, Théo

    2013-01-01

    Background Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km2 of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases. Methodology/Principle Findings the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4). Conclusion/Significance this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle. PMID:24205263

  19. Efficient use of the iron ortho-nitrophenylporphyrin chloride to mimic biological oxidations of dimethylaminoantipyrine.

    PubMed

    Bazin, Marc J; Shi, Helen; Delaney, Jeannine; Kline, Billie; Zhu, Zhendong; Kuhn, Cyrille; Berlioz, Francoise; Farley, Kathleen A; Fate, Gwen; Lam, Wing; Walker, Gregory S; Yu, Linning; Pollastri, Michael P

    2007-10-01

    Major metabolites of dimethylaminoantipyrine have been synthesized using iron ortho-nitrophenylporphyrin chloride as biomimetic catalyst. Reactivity of iron tetrakis-ortho-nitrophenylporphyrin chloride [Fe(TNO2PP)Cl] has been compared with iron tetrakis-pentafluorophenylporphyrin chloride and iron tetrakis-2,6-dichlorophenylporphyrin chloride using various oxidants such as hydrogen peroxide, iodosobenzene, and cumene hydroperoxide in either protic or aprotic solvent. Effect of imidazole has been showed on the reactivity of Fe(TNO2PP)Cl/cumene hydroperoxide system.

  20. ortho-Amidoalkylation of phenols via tandem one-pot approach involving oxazine intermediate.

    PubMed

    Mudududdla, Ramesh; Jain, Shreyans K; Bharate, Jaideep B; Gupta, Ajai P; Singh, Baldev; Vishwakarma, Ram A; Bharate, Sandip B

    2012-10-05

    A new and efficient method for ortho-amidoalkylation of phenols via Mannich-type condensation with formaldehyde and lactams using recyclable solid acid catalyst is described. This is the first report for ortho-amidoalkylation of phenols by lactams via Mannich-type condensation. LC-ESI-MS/MS based mechanistic study revealed that reaction proceeds through o-quinone methide (o-QM) and an oxazine intermediate via tandem Knoevenagel condensation, formal [4 + 2]-Diels-Alder cycloaddition and acid catalyzed oxazine ring-opening.

  1. Intramolecular interactions in ortho-methoxyalkylphenylboronic acids and their catechol esters

    NASA Astrophysics Data System (ADS)

    Adamczyk-Woźniak, Agnieszka; Borys, Krzysztof M.; Czerwińska, Karolina; Gierczyk, Błażej; Jakubczyk, Michał; Madura, Izabela D.; Sporzyński, Andrzej; Tomecka, Ewelina

    2013-12-01

    Catechol esters of ortho-methoxyalkylphenylboronic acids have been synthesized and characterized by 17O NMR spectroscopy. The results were compared with the data for the parent acids. The influence of intramolecular and intermolecular hydrogen bonds on the properties of the boronic acids has been discussed. The 17O NMR data for the boronic esters proved that there are no O → B interactions in the investigated compounds. This fact is connected with weak Lewis acidity of the parent acids and their low sugars' receptors activity. Crystal structure of ortho-methoxyphenylboronic acid catechol ester was determined.

  2. Toxicogenomic response of Pseudomonas aeruginosa to ortho-phenylphenol

    PubMed Central

    Nde, Chantal W; Jang, Hyeung-Jin; Toghrol, Freshteh; Bentley, William E

    2008-01-01

    Background Pseudomonas aeruginosa (P. aeruginosa) is the most common opportunistic pathogen implicated in nosocomial infections and in chronic lung infections in cystic fibrosis patients. Ortho-phenylphenol (OPP) is an antimicrobial agent used as an active ingredient in several EPA registered disinfectants. Despite its widespread use, there is a paucity of information on its target molecular pathways and the cellular responses that it elucidates in bacteria in general and in P. aeruginosa in particular. An understanding of the OPP-driven gene regulation and cellular response it elicits will facilitate more effective utilization of this antimicrobial and possibly lead to the development of more effective disinfectant treatments. Results Herein, we performed a genome-wide transcriptome analysis of the cellular responses of P. aeruginosa exposed to 0.82 mM OPP for 20 and 60 minutes. Our data indicated that OPP upregulated the transcription of genes encoding ribosomal, virulence and membrane transport proteins after both treatment times. After 20 minutes of exposure to 0.82 mM OPP, genes involved in the exhibition of swarming motility and anaerobic respiration were upregulated. After 60 minutes of OPP treatment, the transcription of genes involved in amino acid and lipopolysaccharide biosynthesis were upregulated. Further, the transcription of the ribosome modulation factor (rmf) and an alternative sigma factor (rpoS) of RNA polymerase were downregulated after both treatment times. Conclusion Results from this study indicate that after 20 minutes of exposure to OPP, genes that have been linked to the exhibition of anaerobic respiration and swarming motility were upregulated. This study also suggests that the downregulation of the rmf and rpoS genes may be indicative of the mechanism by which OPP causes decreases in cell viability in P. aeruginosa. Consequently, a protective response involving the upregulation of translation leading to the increased synthesis of

  3. Rhizodeposition flux of competitive versus conservative graminoid: contribution of exudates and root lysates as affected by N loading

    NASA Astrophysics Data System (ADS)

    Kastovska, Eva; Edwards, Keith; Santruckova, Hana

    2017-04-01

    Carbon allocation pattern represents the plant strategy for growth and nutrient capture. Plants exhibit high plasticity in their allocation pattern and belowground C partitioning in response to changes in the availability of nutrients limiting their production, namely nitrogen (N). Any shift in the belowground C fluxes and partitioning between root production, exudation and other rhizodeposits could affect the soil microbial activity and soil organic matter turnover. We studied the influence of N availability on plant allocation patterns with emphasis on belowground C fluxes of two wetland graminoids, the competitive Glyceria maxima and the conservative Carex acuta. Plants were grown in pots under two levels of N availability. We combined pulse-labeling of plants with 13CO2 to track recent assimilates with estimation of the root death rate calculated from the difference between gross and net root growth rates for assessing the rhizodeposition flux to soil, and the contribution of root exudates and lysates from root turnover. We found that higher N supply enhanced root biomass and, subsequently, the total rhizodeposition. Both species shifted partitioning of belowground C towards higher mass-specific root production and turnover, with lower investments into root exudation. Therefore, the rhizodeposition flux was enriched in root-derived lysates over soluble exudates. Root exudates accounted for 50-70% of the rhizodeposition flux in conditions of low N availability, while it was only 20-40% under high N availability. The N fertilization induced changes in belowground C fluxes were species-specific, with more pronounced changes in the conservative Carex than the competitive Glyceria. In summary, soil N loading enhanced total C rhizodeposition and, simultaneously, the proportion of predominantly more complex root lysates over soluble root exudates, with potential implications for soil organic matter dynamics. Our results further stress the importance of species

  4. Dendritic cells loaded with pancreatic Cancer Stem Cells (CSCs) lysates induce antitumor immune killing effect in vitro.

    PubMed

    Yin, Tao; Shi, Pengfei; Gou, Shanmiao; Shen, Qiang; Wang, Chunyou

    2014-01-01

    According to the cancer stem cells (CSCs) theory, malignant tumors may be heterogeneous in which a small population of CSCs drive the progression of cancer. Because of their intrinsic abilities, CSCs may survive a variety of treatments and then lead to therapeutic resistance and cancer recurrence. Pancreatic CSCs have been reported to be responsible for the malignant behaviors of pancreatic cancer, including suppression of immune protection. Thus, development of immune strategies to eradicate pancreatic CSCs may be of great value for the treatment of pancreatic cancer. In this study, we enriched pancreatic CSCs by culturing Panc-1 cells under sphere-forming conditions. Panc-1 CSCs expressed low levels of HLA-ABC and CD86, as measured by flow cytometry analysis. We further found that the Panc-1 CSCs modulate immunity by inhibiting lymphocyte proliferation which is promoted by phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies. The monocyte derived dendritic cells (DCs) were charged with total lysates generated from Panc-1 CSCs obtained from tumor sphere culturing. After co-culturing with lymphocytes at different ratios, the Panc-1 CSCs lysates modified DC effectively promoted lymphocyte proliferation. The activating efficiency reached 72.4% and 74.7% at the ratios of 1∶10 and 1∶20 with lymphocytes. The activated lymphocytes secreted high levels of INF-γ and IL-2, which are strong antitumor cytokines. Moreover, Panc-1 CSCs lysates modified DC induced significant cytotoxic effects of lymphocytes on Panc-1 CSCs and parental Panc-1 cells, respectively, as shown by lactate dehydrogenase (LDH) assay. Our study demonstrates that the development of CSCs-based vaccine is a promising strategy for treating pancreatic cancer.

  5. Measurement of intracellular accumulation of anthracyclines in cancerous cells by direct injection of cell lysate in MEKC/LIF detection.

    PubMed

    Mbuna, Julius; Kaneta, Takashi; Imasaka, Totaro

    2010-04-01

    Anthracyclines are chemotherapeutic drugs that are broadly used in the treatment of various types of solid cancers and leukemia. Herein, we report on a novel analytical method for intracellular accumulation of anthracyclines using MEKC/LIF detection. An aqueous separation system permitted the injection of cell lysates directly into the capillary. The MEKC migrating solution was made up of borate buffer at pH 9.22 containing sodium taurodeoxycholate, (2-hydroxypropyl)-gamma-CD, and SDS. The anthracyclines, Doxorubicin (DOX) and epirubicin (EPI) were detected by LIF using a Nd:YAG laser (532 nm) or an argon ion laser (488 nm) for excitation. Two cell lines, human humerus tumor cells (RDES) and human lung tumor cells (A549), were treated with a mixture of the two anthracyclines for fixed periods of time, and then intracellular concentrations were determined by injecting cell lysates directly. Recovery values of 96.0-100.8% were obtained for DOX and EPI. Reproducibility quantified by RSD was less than 3.9% intraday and 6.7% interday at concentrations ranging between 50 and 500 nM. The uptake of EPI was found to be slightly less than that of DOX for A549, but higher levels of EPI were observed in RDES. Intracellular accumulation of anthracyclines was greater in RDES than in A549, but both types of cells excreted anthracyclines after 12 h. These results indicate that MEKC with an aqueous medium is useful for investigating intracellular uptake and accumulation of drugs, since cell lysates can be used directly with no pretreatment such as deproteination or solvent extraction of analytes.

  6. [Weak coherent radiation of OH and ortho- H2O space masers as a carrier in biocommunication: the ortho/para-conversion of H2O spin isomers?].

    PubMed

    Pershin, S M

    2010-01-01

    A conception of biocommunication based on the principle of radiophysics stating that the carrier modulation takes place at the resonance frequency in the transmission-receiver system has been substantiated and proved. The coherent radiation of space OH-masers (1.6-1.7 GHz) and ortho-H2O-maser (22.3 GHz) is proposed as a source of the carrier frequency. The narrow lines of rotational transition of H2O and OH molecules in liquid water were proposed to be considered as an analog of selective resonances of transmitter and receiver in radiocommunication. The possibility of the ortho-para conversion of H2O spin isomers, induced by weak electromagnetic fields, is discussed.

  7. Isolation and partial characterization of an immunogenic antigen of Giardia lamblia.

    PubMed

    Quintero, Jael; Valdez, Alejandra; Samaniego, Brenda; Lopez-Romero, Gloria; Astiazaran-Garcia, Humberto; Rascon, Lucila; Breci, Linda; Garibay-Escobar, Adriana; Robles-Zepeda, Ramón; Velazquez, Carlos

    2017-06-01

    Humoral and cellular immune responses play an important role during Giardia lamblia infection. Several Giardia proteins have been identified as immunogenic antigens based on their elicited humoral immune response. Poorly is known about Giardia antigens that stimulate a cellular immune response. The main purpose of this study was to isolate and partial characterize an immunogenic antigen (5G8) of G. lamblia. The 5G8 protein was isolated from G. lamblia trophozoite lysates by affinity chromatography using moAb 5G8-coupled CNBr-Sepharose. The isolated protein was analysed by electrospray tandem mass spectrometry (ESI-MS/MS), and by diverse bioinformatics tools (GiardiaDB, BLASTn, BLASTp and ExPASy). Additionally, several biochemical and immunological characteristics of the isolated protein were analysed. By ESI-MS/MS the amino acidic 5G8 sequence was deduced. The 5G8 antigen belongs to the VSP family proteins of G. lamblia. This protein is composed by one polypeptide chain (±71kDa). Using the algorithm SYFPHEITI, we identified candidate CD4(+) T-cell epitopes from the 5G8 antigen, which can elicit cell-mediated immune responses. In this study, we have identified a G. lamblia protein that induces a strong immune response in infected mice. The biochemical and immunological characterization of the immunogenic 5G8 antigen may contribute to the rational design of a Giardia vaccine. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  8. Distinct antigen recognition pattern during zoonotic visceral leishmaniasis in humans and dogs.

    PubMed

    Goto, Yasuyuki; Howard, Randall F; Bhatia, Ajay; Trigo, Joelma; Nakatani, Maria; Netto, Eduardo M; Reed, Steven G

    2009-03-23

    Leishmania infantum is a causative agent of endemic zoonotic visceral leishmaniasis (VL) in regions of South America and the Mediterranean. Dogs are the major reservoirs for L. infantum in these regions, and control of disease in dogs could have a significant impact on human disease. Although dogs share many symptoms of VL with humans as a result of L. infantum infection, they also show some unique clinical manifestations, which are often a combination of visceral and cutaneous leishmaniasis, suggesting different mechanisms of disease development in dogs and humans. Here, we compare antibody responses of dogs and humans with VL to various defined leishmanial antigens. Parasite lysate and K39, the two most commonly used antigens for serodiagnosis of VL, detected the highest levels of antibodies in both humans and dogs with VL, whereas the recognition patterns of these antigens were distinct between the hosts. Among other defined antigens tested, LmSTI1 and CPB detected higher levels of antibodies in dogs and humans, respectively. These results indicate there is a difference between humans and dogs in antigen recognition patterns during VL. We infer that different strategies may need to be used in development of vaccines and diagnostics for humans and for dogs. In addition, we show a correlation between antibody titers to several antigens and severity of clinical symptoms during canine VL.

  9. Injections of adipose tissue-derived stem cells and stem cell lysate improve recovery of erectile function in a rat model of cavernous nerve injury

    PubMed Central

    Albersen, Maarten; Fandel, Thomas M.; Lin, Guiting; Wang, Guifang; Banie, Lia; Lin, Ching-Shwun; Lue, Tom F.

    2013-01-01

    Introduction Erectile dysfunction (ED) remains a major complication after radical prostatectomy. The use of adipose tissue-derived stem cells (ADSC) has shown promising results for the treatment of ED. However, the mechanisms of action for stem cell therapy remain controversial, with increasing evidence pointing to paracrine pathways. Aim To determine the effects and to identify the mechanism of action of ADSC and ADSC-derived lysate in a rat model of cavernous nerve (CN) crush injury. Methods Thirty-two male Sprague-Dawley rats were randomly divided into four equal groups: one group underwent sham operation, while three groups underwent bilateral CN crush. Crush-injury groups were treated at the time of injury with intracavernous injection of ADSC, lysate, or vehicle only (injured controls). Erectile function was assessed by cavernous nerve electrostimulation at 4 weeks. Penile tissue was collected for histology. Main Outcome Measures Intracavernous pressure increase upon CN stimulation; neuronal nitric oxide synthase (nNOS) content in the dorsal penile nerve; smooth muscle content, collagen content, and number of apoptotic cells in the corpus cavernosum. Results Both ADSC and lysate treatments resulted in significant recovery of erectile function, as compared to vehicle treatment. nNOS content was preserved in both the ADSC and lysate group, with significantly higher expression compared to vehicle-treated animals. There was significantly less fibrosis and a significant preservation of smooth muscle content in the ADSC and lysate groups compared to injured controls. The observed functional improvement after lysate injection supports the hypothesis that ADSC act through release of intracellular preformed substances or by active secretion of certain biomolecules. The underlying mechanism of recovery appears to involve neuron preservation and cytoprotection by inhibition of apoptosis. Conclusions Penile injection of both ADSC and ADSC-derived lysate can improve

  10. Antigen injection (image)

    MedlinePlus

    Leprosy is caused by the organism Mycobacterium leprae . The leprosy test involves injection of an antigen just under ... if your body has a current or recent leprosy infection. The injection site is labeled and examined ...

  11. Enantioselective [4 + 1] cycloaddition of ortho-quinone methides and bromomalonates under phase-transfer catalysis.

    PubMed

    Lian, Xiao-Lei; Adili, Alafate; Liu, Bin; Tao, Zhong-Lin; Han, Zhi-Yong

    2017-05-03

    An enantioselective [4 + 1] cycloaddition reaction of ortho-quinone methides and bromomalonates using a quinine and BINOL derived phase-transfer catalyst is described. With high yields and enantioselectivities, the method provided a variety of optically active dihydrobenzofurans, which represent a valuable structural motif present in numerous naturally occurring and biologically active molecules.

  12. Reaction between ortho-semiquinones and oxygen: pulse radiolysis, electron spin resonance, and oxygen uptake studies.

    PubMed

    Kalyanaraman, B; Korytowski, W; Pilas, B; Sarna, T; Land, E J; Truscott, T G

    1988-10-01

    The cytotoxicity to tumor cells or cardiotoxic side effects of certain para-quinone antitumor drugs have been attributed to the corresponding semiquinones and derived superoxide and hydroxyl radicals. It has also been suggested that ortho-semiquinones, including those that arise during melanogenesis, produced via either the one-electron oxidation of catechol(amine)s or the one-electron reduction of the corresponding quinones, react with molecular oxygen to give superoxide and hydrogen peroxide. Furthermore it has been shown that catechol(amine)s which form noncyclizable quinones are more cytotoxic toward melanogenic cells than those forming cyclizable quinones. In order to provide further kinetic information on the interaction of oxygen with ortho-semiquinones, using pulse radiolysis we directly measured the rates of reaction of various ortho-semiquinones with molecular oxygen. The semiquinones of the corresponding catechol(amine)s were also produced by the horseradish peroxidase/hydrogen peroxide system, and detected by electron spin resonance spectroscopy using the spin stabilization method. Oxygen consumption was monitored using a standard Clark oxygen electrode. Our data indicate that while ortho-semiquinones from catechol(amine)s and catechol estrogens do not react with molecular oxygen at a rate equal to or greater than k less than or equal to 10(5) M-1 s-1, semiquinones from hydroxy-substituted catechol(amine)s react with dioxygen with rates in the range k = 10(6)-10(7) M-1 s-1.

  13. Ortho C-H Acylation of Aryl Iodides by Palladium/Norbornene Catalysis.

    PubMed

    Dong, Zhe; Wang, Jianchun; Ren, Zhi; Dong, Guangbin

    2015-10-19

    Reported herein is a palladium/norbornene-catalyzed ortho-arene acylation of aryl iodides by a Catellani-type C-H functionalization. This transformation is enabled by isopropyl carbonate anhydrides, which serve as both an acyl cation equivalent and a hydride source.

  14. OrthoEvidence™: A Clinical Resource for Evidence-Based Orthopedics

    PubMed Central

    Sprague, Sheila; Smith, Chris; Bhandari, Mohit

    2015-01-01

    The prevalence of musculoskeletal issues in clinical practice, and the limited focus placed upon musculoskeletal conditions by current electronic summary resources, highlights the need for a resource that provides access to simple and concise summaries of top-quality orthopedic literature for orthopedic surgeons and allied healthcare professionals. OrthoEvidence™ is an online clinical resource that addresses the paucity of adequate evidence-based summary tools in the field of orthopedic surgery. OrthoEvidence™ uses a rigorous, transparent, and unique process to review, evaluate, and summarize high quality research studies and their implications for orthopedic clinical practice. Randomized controlled trials and meta-analyses are identified and reviewed by an expert medical writing team, who prepare Advanced Clinical Evidence (ACETM) reports: one or two detailed pages including critical appraisals and synopses of key research. These timely and targeted reports provide a clear understanding about the quality of evidence associated with each summarized study, and can be organized by users to identify trending information. OrthoEvidence™ allows members to use their time efficiently and to stay current by having access to a breadth of timely, high-quality research output. OrthoEvidence™ is easily accessible through the internet and is available at the point-of-care, which allows treating orthopedic surgeons and allied health professionals to easily practice the principles of evidence-based medicine within their clinical practices.. PMID:26330990

  15. OrthoDB: a hierarchical catalog of animal, fungal and bacterial orthologs.

    PubMed

    Waterhouse, Robert M; Tegenfeldt, Fredrik; Li, Jia; Zdobnov, Evgeny M; Kriventseva, Evgenia V

    2013-01-01

    The concept of orthology provides a foundation for formulating hypotheses on gene and genome evolution, and thus forms the cornerstone of comparative genomics, phylogenomics and metagenomics. We present the update of OrthoDB-the hierarchical catalog of orthologs (http://www.orthodb.org). From its conception, OrthoDB promoted delineation of orthologs at varying resolution by explicitly referring to the hierarchy of species radiations, now also adopted by other resources. The current release provides comprehensive coverage of animals and fungi representing 252 eukaryotic species, and is now extended to prokaryotes with the inclusion of 1115 bacteria. Functional annotations of orthologous groups are provided through mapping to InterPro, GO, OMIM and model organism phenotypes, with cross-references to major resources including UniProt, NCBI and FlyBase. Uniquely, OrthoDB provides computed evolutionary traits of orthologs, such as gene duplicability and loss profiles, divergence rates, sibling groups, and now extended with exon-intron architectures, syntenic orthologs and parent-child trees. The interactive web interface allows navigation along the species phylogenies, complex queries with various identifiers, annotation keywords and phrases, as well as with gene copy-number profiles and sequence homology searches. With the explosive growth of available data, OrthoDB also provides mapping of newly sequenced genomes and transcriptomes to the current orthologous groups.

  16. Palladium-Catalyzed Heteroarylation and Concomitant ortho-Alkylation of Aryl Iodides.

    PubMed

    Lei, Chuanhu; Jin, Xiaojia; Zhou, Jianrong Steve

    2015-11-02

    Three-component couplings were achieved from common aryl halides, alkyl halides, and heteroarenes under palladium and norbornene co-catalysis. The reaction forges hindered aryl-heteroaryl bonds and introduces ortho-alkyl groups to aryl rings. Various heterocycles such as oxazoles, thiazoles and thiophenes underwent efficient coupling. The heteroarenes were deprotonated in situ by bases without the assistance of palladium catalysts.

  17. Derivatization of (quinolin-8-yl)phosphinimidic amides via ortho-lithiation revisited.

    PubMed

    Fernández Sáez, Nerea; García López, Jesús; Iglesias, María José; López Ortiz, Fernando

    2015-07-07

    5The direct ortho-lithiation of N-H containing (quinolin-8-yl)phosphinimidic amides by reaction with 1 equiv. of n-BuLi described by Wang and co-workers has been re-examined. The multinuclear magnetic resonance ((1)H, (2)H, (7)Li, (13)C, (15)N and (31)P) study of the species formed in the monolithiation of N-(tert-butyl)-P,P-diphenyl-N'-(quinolin-8-yl)phosphinimidic amide 5 with n-BuLi in THF showed that proton abstraction occurred exclusively and quantitatively at the NH. The combination of the NMR results with a DFT study made it possible to describe the structure of the N-lithiated species 9 as a dimer consisting of an eight-membered ring showing two lithium ions triply coordinated to nitrogen atoms corresponding to the deprotonated amine and aminoquinoline moieties of different monomers. The formation of a polymer featuring the same coordination mode couldn't be excluded. In addition, optimized conditions for the efficient derivatization of 5 via ortho-lithiation were realised. The reaction of 5 with 2.4 equiv. of t-BuLi in THF in the temperature range of -80 °C to 25 °C for 3 h afforded a N,C(ortho)-dilithiated species that was trapped with a series of electrophiles leading to new functionalized ortho derivatives of 5 in good yields.

  18. ortho-Fluoroazobenzenes: visible light switches with very long-Lived Z isomers.

    PubMed

    Knie, Christopher; Utecht, Manuel; Zhao, Fangli; Kulla, Hannes; Kovalenko, Sergey; Brouwer, Albert M; Saalfrank, Peter; Hecht, Stefan; Bléger, David

    2014-12-08

    Improving the photochemical properties of molecular photoswitches is crucial for the development of light-responsive systems in materials and life sciences. ortho-Fluoroazobenzenes are a new class of rationally designed photochromic azo compounds with optimized properties, such as the ability to isomerize with visible light only, high photoconversions, and unprecedented robust bistable character. Introducing σ-electron-withdrawing F atoms ortho to the NN unit leads to both an effective separation of the n→π* bands of the E and Z isomers, thus offering the possibility of using these two transitions for selectively inducing E/Z isomerizations, and greatly enhanced thermal stability of the Z isomers. Additional para-electron-withdrawing groups (EWGs) work in concert with ortho-F atoms, giving rise to enhanced separation of the n→π* transitions. A comprehensive study of the effect of substitution on the key photochemical properties of ortho-fluoroazobenzenes is reported herein. In particular, the position, number, and nature of the EWGs have been varied, and the visible light photoconversions, quantum yields of isomerization, and thermal stabilities have been measured and rationalized by DFT calculations.

  19. The Ortho-Syllable as a Processing Unit in Handwriting: The Mute E Effect

    ERIC Educational Resources Information Center

    Lambert, Eric; Sausset, Solen; Rigalleau, François

    2015-01-01

    Some research on written production has focused on the role of the syllable as a processing unit. However, the precise nature of this syllable unit has yet to be elucidated. The present study examined whether the nature of this processing unit is orthographic (i.e., an ortho-syllable) or phonological. We asked French adults to copy three-syllable…

  20. Synthesis and characterization of self-catalyzed poly(ortho ester).

    PubMed

    Sintzel, M B; Heller, J; Ng, S Y; Taylor, M S; Tabatabay, C; Gurny, R

    1998-01-01

    Poly(ortho esters) are currently under investigation as a carrier system for an antiproliferative agent in glaucoma filtering surgery. The present investigation illustrates the development of a series of self-catalyzed poly(ortho ester). These polymers contain short dimer segments of alpha-hydroxy acids in their backbone and are prepared by the addition of different polyols to the diketene acetal 3,9-diethylidene-2,4,8,10-tetra-oxaspiro-[5.5]-undecane. The structures were confirmed by NMR- and FT-IR-spectroscopy. The polymers were characterized by determination of the molecular weight, the glass transition temperature and the rheological behavior. The amount of residual solvents was also analyzed. The characteristics of the polymer can be varied by the type of polyol incorporated in its backbone. Since poly(ortho ester) is susceptible to acid-catalyzed degradation, the polymer hydrolysis can be controlled by the amount of incorporated portion of alpha-hydroxy acid. Due to the high hydrophobicity of the polymer structure, the ester bonds are more susceptible to hydrolysis than the ortho ester bonds in the polymer backbone. The hydrolysis proceeds via initial protonation of the exocyclic alkoxy group to yield pentaerythritol dipropionate and the free diol. In a next step, the pentaerythritol dipropionate hydrolysis to pentaerythritol and propionic acid. The molecular weight decrease, weight loss and the pH profile of the polymer in aqueous medium were monitored during the degradation.

  1. Regioselective desymmetrization of diaryltetrahydrofurans via directed ortho-lithiation: an unexpected help from green chemistry.

    PubMed

    Mallardo, Valentina; Rizzi, Ruggiero; Sassone, Francesca C; Mansueto, Rosmara; Perna, Filippo M; Salomone, Antonio; Capriati, Vito

    2014-08-14

    An efficient functionalization of diaryltetrahydrofurans via a regioselective THF-directed ortho-lithiation is first described. This reaction can be successfully carried out in cyclopentyl methyl ether as a "greener" alternative to Et2O, with better results in terms of yield and selectivity and, surprisingly, also in protic eutectic mixtures competitively with protonolysis.

  2. Palladium-catalyzed decarboxylative ortho-arylation of 2-pyridyl sulfoxides with benzoyl peroxides.

    PubMed

    Sun, Meng; Wang, Zhe; Wang, Jiaxin; Guo, Peiyu; Chen, Xiangxiang; Li, Ya-Min

    2016-12-07

    A palladium catalyzed efficient strategy for regio-selective ortho-arylation of sulfoxides with benzoyl peroxides via decarboxylation has been developed. This reaction proceeds smoothly, tolerates a variety of functional groups, and provides easy access to the synthesis of different biaryl compounds.

  3. Implant assisted ortho-surgery in edentulous jaws: a clinical report

    PubMed Central

    Khojasteh, Arash; Payaminia, Leila; Alikhasi, Marzieh

    2015-01-01

    Key Clinical Message The severely atrophy of jaws often complicates ideally oral reconstruction of esthetics and functionality, and necessitates different preprosthetic surgeries including bone grafting, ortho-surgery, and implant insertion. The mentioned procedures could be done within different approaches. This report describes the management of an edentulous case by implant insertion before orthognathic correction. PMID:26576273

  4. The Ortho-Syllable as a Processing Unit in Handwriting: The Mute E Effect

    ERIC Educational Resources Information Center

    Lambert, Eric; Sausset, Solen; Rigalleau, François

    2015-01-01

    Some research on written production has focused on the role of the syllable as a processing unit. However, the precise nature of this syllable unit has yet to be elucidated. The present study examined whether the nature of this processing unit is orthographic (i.e., an ortho-syllable) or phonological. We asked French adults to copy three-syllable…

  5. Ligand-Accelerated ortho-C–H Alkylation of Arylcarboxylic Acids Using Alkyl Boron Reagents

    PubMed Central

    Thuy-Boun, Peter S.; Villa, Giorgio; Dang, Devin; Richardson, Paul; Su, Shun; Yu, Jin-Quan

    2013-01-01

    A protocol for the Pd(II)-catalyzed ortho-C–H alkylation of phenylacetic and benzoic acids using alkylboron reagents is disclosed. Mono-protected amino acid ligands (MPAA) were found to significantly promote reactivity. Both potassium alkyltrifluoroborates and alkylboronic acids were compatible coupling partners. The possibility of a radical alkyl transfer to Pd(II) was also investigated. PMID:24124892

  6. Development of High-Activity Para- to Ortho-Hydrogen Conversion Catalysts. Volume 2

    DTIC Science & Technology

    1989-09-28

    and Loeb1, E. M., J. Phys. Chem. 73, 894 (1969). G. C. Michael , Ph.D. thesis, The Pennsylvania State Univ., University Park, 1969. Misono, M., and...hydrogen. Zhavoronkova, K. N.; Peshkov, A. V.; Spivak ,, N. A. Tr. - M’osk. Khim.-Tekhnol. Inst. im. 0. I. Mendeleeva, 99, 89-92 (1978). Ortho-para

  7. "Sponge-like" dressings based on biopolymers for the delivery of platelet lysate to skin chronic wounds.

    PubMed

    Rossi, S; Faccendini, A; Bonferoni, M C; Ferrari, F; Sandri, G; Del Fante, C; Perotti, C; Caramella, C M

    2013-01-20

    The aim of the present work was the development of sponge-like dressings, obtained by freeze-drying, based on chitosan glutamate and sodium hyaluronate for platelet lysate (PL) delivery to chronic skin wounds. A first phase of the research focused on the choice of the best dressing composition to obtain formulations endowed with the desired mechanical and hydration properties. In particular glycine amount (cryoprotectant agent), and water content were considered as formulation variables. The addition of glycerophosphate, used to solubilize chitosan at pH close to neutrality, was also investigated. In the second phase of the research, dressings were loaded with different amounts of PL. The influence of freeze-drying process and of excipients on the biological activity of platelet growth factors was investigated by means of a cell proliferation test using human fibroblasts. PDGF AB (platelet derived growth factor) content was assayed by means of ELISA test. Depending on composition, dressings showed different mechanical and hydration properties that make them suitable to wounds with different exudate amounts. Both freeze-drying process and excipients employed did not disturb the activity of platelet growth factors. The dressings loaded with platelet lysate were characterized by % proliferation values on fibroblast cell comparable to those observed for the fresh hemoderivate. The PDGF AB assay confirmed the results obtained from cell proliferation test.

  8. [Biological activity of lipopolysaccharides from clinical Bacteroides fragilis strains isolated in Poland determined in reaction with limulus amoebocyte lysate].

    PubMed

    Rokosz, Alicja; Górska, Paulina; Michałkiewicz, Jacek; Łuczak, Miroslaw

    2003-01-01

    The aim of this study was to determine a biological activity of lipopolysaccharides (LPS) from clinical Bacterioides fragilis strains isolated in Poland by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides were extracted from nine clinical B. fragilis strains by the procedure of Westphal and Jann (1965). Crude LPS preparations were purified with ultracentrifugation. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit). Tests were performed according to the recommendations of the producer (Charles River Endosafe Ltd., USA). E. coli O55:B5 LPS and LPS preparations from reference B. fragilis strains were applied to compare the results of examinations. Activities of endotoxins from clinical B. fragilis strains isolated in Poland determined in reaction with Limulus amoebocyte lysate were differentiated. Among endotoxins of clinical B. fragilis strains the most active was the preparation from strain cultured in the case of pancreatic ulcer (B. fragilis 80/81 LPS). Lipopolysaccharides of examined B. fragilis strains were less active in BET test than E. coli O55:B5 LPS.

  9. Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide

    NASA Astrophysics Data System (ADS)

    Benjamin, Christopher J.; Wright, Kyle J.; Bolton, Scott C.; Hyun, Seok-Hee; Krynski, Kyle; Grover, Mahima; Yu, Guimei; Guo, Fei; Kinzer-Ursem, Tamara L.; Jiang, Wen; Thompson, David H.

    2016-10-01

    We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with Nα, Nα-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.

  10. Inhibition of reticulocyte lysate protein synthesis by heavy metal ions involves eIF-2. cap alpha. phosphorylation

    SciTech Connect

    Matts, R.; Hurst, R.; Schatz, J.

    1987-05-01

    Heavy metal ions inhibit protein synthesis and disaggregate polyribosomes in several tissues and cell types. They have initiated studies to determine the mechanism by which this inhibition occurs utilizing the hemin-supplemented rabbit reticulocyte lysate as a model system. The inhibition of protein synthesis observed in the presence of heavy metal ions occurs with biphasic kinetics in a concentration dependent manner, which correlates with the biological toxicity of the ion. Highly toxic ions (AsO/sub 2//sup -/, Cd/sup + +/, Hg/sup + +/, Pb/sup + +/) inhibit protein synthesis by 50% at concentrations of 5-10 ..mu..M. In comparison, Cu/sup + +/, Fe/sup + +/, Zn/sup + +/ and GSSG inhibited protein synthesis by 50% at concentrations of 60, 250, 300 and 350 ..mu..M, respectively. The inhibition of protein synthesis was accompanied by the phosphorylation of eIF-2..cap alpha.., and was reversed or prevented by the addition of 1 mM DTT, 10 mM cAMP, 2 mM MgGTP, eIF-2 or the reversing factor (RF). Glucose-6-phosphate was found to have no effect. The data indicate that the inhibition of protein synthesis observed with the addition of heavy metal ions to reticulocyte lysates is due to the phosphorylation of eIF-2..cap alpha...

  11. Prevention of respiratory tract infections with bacterial lysate OM-85 bronchomunal in children and adults: a state of the art

    PubMed Central

    2013-01-01

    Respiratory tract infections (RTIs) are a leading cause of morbidity and also represent a cause of death in some parts of the world. The treatment of RTIs implies a continuous search for stronger therapies and represents an economical burden for health services and society. In this context the prevention of infections is absolutely required. The use of bacterial lysates as immuno-modulators to boost immunological response is widely debated. Aim of this review is to summarize the main clinical studies on the effect of the bacterial lysate OM-85 in treating RTIs in susceptible subjects - namely children and chronic obstructive pulmonary disease (COPD)-affected adults. Results from clinical trials and recent systematic reviews are reported. The results show that mean number of RTIs decreases upon treatment with OM-85, as measured by frequency of exacerbations or number of antibiotic courses. Data from systematic reviews indicated that OM-85 is particularly beneficial in children at high risk of RTIs. In COPD-affected adults, clinical studies showed that treatment with OM-85 reduced exacerbations, although systematic reviews did not legitimate the protective effect of OM-85 toward COPD as significant. The use of OM-85 could be efficacious in reducing exacerbation frequency of RTIs in children and adults at risk. However further high-quality studies are needed to better explain the mechanism of action and confirm the beneficial results of OM85. PMID:23692890

  12. Effectiveness of Polyvalent Bacterial Lysate and Autovaccines Against Upper Respiratory Tract Bacterial Colonization by Potential Pathogens: A Randomized Study

    PubMed Central

    Zagólski, Olaf; Stręk, Paweł; Kasprowicz, Andrzej; Białecka, Anna

    2015-01-01

    Background Polyvalent bacterial lysate (PBL) is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by lysis of different strains of bacteria. Autovaccines are individually prepared based on the results of smears obtained from the patient. Both types of vaccine can be used to treat an ongoing chronic infection. This study sought to determine which method is more effective against nasal colonization by potential respiratory tract pathogens. Material/Methods We enrolled 150 patients with aerobic Gram stain culture and count results indicating bacterial colonization of the nose and/or throat by potential pathogens. The participants were randomly assigned to each of the following groups: 1. administration of PBL, 2. administration of autovaccine, and 3. no intervention (controls). Results Reduction of the bacterial count in Streptococcus pneumoniae-colonized participants was significant after the autovaccine (p<0.001) and PBL (p<0.01). Reduction of the bacterial count of other β-hemolytic streptococcal strains after treatment with the autovaccine was significant (p<0.01) and was non-significant after PBL. In Haemophilus influenzae colonization, significant reduction in the bacterial count was noted in the PBL group (p<0.01). Methicillin-resistant Staphylococcus aureus colonization did not respond to either treatment. Conclusions The autovaccine is more effective than PBL for reducing bacterial count of Streptococcus pneumoniae and β-hemolytic streptococci, while PBL was more effective against Haemophilus influenzae colonization. PMID:26434686

  13. Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull.

    PubMed

    Rodgers, Margaret L; Paulson, Joshua; Hoskins, Aaron A

    2015-05-01

    Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments. © 2015 Rodgers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  14. Presence of several cellulose-binding proteins in culture supernatant and cell lysate of Eubacterium cellulosolvens 5.

    PubMed

    Toyoda, Atsushi; Yoda, Kazutoyo; Nakamura, Yutaka; Minato, Hajime

    2001-12-01

    Attempts were made to separate and characterize cellulose-binding proteins (CBPs) from both the culture supernatant and cell lysate of Eubacterium cellulosolvens 5. Once the CBPs were bound to Avicel cellulose, they were then effectively eluted with the solution containing 3.2 or 5% sodium dodecyl sulfate (SDS), but not eluted with the solution containing various kinds of carbohydrates and reagents. Namely, CBPs in both the culture supernatant and cell lysate of the bacterium bound tightly and strongly to cellulose. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted CBPs indicated that the CBPs contained the two major proteins having the molecular weights of approximately 160 and 84 kilodaltons (kDa) and one sub-major protein having a molecular weight of approximately 140 kDa. Zymogram analysis after the SDS-PAGE of the eluted CBPs showed that two proteins exhibited the highest levels of carboxymethyl cellulase (CMCase) activity corresponding to the molecular weights of approximately 160 and 90 kDa. A major protein having the molecular weight of approximately 160 kDa exhibited a distinct CMCase activity and was designated as CBPE1. Western immunoblot analysis indicated that the proteins prepared from 16 representative strains of rumen bacteria did not cross-react with rabbit antiserum raised against CBPE1. Thus, CBPE1 may be a unique CBP that plays an important role in the adhesion of the bacterium to cellulose.

  15. Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing.

    PubMed

    Merk, Helmut; Rues, Ralf-Bernhardt; Gless, Christine; Beyer, Kerstin; Dong, Fang; Dötsch, Volker; Gerrits, Michael; Bernhard, Frank

    2015-09-01

    G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.

  16. Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

    PubMed

    Wang, Chuan; Xiao, Ran; Cao, Yi-Lin; Yin, Hong-Yu

    2017-09-09

    Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Mechanism of activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.

    PubMed Central

    de Herreros, A G; de Haro, C; Ochoa, S

    1985-01-01

    We have reported previously that calcium ions and phospholipid activate the heme-stabilized proinhibitor form (pro-HCI) of the heme-controlled translational inhibitor (HCI) in reticulocyte lysates and promote the first step of the reaction pro-HCI in equilibrium reversible HCI----irreversible HCI. This suggested the possible involvement of a Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the activation. However, further investigation revealed, among other things, that polyunsaturated fatty acids (e.g., arachidonic acid) were as effective as Ca2+/phospholipid in promoting translational inhibition and phosphorylation of the alpha subunit of the chain-initiation factor eIF-2 and, moreover, HCI activation could be prevented or reversed in either case by NADPH-generating systems or by dithiols. Our results suggest that pro-HCI is activated by lipoperoxides produced in reticulocyte lysates from either phospholipid or polyunsaturated fatty acids; the presence of Ca2+ is required in the former but not in the latter case. The reversible activation of HCI by Ca2+ and phospholipid might suggest a possible modulatory role of Ca2+ in translational control. Images PMID:3159012

  18. Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide

    PubMed Central

    Benjamin, Christopher J.; Wright, Kyle J.; Bolton, Scott C.; Hyun, Seok-Hee; Krynski, Kyle; Grover, Mahima; Yu, Guimei; Guo, Fei; Kinzer-Ursem, Tamara L.; Jiang, Wen; Thompson, David H.

    2016-01-01

    We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with Nα, Nα-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM. PMID:27748364

  19. Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull

    PubMed Central

    Rodgers, Margaret L.; Paulson, Joshua; Hoskins, Aaron A.

    2015-01-01

    Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments. PMID:25805862

  20. Of the ortho effect in palladium/norbornene-catalyzed reactions: a theoretical investigation.

    PubMed

    Maestri, Giovanni; Motti, Elena; Della Ca', Nicola; Malacria, Max; Derat, Etienne; Catellani, Marta

    2011-06-08

    Mechanistic questions concerning palladium and norbornene catalyzed aryl-aryl coupling reactions are treated in this paper: how aryl halides react with the intermediate palladacycles, formed by interaction of the two catalysts with an aryl halide, and what is the rational explanation of the "ortho effect" (caused by an ortho substituent in the starting aryl halide), which leads to aryl-aryl coupling with a second molecule of aryl halide rather than to aryl-norbornyl coupling. Two possible pathways have been proposed, one involving aryl halide oxidative addition to the palladacycle, the other passing through a palladium(II) transmetalation, also involving the palladacycle, as previously proposed by Cardenas and Echavarren. Our DFT calculations using M06 show that, in palladium-catalyzed reaction of aryl halides, not containing ortho substituents, and norbornene, the intermediate palladacycle formed has a good probability to undergo transmetalation, energetically favored over the oxidative addition leading to Pd(IV). The unselective sp(2)-sp(2) and sp(2)-sp(3) coupling, experimentally observed in this case, can be explained in the framework of the transmetalation pathway since the energetic difference between aryl attack onto the aryl or norbornyl carbon of the palladacycle intermediate is quite small. On the other hand, according to the experimentally observed "ortho effect", selective aryl-aryl coupling only occurs in the reactions of ortho-substituted metallacycles. The present work offers the first possible rationalization of this finding. These in situ formed palladacycles containing an ortho substituent could more easily undergo oxidative addition of an aryl halide rather than reductive elimination from the transmetalation intermediate as a result of a steric clash in the transition state of the latter. The now energetically accessible Pd(IV) intermediate, featuring a Y-distorted trigonal bipyramidal structure, can account for the reported selective aryl

  1. Neurochondrin is a neuronal target antigen in autoimmune cerebellar degeneration

    PubMed Central

    Miske, Ramona; Gross, Catharina C.; Scharf, Madeleine; Golombeck, Kristin S.; Hartwig, Marvin; Bhatia, Urvashi; Schulte-Mecklenbeck, Andreas; Bönte, Kathrin; Strippel, Christine; Schöls, Ludger; Synofzik, Matthis; Lohmann, Hubertus; Dettmann, Inga Madeleine; Deppe, Michael; Mindorf, Swantje; Warnecke, Tobias; Denno, Yvonne; Teegen, Bianca; Probst, Christian; Brakopp, Stefanie; Wandinger, Klaus-Peter; Wiendl, Heinz; Stöcker, Winfried; Meuth, Sven G.

    2016-01-01

    Objective: To report on a novel neuronal target antigen in 3 patients with autoimmune cerebellar degeneration. Methods: Three patients with subacute to chronic cerebellar ataxia and controls underwent detailed clinical and neuropsychological assessment together with quantitative high-resolution structural MRI. Sera and CSF were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IFA) and immunoblot. Immunoprecipitation with lysates of hippocampus and cerebellum combined with mass spectrometric analysis was used to identify the autoantigen, which was verified by recombinant expression in HEK293 cells and use in several immunoassays. Multiparameter flow cytometry was performed on peripheral blood and CSF, and peripheral blood was subjected to T-cell receptor spectratyping. Results: Patients presented with a subacute to chronic cerebellar and brainstem syndrome. MRI was consistent with cortical and cerebellar gray matter atrophy associated with subsequent neuroaxonal degeneration. IFA screening revealed strong immunoglobulin G1 reactivity in sera and CSF with hippocampal and cerebellar molecular and granular layers, but not with a panel of 30 recombinantly expressed established neural autoantigens. Neurochondrin was subsequently identified as the target antigen, verified by IFA and immunoblot with HEK293 cells expressing human neurochondrin as well as the ability of recombinant neurochondrin to neutralize the autoantibodies' tissue reaction. Immune phenotyping revealed intrathecal accumulation and activation of B and T cells during the acute but not chronic phase of the disease. T-cell receptor spectratyping suggested an antigen-specific T-cell response accompanying the formation of antineurochondrin autoantibodies. No such neurochondrin reactivity was found in control cohorts of various neural autoantibody-associated neurologic syndromes, relapsing-remitting multiple sclerosis, cerebellar type of multiple system atrophy, hereditary

  2. Selective ortho-hydroxylation-defluorination of 2-fluorophenolates with a bis(μ-oxo)dicopper(III) species.

    PubMed

    Serrano-Plana, Joan; Garcia-Bosch, Isaac; Miyake, Ryosuke; Costas, Miquel; Company, Anna

    2014-09-01

    The bis(μ-oxo)dicopper(III) species [Cu(III) 2 (μ-O)2 (m-XYL(MeAN) )](2+) (1) promotes the electrophilic ortho-hydroxylation-defluorination of 2-fluorophenolates to give the corresponding catechols, a reaction that is not accomplishable with a (η(2) :η(2) -O2 )dicopper(II) complex. Isotopic labeling studies show that the incoming oxygen atom originates from the bis(μ-oxo) unit. Ortho-hydroxylation-defluorination occurs selectively in intramolecular competition with other ortho-substituents such as chlorine or bromine. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Ruthenium-Catalyzed Ortho C-H Arylation of Aromatic Nitriles with Arylboronates and Observation of Partial Para Arylation.

    PubMed

    Koseki, Yuta; Kitazawa, Kentaroh; Miyake, Masashi; Kochi, Takuya; Kakiuchi, Fumitoshi

    2016-12-29

    Ruthenium-catalyzed C-H arylation of aromatic nitriles with arylboronates is described. The use of RuH2(CO){P(4-MeC6H4)3}3 as a catalyst provided higher yields of the ortho arylation products than the conventional RuH2(CO)(PPh3)3 catalyst. The arylation takes place mostly at the ortho positions, but unprecedented para arylation was also partially observed to give ortho,para diarylation products. In addition to C-H bond cleavage, the cyano group was also found to function as a directing group for cleavage of C-O bonds in aryl ethers.

  4. AntigenMap 3D: an online antigenic cartography resource

    PubMed Central

    Barnett, J. Lamar; Yang, Jialiang; Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2012-01-01

    Summary: Antigenic cartography is a useful technique to visualize and minimize errors in immunological data by projecting antigens to 2D or 3D cartography. However, a 2D cartography may not be sufficient to capture the antigenic relationship from high-dimensional immunological data. AntigenMap 3D presents an online, interactive, and robust 3D antigenic cartography construction and visualization resource. AntigenMap 3D can be applied to identify antigenic variants and vaccine strain candidates for pathogens with rapid antigenic variations, such as influenza A virus. Availability and implementation: http://sysbio.cvm.msstate.edu/AntigenMap3D Contact: wan@cvm.msstate.edu; wanhenry@yahoo.com PMID:22399675

  5. Diagnostic value of carbohydrate antigens in supernatants and sediments of pleural effusions

    PubMed Central

    TERRACCIANO, DANIELA; MAZZARELLA, CLAUDIA; CICALESE, MARCELLINO; GALZERANO, SONIA; APOSTOLICO, GIANFRANCO; DI CARLO, ANGELINA; MARIANO, ANGELA; CECERE, CIRIACO; MACCHIA, VINCENZO

    2010-01-01

    A panel of tumour markers including carcinoembryonic antigen (CEA), carbohydrate antigen (Ca)15-3, Ca125 and Ca19-9 were measured in the lysate of sediments and in the supernatants of pleural effusions of patients with benign and malignant disease. The tumour markers were also measured in the serum of the same patients. Of these patients, 32 had benign diseases (12 trasudative effusions associated with cirrhosis and 20 with non-malignant exudates: 12 pleuritis and 8 other inflammations) and 103 had malignant effusions (37 breast cancers, 29 lung cancers, 10 ovary cancers, 6 kidney cancers, 11 mesotheliomas and 10 lymphomas). We showed the highest level of CEA in pleural effusions of lung cancer followed by that in pleural effusions of breast cancer; whereas Ca15-3 was very high in the pleural effusions of breast and lung cancer. Concerning the lysate of sediment, CEA was high in the pleural effusions of patients with lung cancer and Ca15-3 in those of patients with breast cancer. The other markers are much less useful. For the remaining tumours, none of the markers tested appear to aid in the diagnosis of disease. In conclusion, our data suggest that the combined determination of tumour markers on supernatants and sediments of pleural effusion may provide additional information on the nature of pleural effusion, especially for cases with negative cytology. PMID:22966327

  6. Production kinetics and immunochemical properties of carcinoembryonic antigen and nonspecific cross-reacting antigen synthesized by various human tumor cell lines.

    PubMed

    Ichiki, S; Kuroki, M; Kuroki, M; Koga, Y; Matsuoka, Y

    1986-05-01

    The production kinetics and immunochemical properties of carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA) in various human tumor cell lines were studied. By radioimmunoassay (RIA), five CEA-producing tumor cell lines tested--2 derived from colonic (M7609 and CCK-81), one from pancreatic (QGP-1) and 2 from lung (HLC-1 and KNS-62) carcinomas--were found to produce NCA simultaneously. The cellular contents of CEA and NCA and the amounts of both antigens released into the culture medium were highly variable among the cell lines. It was a distinct contrast that one cell line (CCK-81) released very large amounts of CEA and NCA into the medium while having the smallest amounts of both antigens in the cells, whereas the others contained much larger amounts of the antigens in the cells as compared with the amounts released into the medium. For most of the cell lines, the production of both CEA and NCA increased in the stationary phase of growth as compared with the exponential phase. The production kinetics of both CEA and NCA appeared to be parallel with each other in all the cell lines, though the amount ratio of CEA to NCA produced was variable. By means of a double immunodiffusion test with polyclonal antibodies, antigenic uniformity with no unique organ-specificity was confirmed for all the CEA preparations from spent media of the cell lines, though some differences in the sugar moiety of CEA were detected by RIA using monoclonal antibodies. No antigenic differences among NCA preparations were observed. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), molecular heterogeneity was observed among CEA or NCA preparations isolated from cell lysates.

  7. Immunogencity of antigens from Mycobacterium tuberculosis self-assembled as particulate vaccines.

    PubMed

    Rubio Reyes, Patricia; Parlane, Natalie A; Wedlock, D Neil; Rehm, Bernd H A

    2016-12-01

    Traditional approaches to vaccine development have failed to identify better vaccines to replace or supplement BCG for the control of tuberculosis (TB). Subunit vaccines offer a safer and more reproducible alternative for the prevention of diseases. In this study, the immunogenicity of bacterially derived polyester beads displaying three different Rv antigens of Mycobacterium tuberculosis was evaluated. Polyester beads displaying the antigens Rv1626, Rv2032, Rv1789, respectively, were produced in an endotoxin-free Escherichia coli strain. Beads were formulated with the adjuvant DDA and subcutaneously administered to C57BL/6 mice. Cytokine responses were evaluated by CBA and antibody responses by ELISA. Specificity of the IgG response was assessed by immunoblotting cell lysates of the vaccine production strains using sera from the vaccinated mice. Mice vaccinated with beads displaying Rv1626 had significantly greater IgG1 responses compared to mice vaccinated with Rv1789 beads and greater IgG2 responses than the group vaccinated with Rv2032 beads (p<0.05). Immunoblotting of antisera from these mice indicated the antibody responses were Rv1626 antigen-specific and there was no detectable immune response to the polyester component of the vaccine. Overall, this study suggested that selected TB antigens derived from reverse vaccinology approaches can be displayed on polyester beads to produce antigen-specific immune responses potentially relevant to the prevention of TB. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. Eliciting cytotoxic T lymphocytes against acute myeloid leukemia-derived antigens: evaluation of dendritic cell-leukemia cell hybrids and other antigen-loading strategies for dendritic cell-based vaccination.

    PubMed

    Galea-Lauri, Joanna; Darling, David; Mufti, Ghulam; Harrison, Phillip; Farzaneh, Farzin

    2002-08-01

    Dendritic cells (DC) have been successfully used in clinical pilot studies to induce tumor-specific immunity as well as clinical response in selected patients. However, DC-based immunotherapy remains a challenge and several parameters need to be examined in order to optimize the induction of anti-tumor immune responses. This study focuses on DC vaccination for leukemia and evaluates the in vitro efficacy of three different strategies for generating antigen-loaded DC-based vaccines for the induction of major histocompatibility complex (MHC) class I-restricted anti-leukemia cytotoxic T lymphocyte (CTL) responses. These included direct fusion of DC with leukemia cells to generate DC-leukemia cell hybrids, and DC pulsed with either apoptotic leukemia cell fragments or whole tumor cell lysates. Using either the U937 cell line or primary human acute myeloid leukemia blasts (AML), DC-leukemia cell hybrids were found to be the most potent in vitro inducers of CTL activity. DC pulsed with apoptotic tumor cell fragments were less efficient, but induced a more potent CTL response compared to tumor lysate-pulsed DC. The CTL responses were both MHC class I-restricted and antigen-specific, as shown by the inability of the CTL to lyse other control targets. The data presented here suggest that the method of antigen loading onto DC may be critical in the design of tumor vaccines.

  9. Lipid antigens in immunity

    PubMed Central

    Dowds, C. Marie; Kornell, Sabin-Christin

    2014-01-01

    Lipids are not only a central part of human metabolism but also play diverse and critical roles in the immune system. As such, they can act as ligands of lipid-activated nuclear receptors, control inflammatory signaling through bioactive lipids such as prostaglandins, leukotrienes, lipoxins, resolvins, and protectins, and modulate immunity as intracellular phospholipid- or sphingolipid-derived signaling mediators. In addition, lipids can serve as antigens and regulate immunity through the activation of lipid-reactive T cells, which is the topic of this review. We will provide an overview of the mechanisms of lipid antigen presentation, the biology of lipid-reactive T cells, and their contribution to immunity. PMID:23999493

  10. Engineering Chimeric Antigen Receptors

    PubMed Central

    Kulemzin, S. V.; Kuznetsova, V. V.; Mamonkin, M.; Taranin, A. V.; Gorchakov, A. A.

    2017-01-01

    Chimeric antigen receptors (CARs) are recombinant protein molecules that redirect cytotoxic lymphocytes toward malignant and other target cells. The high feasibility of manufacturing CAR-modified lymphocytes for the therapy of cancer has spurred the development and optimization of new CAR T cells directed against a broad range of target antigens. In this review, we describe the main structural and functional elements constituting a CAR, discuss the roles of these elements in modulating the anti-tumor activity of CAR T cells, and highlight alternative approaches to CAR engineering. PMID:28461969

  11. Immune response to different fractions of Taenia solium cyst fluid antigens in patients with neurocysticercosis.

    PubMed

    Amit, Prasad; Prasad, Kashi Nath; Kumar, Gupta Rakesh; Shweta, Tripathi; Sanjeev, Jha; Kumar, Paliwal Vimal; Mukesh, Tripathi

    2011-03-01

    The immunopathogenesis of neurocysticercosis (NCC) largely remains unknown. We analyzed the immune response to different fractions of Taenia solium cyst fluid antigens in patients with NCC. Lymphocytes were separated from 48 patients with NCC-related active epilepsy and 30 healthy controls. T. solium (isolated from pig muscles) antigens (crude lysate, CL; cyst wall, CW and cyst fluid, CF) at 20 μg/well concentrations were used to stimulate the cells in a lymphocyte transformation test (LTT). Only CF antigen stimulated cell proliferation significantly greater than control (p<0.001), hence cyst fluid antigens were further studied. The CF antigens were electro-blotted on nitrocellulose membrane (NC), cut at 0.5 cm distance and particulate antigens were prepared. A total of 12 fractions, designated F1 to F12 according to molecular weight were tested in-vitro for LTT. After 72 h of stimulation by the different fractions, Th1 (IL-1β, TNF-α, IL-2) and Th2 (IL-4, IL-10) cytokine responses were determined in culture supernatants by ELISA. Low molecular weight fractions F1 through F4 (Mol. wt.<25 kDa) were found to be potent inducers of cytokines. Fractions F1, F3 and F4 induced the production of Th1 (IL-1β, TNF-α, IL-2), whereas F2 induced the production of Th2 (IL-4 and IL-10) cytokine. The study shows that the low molecular weight fractions of CF antigens are immuno-dominant. Most of these fractions (F1, F3, F4) induce strong Th1 immune response except F2 which induces Th2 response. Further studies are needed to identify the different antigens present in these fractions to determine the molecules responsible for the immune response.

  12. Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis*

    PubMed Central

    Kim, Tae-Hyun; Sebastian, Shite; Pinkham, Jessica T.; Ross, Robin A.; Blalock, LeeAnn T.; Kasper, Dennis L.

    2010-01-01

    The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly176, Asp177, Gly323, and Tyr324) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface. PMID:20605777

  13. Characterization of the O-antigen polymerase (Wzy) of Francisella tularensis.

    PubMed

    Kim, Tae-Hyun; Sebastian, Shite; Pinkham, Jessica T; Ross, Robin A; Blalock, LeeAnn T; Kasper, Dennis L

    2010-09-03

    The O-antigen polymerase of gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly(176), Asp(177), Gly(323), and Tyr(324)) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface.

  14. The Metabolic Fate of ortho-Quinones Derived from Catecholamine Metabolites.

    PubMed

    Ito, Shosuke; Yamanaka, Yuta; Ojika, Makoto; Wakamatsu, Kazumasa

    2016-01-27

    ortho-Quinones are produced in vivo through the oxidation of catecholic substrates by enzymes such as tyrosinase or by transition metal ions. Neuromelanin, a dark pigment present in the substantia nigra and locus coeruleus of the brain, is produced from dopamine (DA) and norepinephrine (NE) via an interaction with cysteine, but it also incorporates their alcoholic and acidic metabolites. In this study we examined the metabolic fate of ortho-quinones derived from the catecholamine metabolites, 3,4-dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethylene glycol (DOPEG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylmandelic acid (DOMA). The oxidation of catecholic substrates by mushroom tyrosinase was followed by UV-visible spectrophotometry. HPLC analysis after reduction with NaBH₄ or ascorbic acid enabled measurement of the half-lives of ortho-quinones and the identification of their reaction products. Spectrophotometric examination showed that the ortho-quinones initially formed underwent extensive degradation at pH 6.8. HPLC analysis showed that DOPE-quinone and DOPEG-quinone degraded with half-lives of 15 and 30 min at pH 6.8, respectively, and >100 min at pH 5.3. The major product from DOPE-quinone was DOPEG which was produced through the addition of a water molecule to the quinone methide intermediate. DOPEG-quinone yielded a ketone, 2-oxo-DOPE, through the quinone methide intermediate. DOPAC-quinone and DOMA-quinone degraded immediately with decarboxylation of the ortho-quinone intermediates to form 3,4-dihydroxybenzylalcohol (DHBAlc) and 3,4-dihydroxybenzaldehyde (DHBAld), respectively. DHBAlc-quinone was converted to DHBAld with a half-life of 9 min, while DHBAld-quinone degraded rapidly with a half-life of 3 min. This study confirmed the fact that ortho-quinones from DOPE, DOPEG, DOPAC and DOMA are converted to quinone methide tautomers as common intermediates, through proton rearrangement or decarboxylation. The unstable quinone

  15. Detection of gram-negative bacteremia by limulus amebocyte lysate assay: evaluation in a rat model of peritonitis.

    PubMed

    du Moulin, G C; Lynch, S E; Hedley-Whyte, J; Broitman, S A

    1985-01-01

    A spectrophotometric Limulus amebocyte lysate assay using lysis filtration and centrifugation has been developed for the detection of gram-negative bacteria in blood. The assay is directed at detection of endotoxin in viable and nonviable bacteria present in the blood-stream and not detection of free endotoxin in plasma. The assay was evaluated in a model of peritonitis in which rats were challenged with an inoculum consisting of sterilized human feces, barium sulfate, and one of eight species of bacteria. This assay was able to detect gram-negative bacteremia due to Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, and Klebsiella pneumoniae in the rat model when compared with sham-inoculated uninfected rats. The assay failed to detect bacteremia due to Bacteroides fragilis or Staphylococcus aureus, nor was there a significant rise in absorbance when a pellet containing sterilized feces was implanted in the rat.

  16. Conversion rate of para-hydrogen to ortho-hydrogen by oxygen: implications for PHIP gas storage and utilization.

    PubMed

    Wagner, Shawn

    2014-06-01

    To determine the storability of para-hydrogen before reestablishment of the room temperature thermal equilibrium mixture. Para-hydrogen was produced at near 100% purity and mixed with different oxygen quantities to determine the rate of conversion to the thermal equilibrium mixture of 75: 25% (ortho: para) by detecting the ortho-hydrogen (1)H nuclear magnetic resonance using a 9.4 T imager. The para-hydrogen to ortho-hydrogen velocity constant, k, near room temperature (292 K) was determined to be 8.27 ± 1.30 L/mol · min(-1). This value was calculated utilizing four different oxygen fractions. Para-hydrogen conversion to ortho-hydrogen by oxygen can be minimized for long term storage with judicious removal of oxygen contamination. Prior calculated velocity rates were confirmed demonstrating a dependence on only the oxygen concentration.

  17. Hydrogen adsorption on two catalysts for the ortho- to parahydrogen conversion: Cr-doped silica and ferric oxide gel.

    PubMed

    Hartl, Monika; Gillis, Robert Chad; Daemen, Luke; Olds, Daniel P; Page, Katherine; Carlson, Stefan; Cheng, Yongqiang; Hügle, Thomas; Iverson, Erik B; Ramirez-Cuesta, A J; Lee, Yongjoong; Muhrer, Günter

    2016-06-29

    Molecular hydrogen exists in two spin-rotation coupled states: parahydrogen and orthohydrogen. Due to the variation of energy with rotational level, the occupation of ortho- and parahydrogen states is temperature dependent, with parahydrogen being the dominant species at low temperatures. The equilibrium at 20 K (99.8% parahydrogen) can be reached by natural conversion only after a lengthy process. With the use of a suitable catalyst, this process can be shortened significantly. Two types of commercial catalysts currently being used for ortho- to parahydrogen conversion are: iron(iii) oxide (Fe2O3, IONEX®), and chromium(ii) oxide doped silica catalyst (CrO·SiO2, OXISORB®). We investigate the interaction of ortho- and parahydrogen with the surfaces of these ortho-para conversion catalysts using neutron vibrational spectroscopy. The catalytic surfaces have been characterized using X-ray absorption fine structure (XAFS) and X-ray/neutron pair distribution function measurements.

  18. Hetero-organic thymus antigens.

    PubMed

    Beletskaya, L V; Gnezditskaya, E V

    1985-01-01

    The use of sera containing antibodies to tissue-specific antigens of highly specialized organs (skeletal muscles, heart, skin, excretory glands) enabled us to detect, by immunofluorescence, cells capable of synthesizing analogous antigens (i.e. hetero-organic thymus antigens) in human and animal thymus. Detection of hetero-organic antigens in the thymus is the basis for the hypothesis that natural immunological tolerance to tissue self antigens is formed within the thymus in the course of T-lymphocyte maturation, with thymus antigens taking part in the process.

  19. Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography.

    PubMed

    Caramelo-Nunes, C; Gabriel, M F; Almeida, P; Marcos, J C; Tomaz, C T

    2012-09-01

    In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. A Catalyst-Controlled Aerobic Coupling of ortho-Quinones and Phenols Applied to the Synthesis of Aryl Ethers.

    PubMed

    Huang, Zheng; Lumb, Jean-Philip

    2016-09-12

    ortho-Quinones are underutilized six-carbon-atom building blocks. We herein describe an approach for controlling their reactivity with copper that gives rise to a catalytic aerobic cross-coupling with phenols. The resulting aryl ethers are generated in high yield across a broad substrate scope under mild conditions. This method represents a unique example where the covalent modification of an ortho-quinone is catalyzed by a transition metal, creating new opportunities for their utilization in synthesis.

  1. Antigen smuggling in tuberculosis.

    PubMed

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells.

  2. Antigen detection systems

    USDA-ARS?s Scientific Manuscript database

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

  3. Antigen detection systems

    USDA-ARS?s Scientific Manuscript database

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissue using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular methodology is chosen ...

  4. Human liver nucleolar antigens.

    PubMed

    Busch, R K; Busch, H

    1981-10-01

    In an extension of previous studies on the antigens in rat liver nucleoli (R. K. Busch, R. C. Reddy, D. H. Henning, and H. Busch, Proc. Soc. Exp. Biol. Med. 160, 185 (1979); R. K. Busch and H. Busch, Tumori 63, 347 (1977); F. M. Davis, R. K. Busch, L. C. Yeoman, and H. Busch, Cancer Res. 38, 1906 (1978), rabbit antibodies were elicited to human liver nucleoli isolated by the sucrose--Mg2+ method (10). Fluorescent nucleoli were found in liver cryostat sections treated with rabbit anti-human liver nucleolar antibodies followed by fluorescein-conjugated goat anti-rabbit antibodies. In HeLa cells, fluorescence was distributed throughout the nucleus and in a nuclear network but was not localized to the nucleolus. In placental cryostat sections, an overall nuclear fluorescence was observed with some localization to nucleoli. Immunodiffusion analysis revealed two immunoprecipitin bands which appeared to be liver specific. Other immunoprecipitin bands were common to liver, placenta, and HeLa nuclear extracts. Rocket immunoelectrophoresis revealed two liver-specific antigens, one migrating to the cathode and the other to the anode Other rockets exhibited identity to antigens of other nuclear extracts. These results demonstrate the presence of human liver nucleolar-specific antigens which were not found in the HeLa and placental cells.

  5. AntigenDB: an immunoinformatics database of pathogen antigens.

    PubMed

    Ansari, Hifzur Rahman; Flower, Darren R; Raghava, G P S

    2010-01-01

    The continuing threat of infectious disease and future pandemics, coupled to the continuous increase of drug-resistant pathogens, makes the discovery of new and better vaccines imperative. For effective vaccine development, antigen discovery and validation is a prerequisite. The compilation of information concerning pathogens, virulence factors and antigenic epitopes has resulted in many useful databases. However, most such immunological databases focus almost exclusively on antigens where epitopes are known and ignore those for which epitope information was unavailable. We have compiled more than 500 antigens into the AntigenDB database, making use of the literature and other immunological resources. These antigens come from 44 important pathogenic species. In AntigenDB, a database entry contains information regarding the sequence, structure, origin, etc. of an antigen with additional information such as B and T-cell epitopes, MHC binding, function, gene-expression and post translational modifications, where available. AntigenDB also provides links to major internal and external databases. We shall update AntigenDB on a rolling basis, regularly adding antigens from other organisms and extra data analysis tools. AntigenDB is available freely at http://www.imtech.res.in/raghava/antigendb and its mirror site http://www.bic.uams.edu/raghava/antigendb.

  6. Comparative biocidal activity of peracetic acid, benzalkonium chloride and ortho-phthalaldehyde on 77 bacterial strains.

    PubMed

    Bridier, A; Briandet, R; Thomas, V; Dubois-Brissonnet, F

    2011-07-01

    Despite numerous reports on biocide activities, it is often difficult to have a reliable and relevant overview of bacterial resistance to disinfectants because each work challenges a limited number of strains and tested methods are often different. The aim of this study was to evaluate the bactericidal activity of three different disinfectants commonly used in industrial or medical environments (peracetic acid, benzalkonium chloride and ortho-phthalaldehyde) against 77 bacterial strains from different origins using one standard test method (NF EN 1040). Results highlight the existence of high interspecific variability of resistance to disinfectants and, contrary to widespread belief, Gram-positive strains generally appeared more resistant than Gram-negative strains. Resistance was also variable among strains of the same species such as Bacillus subtilis to peracetic acid, Pseudomonas aeruginosa to benzalkonium chloride and Staphylococcus aureus to ortho-phthalaldehyde.

  7. orthoFind Facilitates the Discovery of Homologous and Orthologous Proteins.

    PubMed

    Mier, Pablo; Andrade-Navarro, Miguel A; Pérez-Pulido, Antonio J

    2015-01-01

    Finding homologous and orthologous protein sequences is often the first step in evolutionary studies, annotation projects, and experiments of functional complementation. Despite all currently available computational tools, there is a requirement for easy-to-use tools that provide functional information. Here, a new web application called orthoFind is presented, which allows a quick search for homologous and orthologous proteins given one or more query sequences, allowing a recurrent and exhaustive search against reference proteomes, and being able to include user databases. It addresses the protein multidomain problem, searching for homologs with the same domain architecture, and gives a simple functional analysis of the results to help in the annotation process. orthoFind is easy to use and has been proven to provide accurate results with different datasets. Availability: http://www.bioinfocabd.upo.es/orthofind/.

  8. Yangians and Yang-Baxter R-operators for ortho-symplectic superalgebras

    NASA Astrophysics Data System (ADS)

    Fuksa, J.; Isaev, A. P.; Karakhanyan, D.; Kirschner, R.

    2017-04-01

    Yang-Baxter relations symmetric with respect to the ortho-symplectic superalgebras are studied. We start with the formulation of graded algebras and the linear superspace carrying the vector (fundamental) representation of the ortho-symplectic supergroup. On this basis we study the analogy of the Yang-Baxter operators considered earlier for the cases of orthogonal and symplectic symmetries: the vector (fundamental) R-matrix, the L-operator defining the Yangian algebra and its first and second order evaluations. We investigate the condition for L (u) in the case of the truncated expansion in inverse powers of u and give examples of Lie algebra representations obeying these conditions. We construct the R-operator intertwining two superspinor representations and study the fusion of L-operators involving the tensor product of such representations.

  9. H2CS abundances and ortho-to-para ratios in interstellar clouds

    NASA Technical Reports Server (NTRS)

    Minh, Y. C.; Irvine, W. M.; Brewer, M. K.

    1991-01-01

    Several H2CS ortho and para transitions have been observed toward interstellar molecular clouds, including cold, dark clouds and star-forming regions. H2CS fractional abundances f(H2CS) about 1-2 10 to the -9th relative to molecular hydrogen toward TMC-1, Orion A, and NGC 7538, and about 5 10 to the -10th for L134N are derived. The H2CS ortho-to-para ratios in TMC-1 are about 1.8 toward the cyanopolyyne peak and the ammonia peak, which may indicate the thermalization of H2CS on 10 K grains. A ratio of about 3, the statistical value, for Orion (3N, 1E) and NGC 7538 is derived, while a value of about 2 for Orion (KL) is found.

  10. Evidence for disequilibrium of ortho and para hydrogen on Jupiter from Voyager IRIS measurements

    NASA Technical Reports Server (NTRS)

    Conrath, B. J.; Gierasch, P. J.

    1983-01-01

    Preliminary results of an analysis of the ortho state/para state ratio (parallel/antiparallel) for molecular H2 in the Jovian atmosphere using Voyager IR spectrometer (IRIS) data are reported. The study was undertaken to expand the understanding of the thermodynamics of a predominantly H2 atmosphere, which takes about 100 million sec to reach equilibrium. IRIS data provided 4.3/cm resolution in the 300-700/cm spectral range dominated by H2 lines. Approximately 600 spectra were examined to detect any disequilibrium between the hydrogen species. The results indicate that the ortho-para ratio is not in an equilibrium state in the upper Jovian troposphere. A thorough mapping of the para-state molecules in the upper atmosphere could therefore aid in mapping the atmospheric flowfield.

  11. An antibacterial ortho-quinone diterpenoid and its derivatives from Caryopteris mongolica.

    PubMed

    Saruul, Erdenebileg; Murata, Toshihiro; Selenge, Erdenechimeg; Sasaki, Kenroh; Yoshizaki, Fumihiko; Batkhuu, Javzan

    2015-06-15

    To identify antibacterial components in traditional Mongolian medicinal plant Caryopteris mongolica, an ortho-quinone abietane caryopteron A (1) and three its derivatives caryopteron B-D (2-4) were isolated from the roots of the plant together with three known abietanes demethylcryptojaponol (5), 6α-hydroxydemethyl cryptojaponol (6), and 14-deoxycoleon U (7). The chemical structures of these abietane derivatives were elucidated on the basis of spectroscopic data. Compounds 1-4 had C-13 methylcyclopropane substructures, and 2-4 had a hexanedioic anhydride ring C instead of ortho-quinone in 1. The stereochemistry of these compound was assumed from NOE spectra and ECD Cotton effects. Compounds 1 and 5-7 showed antibacterial activities against the Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Micrococcus luteus, being 1 the more potent.

  12. Surface-Eroding Poly(ortho ester amides) for Highly Efficient Oral Chemotherapy.

    PubMed

    Wei, Bing; Tao, Yangyang; Wang, Xin; Tang, Rupei; Wang, Jun; Wang, Rui; Qiu, Liying

    2015-05-20

    Two new poly(ortho ester amide) copolymers (POEA-4 and POEA-5) were synthesized via polycondensation of a new ortho ester diamine monomer with active esters of different aliphatic diacids. The kinetics of POEA mass loss and release of 5-FU were both nearly zero-order, suggesting predominantly surface-restricted polymer erosion and drug release. In vitro cytotoxicity tests demonstrated that both copolymers have excellent biocompatibility. In vivo acute toxicity tests suggested that oral administration of POEA-4 and POEA-5 did not cause any adverse effects on mice even at a very high dose (2000 mg/kg). In vivo antitumor efficacy against H22 transplanted tumors of 5-FU-loaded POEA tablets were fully examined. We envision that, with further optimization, POEA-based materials could have great potential as drug carriers for oral chemotherapy.

  13. Gold-Catalyzed Suzuki Coupling of ortho-Substituted Hindered Aryl Substrates.

    PubMed

    Dwadnia, Nejib; Roger, Julien; Pirio, Nadine; Cattey, Hélène; Ben Salem, Ridha; Hierso, Jean-Cyrille

    2017-02-16

    A method that allows hindered ortho-substituted aryl iodides to be efficiently coupled to phenylboronic acid using a gold-catalyzed C-C bond formation is presented. The use of a molecularly-defined dinuclear gold chloride catalytic precursor that is stabilized by a new tetradentate (N,N')-diamino-(P,P')-diphosphino ferrocene hybrid ligand in a Suzuki-type reaction is described for the first time. Electron-rich isopropyl groups on phosphorus were found essential for a superior activity, while the performances of a set of analogous gold dinuclear complexes that were fully characterized by multinuclear NMR spectroscopy and XRD analysis, were investigated. Therefore, arylation of para and ortho-substituted iodoarenes bearing electron-rich, electron-poor functional groups, and even hindered polycyclic aromatic compounds is described.

  14. Evidence for disequilibrium of ortho and para hydrogen on Jupiter from Voyager IRIS measurements

    NASA Technical Reports Server (NTRS)

    Conrath, B. J.; Gierasch, P. J.

    1983-01-01

    Preliminary results of an analysis of the ortho state/para state ratio (parallel/antiparallel) for molecular H2 in the Jovian atmosphere using Voyager IR spectrometer (IRIS) data are reported. The study was undertaken to expand the understanding of the thermodynamics of a predominantly H2 atmosphere, which takes about 100 million sec to reach equilibrium. IRIS data provided 4.3/cm resolution in the 300-700/cm spectral range dominated by H2 lines. Approximately 600 spectra were examined to detect any disequilibrium between the hydrogen species. The results indicate that the ortho-para ratio is not in an equilibrium state in the upper Jovian troposphere. A thorough mapping of the para-state molecules in the upper atmosphere could therefore aid in mapping the atmospheric flowfield.

  15. Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase

    SciTech Connect

    Volarevic, S.; Burns, C.M.; Sussman, J.J.; Ashwell, J.D. )

    1990-09-01

    Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least first major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a trasmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and {beta}{sub 2}-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculations based upon the amounts of coprecipitated enzymatic activity or TCR {zeta} chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.

  16. 1,2-Azaborine: The Boron-Nitrogen Derivative of ortho-Benzyne.

    PubMed

    Edel, Klara; Brough, Sarah A; Lamm, Ashley N; Liu, Shih-Yuan; Bettinger, Holger F

    2015-06-26

    The BN analogue of ortho-benzyne, 1,2-azaborine, is generated by flash vacuum pyrolysis, trapped under cryogenic conditions, and studied by direct spectroscopic techniques. The parent BN aryne spontaneously binds N2 and CO2, thus demonstrating its highly reactive nature. The interaction with N2 is photochemically reversible. The CO2 adduct of 1,2-azaborine is a cyclic carbamate which undergoes photocleavage, thus resulting in overall CO2 splitting.

  17. Branch-Selective Alkene Hydroarylation by Cooperative Destabilization: Iridium-Catalyzed ortho-Alkylation of Acetanilides

    PubMed Central

    Crisenza, Giacomo E M; Sokolova, Olga O; Bower, John F

    2015-01-01

    An iridium(I) catalyst system, modified with the wide-bite-angle and electron-deficient bisphosphine dFppb (1,4-bis(di(pentafluorophenyl)phosphino)butane) promotes highly branch-selective hydroarylation reactions between diverse acetanilides and aryl- or alkyl-substituted alkenes. This provides direct and ortho-selective access to synthetically challenging anilines, and addresses long-standing issues associated with related Friedel–Crafts alkylations. PMID:26490739

  18. 1,2-Azaborine, the BN derivative of ortho-benzyne

    PubMed Central

    Edel, Klara; Brough, Sarah; Lamm, Ashley N.; Liu, Shih-Yuan

    2015-01-01

    The BN analogue of ortho-benzyne, 1,2-azaborine, is generated by flash vacuum pyroylsis, trapped under cryogenic conditions, and studied by direct spectroscopic techniques. The parent BN-aryne spontaneously binds N2 and CO2, demonstrating its highly reactive nature. The interaction with N2 is photochemically reversible. The CO2 adduct of 1,2-azaborine is a cyclic lactam that undergoes photocleavage thus resulting in overall CO2 splitting. PMID:26095444

  19. Construction of Escherichia coli Strains for Conversion of Nitroacetophenones to ortho-Aminophenols

    DTIC Science & Technology

    2003-11-01

    No attempt was made to optimize the transformation, extraction , and recovery procedure. The only other detectable product, accounting for 10% of the...transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and... recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic

  20. Mild Palladium Catalyzed ortho C-H Bond Functionalizations of Aniline Derivatives.

    PubMed

    Tischler, Ms Orsolya; Tóth, Mr Balázs; Novák, Zoltán

    2017-02-01

    This account collects the developments and transformations which avoid the utilization of harsh reaction conditions in the field of palladium catalyzed, ortho-directed C-H activation of aniline derivatives from the first attempts to up-to-date results, including the results of our research laboratory. The discussed functionalizations performed under mild conditions include acylation, olefination, arylation, alkylation, alkoxylation reactions. Beside the optimization studies and the synthetic applications mechanistic investigations are also presented.

  1. Active metabolic weight estimation using bioimpedance, indirect calorimetry and the clino-ortho maneuver.

    PubMed

    Cadena, Miguel; Azpiroz, Joaquin; Borja, Gisella; Medel, Humberto; Sandoval, Hector; Rodriguez, Fausto; Flores, Francisco; Flores, Pedro

    2010-01-01

    The resting energy expenditure (REE) and substrate utilization are computed by indirect calorimetry technique (ICT). The REE represents 80-85% of the total energy expenditure (TEE) but only accounts for the 7% of the actual body weight (ABW). The TEE is produced by the organs plus muscles, whereas the REE accounts only for the main organs. An important problem comes up when the REE is computed throughout the fat free mass (FFM) computation or anthropometric measurements because they do not explain the tremendous catabolic variability by ICT when subjects show the same body composition. Therefore, the aim of this work is to develop a method to compute the metabolic active weight (MAW) as a new form that may help to understand the catabolic activity of the body composition. The premise was the clino-ortho maneuver can split the ABW in two parts: one in which the MAW reflects the FFM catabolism while the second part was not considered since there is not energy requirement in it. The experiment design studied 37 young volunteers undergoing the clino-ortho maneuver during fast and postprandial conditions. The results showed REE increments of 21% during phase I (fast), while in phase II (postprandial) only 14% was achieved in ortho-postprandial. Therefore, the computed MAWs were 65.5Kg and 58Kg, respectively, when the ABW average was 70 Kg and the FFM was 50 Kg. One first conclusion was that the 15.5 Kg of the MAW above the FFM could explain a catabolic equivalence which can be exclusively related to the fast-ortho position which can help to classify exclusively the dynamic over activity of the FFM.

  2. Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

    PubMed

    Johnson, David K; Schillinger, Kurt J; Kwait, David M; Hughes, Chambers V; McNamara, Erin J; Ishmael, Fauod; O'Donnell, Robert W; Chang, Ming-Mei; Hogg, Michael G; Dordick, Jonathan S; Santhanam, Lakshmi; Ziegler, Linda M; Holland, James A

    2002-01-01

    NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.

  3. A unified strategy for iron-catalyzed ortho-alkylation of carboxamides.

    PubMed

    Fruchey, Erin R; Monks, Brendan M; Cook, Silas P

    2014-09-24

    Using 8-aminoquinoline-based aryl carboxamides, the direct ortho-alkylation can be achieved in high yields in the presence of an iron source, 1,2-bis(diphenylphosphino)ethane (dppe) and phenylmagnesium bromide. The reactions proceed without overalkylation and provide high levels of regioselectivity. The benzylation reactions can be performed in air with reagent-grade THF, while the alkylation works well with unactivated secondary bromides and iodides in 2-methyltetrahydrofuran. Moreover, the reactions only require 5-10 min.

  4. A note: ortho-phthalaldehyde: proposed mechanism of action of a new antimicrobial agent.

    PubMed

    Simons, C; Walsh, S E; Maillard, J Y; Russell, A D

    2000-10-01

    Ortho-phthalaldehyde (OPA) is a new aromatic dialdehyde antimicrobial agent, the mechanism of action of which has been little studied. The aims of this paper are to examine what is currently known about its mechanism of action, to compare the action with that of a widely investigated aliphatic dialdehyde, glutaraldehyde (GTA), and to put forward a hypothesis that would, in the light of current knowledge, explain how OPA inactivates micro-organisms, including GTA-resistant Mycobacterium chelonae.

  5. ortho-Lithium/magnesium carboxylate-driven aromatic nucleophilic substitution reactions on unprotected naphthoic acids.

    PubMed

    Aissaoui, Regadia; Nourry, Arnaud; Coquel, Ariane; Dao, Thi Thanh Hà; Derdour, Aicha; Helesbeux, Jean-Jacques; Duval, Olivier; Castanet, Anne-Sophie; Mortier, Jacques

    2012-01-06

    Substitution of an ortho-fluoro or methoxy group in 1- and 2-naphthoic acids furnishing substituted naphthoic acids occurs in good to excellent yields upon reaction with alkyl/vinyl/aryl organolithium and Grignard reagents, in the absence of a metal catalyst without the need to protect the carboxyl (CO(2)H) group. This novel nucleophilic aromatic substitution is presumed to proceed via a precoordination of the organometallic with the substrate, followed by an addition/elimination.

  6. Ortho-TMS Benzaldehyde: An Effective Linchpin for Type II Anion Relay Chemistry (ARC)

    PubMed Central

    Smith, Amos B.; Kim, Won-Suk; Wuest, William M.

    2009-01-01

    Ortho-TMS benzaldehyde, an effective bifunctional linchpin for Type II Anion Relay Chemistry (ARC), permits efficient multi-component union of a variety of nucleophiles and electrophiles, including the first example of a Pd-mediated ARC Type II process. To demonstrate the utility of the Type II ARC protocol, a “proof of concept” synthetic sequence was designed and implemented for construction of a focused library of “natural product-like” compounds. PMID:18666303

  7. Ruthenium-Catalyzed Peri- and Ortho-Alkynylation with Bromoalkynes via Insertion and Elimination.

    PubMed

    Tan, Eric; Konovalov, Andrey I; Fernández, Gabriela A; Dorel, Ruth; Echavarren, Antonio M

    2017-10-04

    The alkynylation of naphthols takes place with total regiocontrol at the peri position of the hydroxyl group in the presence of [RuCl2(p-cymene)]2 as the catalyst. This reaction features high functional group tolerance. The related ortho-alkynylation of benzoic acids proceeds under similar conditions and also shows wide functional group tolerance. Both reactions proceed through metalation, insertion of the alkyne, and bromide elimination.

  8. Effect of mono-ortho and di-ortho substituted polychlorinated biphenyl (PCB) congeners on leopard frog survival and sexual development.

    PubMed

    Jofré, Mariana Beatriz; Karasov, William H

    2008-02-01

    We tested the effect of mono-ortho and di-ortho PCB congeners on northern leopard frog (Rana pipiens) hatching success, survival and sexual development. Embryos and tadpoles were exposed to two levels (0.5 and 50 microg/l) of two PCBs. PCBs 101 and 70 were selected because they were present in amphibians collected in the Fox River-Green Bay ecosystem and they have the theoretical structural requirements to be able to bind to the estrogen receptor and mediate estrogenic responses. The exposure of leopard frog embryos and tadpoles to PCB 70 and 101 did not significantly affect hatchability, survival, deformities or growth. There were significant departures from the expected 50:50 sex ratio in tadpoles/froglets exposed to PCB 101 and PCB 70. In all the cases of significant departure, the bias was towards higher number of females. Decrease in the proportion of male gonads and increase in the proportion of intersex gonads were observed with increasing PCB tissue concentrations. The effects of PCB congeners on sexual differentiation occur at concentrations higher than observed in frogs in the Fox River/Green Bay ecosystem.

  9. The ortho:para-H_2 ratio in C- and J-type shocks

    NASA Astrophysics Data System (ADS)

    Wilgenbus, D.; Cabrit, S.; Pineau des Forêts, G.; Flower, D. R.

    2000-04-01

    We have computed extensive grids of models of both C- and J-type planar shock waves, propagating in dark, cold molecular clouds, in order to study systematically the behaviour of the ortho:para-H_2 ratio. Careful attention was paid to both macroscopic (dynamical) and microscopic (chemical reactions and collisional population transfer in H_2) aspects. We relate the predictions of the models to observational determinations of the ortho:para-H_2 ratio using both pure rotational lines and rovibrational lines. As an illustration, we consider ISO and ground-based H_2 observations of HH 54. Neither planar C-type nor planar J-type shocks appear able to account fully for these observations. Given the additional constraints provided by the observed ortho:para H_2 ratios, a C-type bowshock, or a C-type precursor followed by a J-type shock, remain as plausible models. Tables~2a-f and 4a-f are only available in electronic form at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsweb.u-strasbg.fr/Abstract.html

  10. Theoretical study of the design of a catalyst for para to ortho hydrogen conversion

    NASA Technical Reports Server (NTRS)

    Coffman, Robert E.

    1992-01-01

    The theory of Petzinger and Scalapino (1973) was thoroughly reviewed, and all of the basic equations for paramagnetic para to ortho hydrogen catalysis re-derived. There are only a few minor phase errors and errors of omission in the description of the theory. Three models (described by Petzinger and Scalapino) for the rate of para to ortho H2 catalysis were worked out, and uniform agreement obtained to within a constant factor of 2 pi. The analytical methods developed in the course of this study were then extended to two new models, which more adequately describe the process of surface catalysis including transfer of hydrogen molecules onto and off of the surface. All five equations for the para to ortho catalytic rate of conversion are described. The two new equations describe the catalytic rate for these models: H2 on the surface is a 2-D gas with lifetime tau; and H2 on the surface is a 2-D liquid undergoing Brownian motion (diffusion) with surface lifetime tau.

  11. Dielectric property of NiTiO{sub 3} doped substituted ortho-chloropolyaniline composites

    SciTech Connect

    Lakshmi, Mohana; Faisal, Muhammad; Roy, Aashish S.; Khasim, Syed; Sajjan, K. C.; Revanasiddappa, M.

    2013-11-15

    Ortho-chloropolyaniline (OCP)-NiTiO{sub 3} composites have been synthesized via in-situ polymerization of ortho-chloroaniline with various weight percentages of NiTiO{sub 3.} Fourier Transform Infrared spectroscopic studies of Ortho-chloropolyaniline and its composites indicated the formation of composites as a result of Vander Waal's interaction between OCP and NiTiO{sub 3} particles. Surface morphology of OCP and OCP-NiTiO{sub 3} composites were studied using Scanning Electron Microscope (SEM). The SEM micrographs indicated a modified morphology after the composite formation. Dielectric properties and electric modulus of OCP and OCP-NiTiO{sub 3} composites have been investigated in the frequency range of 50 Hz – 5 MHz. It has been noticed that electrical resistance decreases with increase in weight percentage of NiTiO{sub 3} particles in polymer matrix as well as with applied frequency. The display of semicircular arcs in Cole-Cole plots indicates the formation of series resistor and capacitor in network causing a decrease in the relaxation time and as a result conductivity enhances in these composites. The facile and cost effective synthesis process and excellent dielectric and conductivity response of these materials makes them promising materials for practical applications.

  12. Bactericidal effects and cytotoxicity of new aromatic dialdehyde disinfectants (ortho-phthalaldehyde).

    PubMed

    Iwasawa, Atsuo; Niwano, Yoshimi; Kohno, Masahiro; Ayaki, Masahiko

    2011-12-01

    We investigated the bactericidal effects and cytotoxicity of an ortho-phthalaldehyde product in comparison with those of its predecessor glutaraldehyde products. Bactericidal effects ware examined on Mycobacterium terrae, a standard organism used for investigating the bactericidal effect of high-level disinfectants. Cytotoxicity as determined by the MTT assay was examined by using four cell lines. The colony forming test, a method to examine residual toxicity, and the evaporation test, a newly developed method to examine the toxicity of the evaporated ingredients, were performed. Test solutions were 2.25% and 3.5% glutaraldehyde (GA) products and a 0.55% ortho-phthalaldehyde (OPA) product, and glutaraldehyde itself. All the disinfectants showed sufficient bactericidal effects on M. terrae. Meanwhile, the OPA product was less toxic than GA products and GA itself to all the cell lines tested. The colony forming test showed that GA products and GA itself exerted residual cytotoxicity more potently than did the OPA product. The evaporation test showed that GA products and GA itself exerted cytotoxicity via evaporation more potently than did the OPA product. In conclusion, OPA appears to be less cytotoxic than GA even though bactericidal effects were comparable. This may be due to the lower concentration of the active ingredient (ortho-phthalaldehyde) in the OPA product.

  13. Dielectric property of NiTiO{sub 3} doped substituted ortho-chloropolyaniline composites

    SciTech Connect

    Lakshmi, Mohana; Faisal, Muhammad; Roy, Aashish S.; Khasim, Syed; Sajjan, K. C.; Revanasiddappa, M.

    2013-11-15

    Ortho-chloropolyaniline (OCP)-NiTiO{sub 3} composites have been synthesized via in-situ polymerization of ortho-chloroaniline with various weight percentages of NiTiO{sub 3.} Fourier Transform Infrared spectroscopic studies of Ortho-chloropolyaniline and its composites indicated the formation of composites as a result of Vander Waal's interaction between OCP and NiTiO{sub 3} particles. Surface morphology of OCP and OCP-NiTiO{sub 3} composites were studied using Scanning Electron Microscope (SEM). The SEM micrographs indicated a modified morphology after the composite formation. Dielectric properties and electric modulus of OCP and OCP-NiTiO{sub 3} composites have been investigated in the frequency range of 50 Hz – 5 MHz. It has been noticed that electrical resistance decreases with increase in weight percentage of NiTiO{sub 3} particles in polymer matrix as well as with applied frequency. The display of semicircular arcs in Cole-Cole plots indicates the formation of series resistor and capacitor in network causing a decrease in the relaxation time and as a result conductivity enhances in these composites. The facile and cost effective synthesis process and excellent dielectric and conductivity response of these materials makes them promising materials for practical applications.

  14. The ortho:para ratio of H{sub 3}{sup +} in laboratory and astrophysical plasmas

    SciTech Connect

    Crabtree, Kyle N.; Indriolo, Nick; Kreckel, Holger; McCall, Benjamin J.

    2015-01-22

    The discovery of H{sub 3}{sup +} in the diffuse interstellar medium has dramatically changed our view of the cosmic-ray ionization rate in diffuse molecular clouds. However, another surprise has been that the ortho:para ratio of H{sub 3}{sup +} in these clouds is inconsistent with the temperature derived from the excitation of H{sub 2}, the dominant species in these clouds. In an effort to understand this discrepancy, we have embarked on an experimental program to measure the nuclear spin dependence of the dissociative electron recombination rate of H{sub 3}{sup +} using the CRYRING and TSR ion storage rings. We have also performed the first measurements of the reaction H{sub 3}{sup +}+H{sub 2}→H{sub 2}+H{sub 3}{sup +} below room temperature. This reaction is likely the most common bimolecular reaction in the universe, and plays an important role in interconverting ortho- and para-H{sub 3}{sup +}. Finally, we have constructed a steady-state chemical model for diffuse clouds, which takes into account the spin-dependence of the formation of H{sub 3}{sup +}, its electron recombination, and its reaction with H{sub 2}. We find that the ortho:para ratio of H{sub 3}{sup +} in diffuse clouds is likely governed by a competition between dissociative recombination and thermalization by reactive collisions.

  15. Influence of the ortho-methoxyalkyl substituent on the properties of phenylboronic acids

    NASA Astrophysics Data System (ADS)

    Adamczyk-Woźniak, Agnieszka; Brzózka, Zbigniew; Dąbrowski, Marek; Madura, Izabela D.; Scheidsbach, Roy; Tomecka, Ewelina; Żukowski, Kamil; Sporzyński, Andrzej

    2013-03-01

    Novel phenylboronic acids with methoxyalkyl groups at ortho position were synthesized. Molecular and crystal structures for two compounds were determined by single crystal X-ray diffraction. In both cases the O-H⋯O hydrogen-bonded dimers are the primary supramolecular motives in which the relatively short intramolecular B-O-H⋯O hydrogen bonds are observed between boronic group and oxygen atom of the ortho-substituent. Based on the CSD data for ortho-substitued boronic acids, the relation between the twist of the boronic moiety towards phenyl ring and the intramolecular H-bond angle is discussed. The intermolecular interactions between dimeric motives were investigated with the aid of Hirshfeld surface analysis. The weak C-H⋯O and C-H⋯π interactions were detected together with the agostic B⋯H ones. Sugar-binding ability of the methoxyalkyl compounds was evaluated for D-glucose, D-fructose and D-galactose by the competition assay with Alizarin Red S.

  16. Purification of Proteins From Cell-Culture Medium or Cell-Lysate by High-Speed Counter-Current Chromatography Using Cross-Axis Coil Planet Centrifuge

    PubMed Central

    Shibusawa, Yoichi; Ito, Yoichiro

    2014-01-01

    This review describes protein purifications from cell culture medium or cell-lysate by high speed counter-current chromatography using the cross-axis coil planet centrifuge. Purifications were performed using aqueous two phase systems composed of polyethylene glycols and dextrans. PMID:25360182

  17. Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

    PubMed

    Forte, Dorian; Ciciarello, Marilena; Valerii, Maria Chiara; De Fazio, Luigia; Cavazza, Elena; Giordano, Rosaria; Parazzi, Valentina; Lazzari, Lorenza; Laureti, Silvio; Rizzello, Fernando; Cavo, Michele; Curti, Antonio; Lemoli, Roberto M; Spisni, Enzo; Catani, Lucia

    2015-09-09

    Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of

  18. Evaluation of recombinant antigens in combination and single formula for diagnosis of feline toxoplasmosis.

    PubMed

    Abdelbaset, Abdelbaset Eweda; Alhasan, Hend; Salman, Doaa; Karram, Mohamed Hassan; Ellah Rushdi, Mahmoud Abd; Xuenan, Xuan; Igarashi, Makoto

    2017-01-01

    Cats are the only definitive hosts of Toxoplasma gondii and constitute an essential source of infection to all warm blooded animals and humans. Diagnosis of T. gondii infection in cats is fundamental for proper management and control of infection in humans and animals. In the current study, we have evaluated the diagnostic performance of tachyzoite lysate antigen (TLA) and different T. gondii recombinant antigens including surface antigen 2 (SAG2), dense granule proteins 2, 6, 7, 15 (GRA2, GRA6, GRA7, GRA15) and microneme 10 protein (MIC10) in immunoglobulin G enzyme linked-immunosorbent assay (IgG ELISA) using cat serum samples, with reference to latex agglutination test (LAT). Remarkably, TLA showed better performance than other recombinant antigens in IgG ELISAs as compared to LAT, with concordance and Kappa values of 94.27% and 0.93, respectively. Furthermore, to improve the reactivity of the recombinant antigens, we have developed IgG ELISAs using different combinations with these recombinant antigens. Strikingly, a combination of SAG2 and GRAs has relatively similar performance as TLA evidenced by concordance and Kappa values of 94.27% and 0.81, respectively. The developed ELISA with a combination of recombinant antigens can be used as a promising diagnostic tool for routine testing of T. gondii infection and mass screening in cats. The major advantages of this assay are the high sensitivity and specificity, lower cost, safer production and easiness of standardization in various laboratories worldwide. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Biochemical characterization of PECAM-1 (CD31 antigen) on human platelets.

    PubMed

    Metzelaar, M J; Korteweg, J; Sixma, J J; Nieuwenhuis, H K

    1991-12-02

    The platelet plasma membrane expresses several membrane glycoproteins with a high molecular weight. In this study we have investigated the properties of the CD31 antigen on platelets and endothelial cells using the monoclonal antibody (MoAb) RUU-PL 7E8. Comparative studies revealed that the CD31 antigen, PECAM-1 and endoCAM are the same protein. The CD31 antigen was immunoprecipitated with a molecular mass of 125 kDa nonreduced and 135 kDa reduced from Nonidet-P40 lysates of surface labeled human platelets. The relative position in two-dimensional nonreduced/reduced SDS-PAGE and IEF-PAGE, compared to other glycoproteins of similar molecular weight, was elucidated. The position of the CD31 antigen was clearly distinct from the position of the platelet membrane glycoproteins Ia, Ib, IIa, IIb, IIIa and the granule membrane protein GMP-140. Native resting platelets bound 7,760 +/- 1,670 molecules/platelet, whereas thrombin-stimulated platelets bound 14,500 +/- 3,790 molecules/platelet. Immunoelectron microscopy revealed the presence of the CD31 antigen on the membrane of both resting and thrombin-activated platelets. Immunofluorescence studies showed the presence of the CD31 antigen in the membrane of endothelial cells on sites of cell-cell contact, suggesting that the CD31 antigen might be involved in cell-cell interaction. In functional studies, MoAb RUU-PL 7E8 did not inhibit platelet aggregation, platelet adherence to the extracellular matrix of endothelial cells and purified collagen fibrils under flow conditions, nor was any influence found on endothelial cell detachment and growth.

  20. Antigenic proteins of Lactobacillus acidophilus that are recognised by serum IgG antibodies in children with type 1 diabetes and coeliac disease.

    PubMed

    Prangli, Anna-Liisa; Utt, Meeme; Talja, Ija; Sepp, Epp; Mikelsaar, Marika; Rajasalu, Tarvo; Uibo, Oivi; Tillmann, Vallo; Uibo, Raivo

    2010-06-01

    Immune responses to lactobacilli have been so far insufficiently investigated in patients with autoimmune diseases. We used whole-cell lysate of an indigenous Lactobacillus acidophilus strain isolated from an Estonian child to study serum IgG antibodies in children groups with type 1 diabetes [insulin dependent diabetes mellitus (IDDM)] (n = 21, age 4-18 yr) and with acute coeliac disease (CD) (n = 20, age 0.6-15 yr) and to compare the results with the controls (n = 24, age 2-17 yr). We found that our developed 1-D immunoblot assay readily enables to reveal antibodies against 28 L. acidophilus antigenic proteins in patients' and controls' sera. As verified by immunoproteomics analysis with 2-D and LC ESI-MS/MS the antigens of L. acidophilus were mainly common cytoplasmic proteins GroEL (HSP60), enolase, transcription factor EF-Ts and EF-Tu. However, in addition we identified formyl-CoA transferase being target for antibodies in every tested IDDM patients' serum. We have characterized for the first time the antigenic profile of L. acidophilus whole-cell lysate using sera from children with IDDM, CD, and controls. The different prevalence of reactions against tested antigens in patients and controls sera may indicate significant differences in immune system and commensal bacteria cross-talk in these groups.

  1. Association of plasma ortho-tyrosine/para-tyrosine ratio with responsiveness of erythropoiesis-stimulating agent in dialyzed patients.

    PubMed

    Kun, Szilárd; Mikolás, Esztella; Molnár, Gergo A; Sélley, Eszter; Laczy, Boglárka; Csiky, Botond; Kovács, Tibor; Wittmann, István

    2014-09-01

    Objectives Patients with end-stage renal failure (ESRF) treated with erythropoiesis-stimulating agents (ESAs) are often ESA-hyporesponsive associated with free radical production. Hydroxyl free radical converts phenylalanine into ortho-tyrosine, while physiological isomer para-tyrosine is formed enzymatically, mainly in the kidney. Production of 'para-tyrosine' is decreased in ESRF and it can be replaced by ortho-tyrosine in proteins. Our aim was to study the role of tyrosines in ESA-responsiveness. Methods Four groups of volunteers were involved in our cross-sectional study: healthy volunteers (CONTR; n = 16), patients on hemodialysis without ESA-treatment (non-ESA-HD; n = 8), hemodialyzed patients with ESA-treatment (ESA-HD; n = 40), and patients on continuous peritoneal dialysis (CAPD; n = 21). Plasma ortho-, para-tyrosine, and phenylalanine levels were detected using a high performance liquid chromatography (HPLC)-method. ESA-demand was expressed by ESA-dose, ESA-dose/body weight, and erythropoietin resistance index1 (ERI1, weekly ESA-dose/body weight/hemoglobin). Results We found significantly lower para-tyrosine levels in all groups of dialyzed patients when compared with control subjects, while in contrast ortho-tyrosine levels and ortho-tyrosine/para-tyrosine ratio were comparatively significantly higher in dialyzed patients. Among groups of dialyzed patients the ortho-tyrosine level and ortho-tyrosine/para-tyrosine ratio were significantly higher in ESA-HD than in the non-ESA-HD and CAPD groups. There was a correlation between weekly ESA-dose/body weight, ERI1, and ortho-tyrosine/para-tyrosine ratio (r = 0.441, P = 0.001; r = 0.434, P = 0.001, respectively). Our most important finding was that the ortho-tyrosine/para-tyrosine ratio proved to be an independent predictor of ERI1 (β = 0.330, P = 0.016). In these multivariate regression models most of the known predictors of ESA-hyporesponsiveness were included. Discussion Our findings may

  2. The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates.

    PubMed

    Vaughn, James P; Creacy, Steven D; Routh, Eric D; Joyner-Butt, Christi; Jenkins, G Scott; Pauli, Sandra; Nagamine, Yoshikuni; Akman, Steven A

    2005-11-18

    G4-DNA is a highly stable alternative DNA structure that can form spontaneously in guanine-rich regions of single-stranded DNA under physiological conditions. Since a number of biological processes create such single-stranded regions, G4-DNA occurrence must be regulated. To date, resolution of tetramolecular G4-DNA into single strands (G4-resolvase activity) has been observed only in recombinant RecQ DNA helicases. We previously reported that human cell lysates possess tetramolecular G4-DNA resolving activity (Harrington, C., Lan, Y., and Akman, S. (1997) J. Biol Chem. 272, 24631-24636). Here we report the first complete purification of a major non-RecQ, NTP-dependent G4-DNA resolving enzyme from human cell lysates. This enzyme is identified as the DEXH helicase product of gene DHX36 (also known as RHAU). G4-DNA resolving activity was captured from HeLa cell lysates on G4-DNA affinity beads and further purified by gel filtration chromatography. The DHX36 gene product was identified by mass spectrometric sequencing of a tryptic digest from the protein band on SDS-PAGE associated with activity. DHX36 was cloned within a His(6)-tagging vector, expressed, and purified from Escherichia coli. Inhibition and substrate resolution assays showed that recombinant DHX36 protein displayed robust, highly specific G4-DNA resolving activity. Immunodepletion of HeLa lysates by a monoclonal antibody to the DHX36 product removed ca. 77% of the enzyme from lysates and reduced G4-DNA resolving activity to 46.0 +/- 0.4% of control, demonstrating that DHX36 protein is responsible for the majority of tetramolecular G4-DNA resolvase activity.

  3. Characterization of ribonuclease H activities present in two cell-free protein synthesizing systems, the wheat germ extract and the rabbit reticulocyte lysate.

    PubMed

    Cazenave, C; Frank, P; Büsen, W

    1993-01-01

    Experimental evidence accumulated to date by several research groups indicates that antisense oligodeoxynucleotides targeted against messenger RNA (mRNA) sequences located downstream of the initiation codon fail to inhibit the translation of this mRNA unless the hybrid is cleaved by RNase H. It has previously been shown that exogenous RNase H has to be added to rabbit reticulocyte lysate to obtain translational arrest (unless freshly prepared lysates are used). In contrast there is no need of exogenous RNase H by using wheat germ extract for translation because the level of endogenous RNase H is high enough to ensure cleavage of the hybrid formed between the antisense oligodeoxyribonucleotide and its complementary sequence on the mRNA. Surprisingly, we found that these two cell-free translation systems display similar amounts of RNase H activities when tested under standard conditions (extract diluted 500 times in the RNase H reaction mix). The RNase H activity of the rabbit reticulocyte lysate has a divalent cation requirement and sensitivity to inhibitors similar to class I ribonuclease H, whereas the activity of the wheat germ extract shows similarities to class II ribonuclease H. However, when these activities were assayed under conditions similar to those used for translation experiments, only highly reduced levels of activity were found in comparison to the standard assays. This reduction is due in part to sub-optimal ionic conditions for the endogenous RNase H activities in these extracts, and, for the other part, likely due to interactions with other proteins present in the lysates. In these conditions, however, the remaining activity found in the wheat germ extract was three times higher than the activity found in the rabbit reticulocyte lysate. Whether this difference can by itself explain the indicated differences in the two systems observed in hybrid-arrest of translation experiments remains open to discussion.

  4. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  5. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    PubMed

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S; Greenblatt, Jack F; Marcon, Edyta; Arrowsmith, Cheryl H; Edwards, Aled M; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.

  6. Beneficial effect of a symbiotic preparation with S. boulardii lysate in mild stress-induced gut hyper-permeability.

    PubMed

    Takadanohara, Hiroshi; Catanzaro, Roberto; Chui, De Hua; He, Fengtian; Yadav, Hariom; Ganguli, Abhijit; Sakata, Yasuhiko; Solimene, Umberto; Minelli, Emilio; Kobayashi, Riyichi; Nagamachi, Yoko; Marotta, Francesco

    2012-12-01

    Increased intestinal permeability has been advocated as one of the likely causes of various pathologies, such as allergies and metabolic or even cardiovascular disturbances. Thus, the aim of the present study was to test a symbiotic preparation containing microbial lysates (KC-1317, Named, Italy) against stress-induced derangement of gut mucosa permeability. Sprague Dawley rats were allocated into control (n=20) and stress (n=20) group. Stress was implemented by 1h of water avoidance stress daily for 10 days. Body weight, food and water intake and passage of stool pellet during stress session were recorded throughout the experiment. On the 11th day, fluorescent iso-thiocyanate dextran solution was injected into small intestinal loops. One hour after the injection, rats were sacrificed. Jejunum and ileum were taken for histopathology. Blood was collected from the abdominal aorta to measure intestinal permeability. In stress group, stool pellets during stress session was significantly higher than control group (p < 0.01). Villus height (p < 0.01), crypt depth (p < 0.01), number of goblet cells in villus (p < 0.01) and crypt (p < 0.05) decreased significantly in jejunum as compared to control. These phenomena were significantly prevented by KC-1317 (p < 0.05). Ileum also showed atrophy but villus height and the number of goblet cells in the villi did not significantly differ. Plasma-concentration of brain-gut peptides (substance P, thyrotropin-releasing hormone, cholecystokinin and motilin) were affected by stress (p < 0.001) and this effect did not change during supplementation with KC-1317. Polymorphonuclear neutrophil counting was significantly higher in stress group as compared to control (p < 0.01) but this phenomenon was abolished in the ileum (p < 0.01) or partly but significantly reduced by KC-1317 supplementation (p < 0.05). Accordingly, intestinal permeability was significantly enhanced in stress group as compared to control (p < 0.01) and prevented by KC

  7. A nontetrameric species is the major soluble form of keratin in Xenopus oocytes and rabbit reticulocyte lysates

    PubMed Central

    1996-01-01

    Inside the interphase cell, approximately 5% of the total intermediate filament protein exists in a soluble form. Past studies using velocity gradient sedimentation (VGS) indicate that soluble intermediate filament protein exists as an approximately 7 S tetrameric species. While studying intermediate filament assembly dynamics in the Xenopus oocyte, we used both VGS and size-exclusion chromatography (SEC) to analyze the soluble form of keratin. Previous studies (Coulombe, P. A., and E. Fuchs. 1990. J. Cell Biol. 111:153) report that tetrameric keratins migrate on SEC with an apparent molecular weight of approximately 150,000; the major soluble form of keratin in the oocyte, in contrast, migrates with an apparent molecular weight of approximately 750,000. During oocyte maturation, the keratin system disassembles into a soluble form (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787) and the amount of the 750-kD keratin complex increases dramatically. Immunoprecipitation analysis of soluble keratin from matured oocytes revealed the presence of type I and type II keratins, but no other stoichiometrically associated polypeptides, suggesting that the 750-kD keratin complex is composed solely of keratin. To further study the formation of the 750-kD keratin complex, we used rabbit reticulocyte lysates (RRL). The 750-kD keratin complex was formed in RRLs contranslating type I and type II Xenopus keratins, but not when lysates translated type I or type II keratin RNAs alone. The 750-kD keratin complex could be formed posttranslationally in an ATP-independent manner when type I and type II keratin translation reactions were mixed. Under conditions of prolonged incubation, such as occur during VGS analysis, the 750-kD keratin complex disassembled into a 7 S (by VGS), 150-kD (by SEC) form. In urea denaturation studies, the 7 S/150-kD form could be further disassembled into an 80-kD species that consists of cofractionating dimeric and monomeric

  8. Where does the electron go? The nature of ortho/para and meta group directing in electrophilic aromatic substitution

    SciTech Connect

    Liu, Shubin

    2014-11-21

    Electrophilic aromatic substitution as one of the most fundamental chemical processes is affected by atoms or groups already attached to the aromatic ring. The groups that promote substitution at the ortho/para or meta positions are, respectively, called ortho/para and meta directing groups, which are often characterized by their capability to donate electrons to or withdraw electrons from the ring. Though resonance and inductive effects have been employed in textbooks to explain this phenomenon, no satisfactory quantitative interpretation is available in the literature. Here, based on the theoretical framework we recently established in density functional reactivity theory (DFRT), where electrophilicity and nucleophilicity are simultaneously quantified by the Hirshfeld charge, the nature of ortho/para and meta group directing is systematically investigated for a total of 85 systems. We find that regioselectivity of electrophilic attacks is determined by the Hirshfeld charge distribution on the aromatic ring. Ortho/para directing groups have most negative charges on the ortho/para positions, while meta directing groups often possess the largest negative charge on the meta position. Our results do not support that ortho/para directing groups are electron donors and meta directing groups are electron acceptors. Most neutral species we studied here are electron withdrawal in nature. Anionic systems are always electron donors. There are also electron donors serving as meta directing groups. We predicted ortho/para and meta group directing behaviors for a list of groups whose regioselectivity is previously unknown. In addition, strong linear correlations between the Hirshfeld charge and the highest occupied molecular orbital have been observed, providing the first link between the frontier molecular orbital theory and DFRT.

  9. Where does the electron go? The nature of ortho/para and meta group directing in electrophilic aromatic substitution.

    PubMed

    Liu, Shubin

    2014-11-21

    Electrophilic aromatic substitution as one of the most fundamental chemical processes is affected by atoms or groups already attached to the aromatic ring. The groups that promote substitution at the ortho/para or meta positions are, respectively, called ortho/para and meta directing groups, which are often characterized by their capability to donate electrons to or withdraw electrons from the ring. Though resonance and inductive effects have been employed in textbooks to explain this phenomenon, no satisfactory quantitative interpretation is available in the literature. Here, based on the theoretical framework we recently established in density functional reactivity theory (DFRT), where electrophilicity and nucleophilicity are simultaneously quantified by the Hirshfeld charge, the nature of ortho/para and meta group directing is systematically investigated for a total of 85 systems. We find that regioselectivity of electrophilic attacks is determined by the Hirshfeld charge distribution on the aromatic ring. Ortho/para directing groups have most negative charges on the ortho/para positions, while meta directing groups often possess the largest negative charge on the meta position. Our results do not support that ortho/para directing groups are electron donors and meta directing groups are electron acceptors. Most neutral species we studied here are electron withdrawal in nature. Anionic systems are always electron donors. There are also electron donors serving as meta directing groups. We predicted ortho/para and meta group directing behaviors for a list of groups whose regioselectivity is previously unknown. In addition, strong linear correlations between the Hirshfeld charge and the highest occupied molecular orbital have been observed, providing the first link between the frontier molecular orbital theory and DFRT.

  10. Orthopedic Multidimensional Prognostic Index (Ortho-MPI) in the elderly with hip or neck femur fracture: a pilot study.

    PubMed

    Vitale, Elsa; Notarnicola, Angela; Tafuri, Silvio; Vicenti, Giovanni; Cassano, Maria; Moretti, Biagio

    2014-01-01

    The Orthopedic Multidimensional Prognostic Index (Ortho-MPI) was performed and validated in order to ameliorate the decision-making process as regards the elderly with hip or neck femur fractures. A retrospective study was performed. 95 patients 65 years old and over with a diagnosis of hip or femur fracture were enrolled. A standardized comprehensive orthopedic geriatric assessment was performed. It included information on: depressive symptoms, functional and instrumental activities of daily living, cognitive and nutritional status, laboratory tests, risk of pressure sore, comorbidities and comorbidity. The Ortho-MPI was calculated. After six months their initial assessment, patients were recalled in order to know if they live too or not. The survival condition was associated to the prognostic capacity calculated by the Ortho-MPI. Results showed that higher Ortho-MPI Index value was associated with higher six months-later mortality. In an unvaried analysis model the Ortho-MPI index was associated with death event of the elderly patients enrolled (OR=1.05; 95% CI, 1.01-1.10; z=2.27; p=0.023). This association was also validated by considering different ages between participants (OR=1.05; 95% CI, 1.004-1.11; z=2.13; p=0.033). Furthermore, each specific index considered in the total Ortho-MPI was associated with the death event of the elderly patients. In conclusion it was shown that the Ortho-MPI Index could be used to predict outcome in the elderly with hip or femur fracture.

  11. Levels of non-ortho-substituted (coplanar), mono- and di-ortho-substituted polychlorinated biphenyls, dibenzo-p-dioxins, and dibenzofurans in human serum and adipose tissue.

    PubMed Central

    Patterson, D G; Todd, G D; Turner, W E; Maggio, V; Alexander, L R; Needham, L L

    1994-01-01

    We have measured non-ortho-substituted (coplanar) polychlorinated biphenyl (PCB) levels as well as polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) levels in human adipose tissue and serum collected in Atlanta, Georgia. The results show that the concentrations of the coplanar PCBs can be more than an order of magnitude higher than the concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Our measurements in pooled serum collected in 1982, 1988, and 1989 show a decrease in coplanar PCB levels from 1982 to 1989. We found that the pattern of relative amounts of coplanar PCBs in adipose tissue varied greatly from person to person unlike the PCDD and PCDF patterns, which were more nearly the same. Age was significantly correlated with the concentrations of 2,3,7,8-TCDD,3,3'4,4'-PCB, 3,3',4,4',5-PCB, and 3,3'4,4',5,5'-PCB in adipose tissue. We also measured levels of the mono- and di-ortho chlorine-substituted PCBs in human serum. The levels for some of these PCB congeners were three orders of magnitude higher than the coplanar PCBs, PCDDs, and PCDFs. We used the international toxicity equivalency factors (TEFs) for PCDDs and PCDFs and the TEFs proposed by Safe for PCBs to calculate the 2,3,7,8-TCDD equivalents. Four PCBs (3,3',4,4',5-; 2,3',4,4',5-;2,3,3',4,4'-;2,3,3',4,4',5-) make a larger contribution than 2,3,7,8-TCDD, while four other PCBs (3,3',4,4'5,5'-; 2,2',3,4,4',5'-;2,2',4,4',5,5'-;2,2',3,4,4',5,5'-) make nearly the same contribution as 2,3,7,8-TCDD. The mono-ortho-chlorine-substituted 2,3',4,4',5-PCB, however, is the major contributor to the total 2,3,7,8-TCDD equivalents in general population samples from the United States, Sweden, and Japan.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8187709

  12. Factors important in the extraction, stability and in vitro assembly of the hepatitis B surface antigen derived from recombinant plant systems.

    PubMed

    Smith, Mark L; Keegan, Mark E; Mason, Hugh S; Shuler, Michael L

    2002-01-01

    The expression of vaccine antigens in edible plant material together with their delivery by the oral route constitutes a powerful paradigm, with the potential to dramatically reduce the cost of vaccine production and administration, in addition to improving distribution and patient compliance. These products will be subject to many of the same regulations applied to current injectable vaccines, so reliable methods to quantify antigen and ensure stability in crude plant extracts are required. As a model system the hepatitis B surface antigen (HBsAg) was expressed in soybean and tobacco cell cultures. This complex antigen consists of membrane-associated small surface antigen proteins (p24(s)), disulfide cross-linked to yield dimers and higher multimers. Although the total p24(s) extracted from plant cells was relatively unaffected by detergent concentration, the quantification of antigenically reactive product depended strongly on the ratio of detergent to cell concentration. Furthermore, 1-20% w/v sodium ascorbate improved the measured levels of monoclonal-reactive antigen 4- to 12-fold. Detergent also influenced antigen stability in cell lysates stored at 4 degrees C; under optimum conditions stability was maintained for at least 1 month, whereas excess detergent rendered the antigen susceptible to proteolytic degradation. This proteolysis could be counteracted by the addition of skim milk or its protein component, which stabilized antigenically reactive p24(s) for up to 2 months. The immunologically relevant epitopes of HBsAg are critically dependent on disulfide bonding. By altering the sodium ascorbate concentration or buffer pH the proportion of HBsAg displaying the monoclonal reactive epitopes was increased between 8- and 20-fold. In addition, under certain conditions the dimerized p24(s) could be converted to oligomeric aggregates, resembling the form of the serum-derived antigen. These simple in vitro manipulations, compatible with the goal of a minimally

  13. HEMOXCell, a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate-Supplemented Media.

    PubMed

    Le Pape, Fiona; Cosnuau-Kemmat, Lucie; Richard, Gaëlle; Dubrana, Frédéric; Férec, Claude; Zal, Franck; Leize, Elisabeth; Delépine, Pascal

    2017-03-22

    Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds.

  14. Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system.

    PubMed

    Kepka, Cecilia; Rhodin, Jenny; Lemmens, Raf; Tjerneld, Folke; Gustavsson, Per-Erik c

    2004-01-23

    The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA.

  15. Platelet lysate coating on scaffolds directly and indirectly enhances cell migration, improving bone and blood vessel formation.

    PubMed

    Leotot, Julie; Coquelin, Laura; Bodivit, Gwellaouen; Bierling, Philippe; Hernigou, Philippe; Rouard, Helene; Chevallier, Nathalie

    2013-05-01

    Suitable colonization and vascularization of tissue-engineered constructs after transplantation represent critical steps for the success of bone repair. Human platelet lysate (hPL) is composed of numerous growth factors known for their proliferative, differentiative and chemo-attractant effects on various cells involved in wound healing and bone growth. The aim of this study was to determine whether the delivery of human mesenchymal stromal cells (hMSC) seeded on hPL-coated hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) scaffolds could enhance vascularization and bone formation, as well as to investigate the mechanisms by which hMSC participate in tissue regeneration. Our study demonstrates that hPL can be coated on HA/β-TCP scaffolds, which play direct and indirect effects on implanted and/or resident stem cells. Effectively, we show that hPL coating directly increases chemo-attraction to and adhesion of hMSC and endothelial cells on the scaffold. Moreover, we show that hPL coating induces hMSC to produce and secrete pro-angiogenic proteins (placental growth factor and vascular endothelial growth factor) which allow the proliferation and specific chemo-attraction of endothelial cells in vitro, thus improving in vivo neovascularization and new bone formation. This study highlights the potential of functionalizing biomaterials with hPL and shows that this growth factor combination can have synergistic effects leading to enhanced bone and blood vessel formation.

  16. The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

    PubMed

    Ruggiu, Alessandra; Ulivi, Valentina; Sanguineti, Francesca; Cancedda, Ranieri; Descalzi, Fiorella

    2013-12-01

    Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair.

  17. Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    PubMed

    Xia, Wenjie; Li, Hui; Wang, Zhen; Xu, Ru; Fu, Yongshui; Zhang, Xiuming; Ye, Xin; Huang, Yingfeng; Xiang, Andy Peng; Yu, Weihua

    2011-06-01

    MSCs (mesenchymal stem cells) with their versatile growth and differentiation potential are ideal candidates for use in regenerative medicine and are currently making their way into clinical trials, which requires the development of xeno-free protocols for their culture. In this study, MSCs were cultured in 10% FCS or 7.5% HPL (human platelet lysate)-supplemented media. We found that both groups of MSCs showed a comparable morphology, phenotype and proliferation. The percentage of cells in the S- and G2-/M-phases, however, was slightly up-regulated (P<0.01) in HPL group. HPL contains PDGF (platelet derived growth factor)-AB and IGF (insulin-like growth factor)-1. In addition, compared with FCS group, MSCs in HPL group showed an increase in osteogenic differentiation and a decrease in adipogenic differentiation. In conclusion, MSCs in HPL-supplemented media maintained similar growing potential and phenotype, while osteogenic potential was enhanced. HPL offers a promising alternative to FCS for MSC expansion for clinical application, especially in bone injury diseases.

  18. Fast and mild strategy, using superhydrophobic surfaces, to produce collagen/platelet lysate gel beads for skin regeneration.

    PubMed

    Lima, Ana Catarina; Mano, João F; Concheiro, Angel; Alvarez-Lorenzo, Carmen

    2015-02-01

    Platelet lysate (PL) was encapsulated in collagen (Coll) millimetric gel beads, on biomimetic superhydrophobic surfaces, under mild conditions, with the aim of obtaining easy-to-handle formulations able to provide sustained release of multiple growth factors for skin ulcers treatment. The gel particles were prepared with various concentrations of PL incorporating or not stem cells, and tested as freshly prepared or after being freeze-dried or cryopreserved. Coll + PL particles were evaluated regarding degradation in collagenase-rich environment (simulating the aggressive environment of the chronic ulcers), sustained release of total protein, PDGF-BB and VEGF, cell proliferation (using particles as the only source of growth factors), scratch wound recovery and angiogenic capability. Compared to Coll solely particles, incorporation of PL notably enhanced cell proliferation (inside and outside gels) and favored scratch wound recovery and angiogenesis. Moreover, cell-laden gel particles containing PL notably improved cell proliferation and even migration of cells from one particle towards a neighbor one, which led to cell-cell contacts and the spontaneous formation of tissue layers in which the spherical gels were interconnected by the stem cells.

  19. Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells.

    PubMed

    Walenda, Gudrun; Hemeda, Hatim; Schneider, Rebekka K; Merkel, Rudolf; Hoffmann, Bernd; Wagner, Wolfgang

    2012-12-01

    Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that-apart from heparin-consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix-this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion of MSCs.

  20. A clinically-feasible protocol for using human platelet lysate and mesenchymal stem cells in regenerative therapies.

    PubMed

    Warnke, Patrick H; Humpe, Andreas; Strunk, Dirk; Stephens, Sebastien; Warnke, Frauke; Wiltfang, Joerg; Schallmoser, Katharina; Alamein, Mohammad; Bourke, Robert; Heiner, Peter; Liu, Qin

    2013-03-01

    The transplantation of human stem cells seeded on biomaterials holds promise for many clinical applications in cranio-maxillo-facial tissue engineering and regenerative medicine. However, stem cell propagation necessary to produce sufficient cell numbers currently utilizes fetal calf serum (FCS) as a growth supplement which may subsequently transmit animal pathogens. Human platelet lysate (HPL) could potentially be utilized to produce clinical-grade stem cell-loaded biomaterials as an appropriate FCS substitute that is in line with clinically-applicable practice. The goal of this study was to investigate whether HPL can be successfully used to propagate human mesenchymal stem cells (HMSCs) seeded on clinically-approved collagen materials under clinically-applicable conditions using FCS as a control. HMSCs were isolated from bone marrow and cultured in the presence of 10% FCS or 10% HPL. Characterization of HMSCs was performed by flow cytometry and through osteogenic and adipogenic differentiation assays. Proliferative capacity of HMSCs on both matrices was investigated by mitochondrial dehydrogenase assays (WST) and tissue coverage scanning electron microscopy (SEM). The isolated HMSC differentiated into osteogenic and adipogenic cells authenticating the multipotentiality of the HMSCs. WST tests and the SEM images demonstrated that HPL was generally superior to FCS in promoting growth of seeded HMSCs. For all other tests HPL supported HMSCs at least equal to FCS. In conclusion, HPL is an effective growth factor to allow expansion of clinical-grade HMSCs on clinically-approved biomaterials for maxillofacial and oral implantology applications.

  1. Calcium alginate particles for the combined delivery of platelet lysate and vancomycin hydrochloride in chronic skin ulcers.

    PubMed

    Mori, Michela; Rossi, Silvia; Bonferoni, Maria Cristina; Ferrari, Franca; Sandri, Giuseppina; Riva, Federica; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla

    2014-01-30

    The aim of the present work was the development of a powder formulation for the combined delivery of platelet lysate and of a model antibiotic drug, vancomycin hydrochloride (VCM), in chronic skin ulcers. Calcium alginate particles were prepared by freeze-drying beads obtained by ionic gelation method. The experimental conditions adopted permitted the complete loading of VCM and of PDGF AB, the growth factor chosen as representative of those contained in PL. Such particles where able to absorb PBS (mimicking wound exudate), to form a gel and to modulate the release of VCM and of PDGF AB. They are characterized by enhancement properties of human fibroblast proliferation due to PL presence. In particular, PL, when loaded in alginate particles, was able not only to increase the number of viable cells, but also the number of cells in proliferative phase. Such properties were comparable to those of fresh PL indicating the capability of calcium alginate particles to load PL bioactive substances without altering their activity. The formulation developed is characterized by an easier and a less painful administration with respect to traditional gauzes and semisolid preparations and permits the loading in the same dosage form of active substances of different nature avoiding eventual incompatibility problems.

  2. The Limulus Amebocyte Lysate assay may be unsuitable for detecting endotoxin in blood of healthy female subjects.

    PubMed

    Gnauck, Anne; Lentle, Roger G; Kruger, Marlena C

    2015-01-01

    We examined the factors that may influence the outcome of the Limulus Amebocyte Lysate (LAL) assay, when it is used for quantifying Gram-negative bacterial endotoxin, also referred to as lipopolysaccharide (LPS), in samples of human blood. We found that the method recommended by the manufacturers, based on the reaction time, was inaccurate with any type of serum samples due to the slowing of the initial phase of reaction, likely by serum proteins. We describe an alternative method that is more accurate for use with heated serum samples. Further, we found that components of fresh serum irreversibly sequester endotoxin but that this action may be largely prevented by dilution and heating, but only if this occurs prior to the addition of endotoxin. The tests also indicated that a number of types of proprietary plastic vacutainers appeared to contain significant amounts of endotoxin. However, even when appropriate blood collection containers and calculation methods were used, the levels of endotoxin in serum samples detected by LAL assay were unlikely to reflect the total quantities of endotoxin in that sample and more likely to reflect the capacity of a given serum sample to sequester endotoxin.

  3. Endotoxin Detection in Pharmaceuticals and Medical Devices with Kinetic-QCL, a Kinetic-Quantitative Chromogenic Limulus Amebocyte Lysate Assay.

    PubMed

    Berzofsky, Ronald N.

    1995-01-01

    The observation that endotoxin caused gelation in extracts of Limulus amebocytes has been expanded to the development of an in vitro kinetic, quantitative chromogenic LAL assay (Kinetic-QCL) for the detection of endotoxin in aqueous fluids. Within the last 15 years, the use of Limulus amebocyte lysate to detect and control the presence of pyrogenic substances in pharmaceuticals and medical devices has gained wide international acceptance. Both the United States and European Pharmacopoeias contain descriptions of and requirements for the LAL Bacterial Endotoxin Test. Both pharmacopoeias have begun to remove the rabbit pyrogen test requirement in a majority of drug monographs and have substituted endotoxin limits to be determined by LAL. The use of LAL has proved invaluable in controlling the level of endotoxin in finished product. The endotoxin contribution of raw materials and packaging material can be monitored as well. In-process testing at critical production steps can identify additional sources of endotoxin contamination, and depyrogenation processes can be validated by quantitating the degradation of endotoxin challenges. The speed, reproducibility, sensitivity, and economics of the Kinetic-QCL assay, in conjunction with the ppropriate equipment and software, over both the in vivo rabbit pyrogen test and the more traditional LAL gel-clot assay allow a more in-depth approach to the control of endotoxin in pharmaceuticals and medical devices.

  4. Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers

    PubMed Central

    Pedrero, María; Manuel de Villena, F. Javier; Muñoz-San Martín, Cristina; Campuzano, Susana; Garranzo-Asensio, María; Barderas, Rodrigo; Pingarrón, José M.

    2016-01-01

    An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation. PMID:27879639

  5. Identification of a bacteriolysis-associated virulence factor against lung epithelial cells in Pseudomonas aeruginosa PAO-1 cell lysate.

    PubMed

    Shinagawa, Masaaki; Kobayashi, Daisuke; Goto, Maki; Tanaka, Maki; Kuribayashi, Kageaki; Yanagihara, Nozomi; Watanabe, Naoki

    2014-10-01

    The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. A therapy-grade protocol for differentiation of pluripotent stem cells into mesenchymal stem cells using platelet lysate as supplement.

    PubMed

    Luzzani, Carlos; Neiman, Gabriel; Garate, Ximena; Questa, María; Solari, Claudia; Fernandez Espinosa, Darío; García, Marcela; Errecalde, Ana Lía; Guberman, Alejandra; Scassa, María Elida; Sevlever, Gustavo Emilio; Romorini, Leonardo; Miriuka, Santiago Gabriel

    2015-01-12

    Mesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes. In this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement. We showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum. This protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.

  7. Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.

    PubMed

    Doucet, Christelle; Ernou, Isabelle; Zhang, Yizhou; Llense, Jean-Roch; Begot, Laurent; Holy, Xavier; Lataillade, Jean-Jacques

    2005-11-01

    Mesenchymal stem cells (MSCs) are considered as emergent "universal" cells and various tissue repair programs using MSCs are in development. In vitro expansion of MSCs is conventionally achieved in medium containing fetal calf serum (FCS) and is increased by addition of growth factors. However, for widespread clinical applications, contact of MSCs with FCS must be minimized since it is a putative source of prion or virus transmission. Therefore, because platelets are a natural source of growth factors, we sought to investigate in vitro MSC expansion in response to platelet lysates (PL) obtained from platelet-rich plasma. Human MSCs were expanded in FCS (+/-bFGF)- or PL-supplemented medium through a process of subculture. We demonstrated that PL-containing medium is enriched by growth factors (platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), insulin-like growth factor-1 (IGF-1) ...) and showed that PL is able to promote MSC expansion, to decrease the time required to reach confluence, and to increase CFU-F size, as compared to the FCS medium. Furthermore, we demonstrated that MSCs cultured in the presence of PL maintain their osteogenic, chondrogenic, and adipogenic differentiation properties and retain their immunosuppressive activity. Therefore, we propose that PL may be a powerful and safe substitute for FCS in development of tissue- and cellular-engineered products in clinical settings using MSCs.

  8. A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates.

    PubMed

    Buntru, Matthias; Vogel, Simon; Stoff, Katrin; Spiegel, Holger; Schillberg, Stefan

    2015-05-01

    Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins.

  9. Manipulation and extraction of genomic DNA from cell lysate by functionalized magnetic particles for lab on a chip applications.

    PubMed

    Yeung, Siu Wai; Hsing, I-Ming

    2006-01-15

    A novel approach for extracting living cells' genomic DNA materials utilizing functionalized magnetic particles (MPs) is reported in this investigation. This strategy is amenable to handle bio-samples in a miniaturized environment and it offers a possibility to separate and purify DNA from other cell lysate mixtures "on-chip", which is known to be a bottle-neck step in an integrated micro-total-analysis-system (muTAS). "Species-specific" genomic DNA of interest is captured by the MPs based on the hybridization interaction between the biotinylated probes modified MPs and a complementary region of the targeted genome. The genome DNA anchored on the particles can be separated from the rest of cellular mixtures by a simple buffer washing upon the exertion of external magnetic force. Surface modifications of MPs and hybridization conditions affecting the genome capturing efficiency are investigated. Extraction of genomic DNA from E. coli is demonstrated in a silicon/glass-based micro-reactor patterned with a platinum heater and sensors. On-chip extraction and manipulation of genomic DNAs illustrated in this study is a step forward toward a total integrated bioanalytical microsystem for crude cells/sample analysis.

  10. Cytokine-induced killer cells interact with tumor lysate-pulsed dendritic cells via CCR5 signaling.

    PubMed

    Lee, Hong Kyung; Kim, Yong Guk; Kim, Ji Sung; Park, Eun Jae; Kim, Boyeong; Park, Ki Hwan; Kang, Jong Soon; Hong, Jin Tae; Kim, Youngsoo; Han, Sang-Bae

    2016-08-10

    The antitumor activity of cytokine-induced killer (CIK) cells can be increased by co-culturing them with tumor lysate-pulsed dendritic cells (tDCs); this phenomenon has been studied mainly at the population level. Using time-lapse imaging, we examined how CIK cells gather information from tDCs at the single-cell level. tDCs highly expressed CCL5, which bound CCR5 expressed on CIK cells. tDCs strongly induced migration of Ccr5(+/+) CIK cells, but not that of Ccr5(-/-) CIK cells or Ccr5(+/+) CIK cells treated with the CCR5 antagonist Maraviroc. Individual tDCs contacted Ccr5(+/+) CIK cells more frequently and lengthily than with Ccr5(-/-) CIK cells. Consequently, tDCs increased the antitumor activity of Ccr5(+/+) CIK cells in vitro and in vivo, but did not increase that of Ccr5(-/-) CIK cells. Taken together, our data provide insight into the mechanism of CIK cell activation by tDCs at the single-cell level.

  11. Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation

    PubMed Central

    Palmieri, Francesca; Marrelli, Massimo

    2016-01-01

    Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H2O2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use. PMID:27774106

  12. Setting a standard: the limulus amebocyte lysate assay and the assessment of microbial contamination on spacecraft surfaces.

    PubMed

    Morris, Heather C; Monaco, Lisa A; Steele, Andrew; Wainwright, Norm

    2010-10-01

    Historically, colony-forming units as determined by plate cultures have been the standard unit for microbiological analysis of environmental samples, medical diagnostics, and products for human use. However, the time and materials required make plate cultures expensive and potentially hazardous in the closed environments of future NASA missions aboard the International Space Station and missions to other Solar System targets. The Limulus Amebocyte Lysate (LAL) assay is an established method for ensuring the sterility and cleanliness of samples in the meat-packing and pharmaceutical industries. Each of these industries has verified numerical requirements for the correct interpretation of results from this assay. The LAL assay is a rapid, point-of-use, verified assay that has already been approved by NASA Planetary Protection as an alternate, molecular method for the examination of outbound spacecraft. We hypothesize that standards for molecular techniques, similar to those used by the pharmaceutical and meat-packing industries, need to be set by space agencies to ensure accurate data interpretation and subsequent decision making. In support of this idea, we present research that has been conducted to relate the LAL assay to plate cultures, and we recommend values obtained from these investigations that could assist in interpretation and analysis of data obtained from the LAL assay.

  13. Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[S

    PubMed Central

    Pais de Barros, Jean-Paul; Gautier, Thomas; Sali, Wahib; Adrie, Christophe; Choubley, Hélène; Charron, Emilie; Lalande, Caroline; Le Guern, Naig; Deckert, Valérie; Monchi, Mehran; Quenot, Jean-Pierre; Lagrost, Laurent

    2015-01-01

    Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo. PMID:26023073

  14. TOP mRNAs are translationally inhibited by a titratable repressor in both wheat germ extract and reticulocyte lysate.

    PubMed

    Biberman, Y; Meyuhas, O

    1999-08-13

    Vertebrate TOP mRNAs contain a 5' terminal oligopyrimidine tract (5' TOP), which is subject to selective translational repression in non-growing cells or in cell-free translation systems. In the present study, we monitored in vitro the effect of increasing amounts of a 16 nucleotides long oligoribonucleotide representing the 5' terminus of mouse ribosomal protein S16 mRNA on the translation of TOP and non-TOP mRNAs. Our results demonstrate that the wild-type sequence (but not its mutant counterparts) derepresses the translation of mRNAs containing 5' TOP motifs, but failed to stimulate the translation of non-TOP mRNAs, even if the latter differed only by a single nucleotide from their 5' TOP-containing counterparts. Similar results have been obtained with both wheat germ extract and rabbit reticulocyte lysate. It appears, therefore, that translational repression of TOP mRNAs is achieved in vitro by the accumulation of a titratable repressor rather than by the loss of an activator and that this repressor recognizes multiple TOP mRNAs with a diverse set of 5' TOP motifs.

  15. Acute on-chip HIV detection through label-free electrical sensing of viral nano-lysate.

    PubMed

    Shafiee, Hadi; Jahangir, Muntasir; Inci, Fatih; Wang, Shuqi; Willenbrecht, Remington B M; Giguel, Francoise F; Tsibris, Athe M N; Kuritzkes, Daniel R; Demirci, Utkan

    2013-08-12

    Development of portable biosensors has broad applications in environmental monitoring, clinical diagnosis, public health, and homeland security. There is an unmet need for pathogen detection at the point-of-care (POC) using a fast, sensitive, inexpensive, and easy-to-use method that does not require complex infrastructure and well-trained technicians. For instance, detection of Human Immunodeficiency Virus (HIV-1) at acute infection stage has been challenging, since current antibody-based POC technologies are not effective due to low concentration of antibodies. In this study, we demonstrated for the first time a label-free electrical sensing method that can detect lysed viruses, i.e. viral nano-lysate, through impedance analysis, offering an alternative technology to the antibody-based methods such as dipsticks and Enzyme-linked Immunosorbent Assay (ELISA). The presented method is a broadly applicable platform technology that can potentially be adapted to detect multiple pathogens utilizing impedance spectroscopy for other infectious diseases including herpes, influenza, hepatitis, pox, malaria, and tuberculosis. The presented method offers a rapid and portable tool that can be used as a detection technology at the POC in resource-constrained settings, as well as hospital and primary care settings.

  16. A new application of aptamer: One-step purification and immobilization of enzyme from cell lysates for biocatalysis.

    PubMed

    Qiao, Lifeng; Lv, Bo; Feng, Xudong; Li, Chun

    2015-06-10

    Aptamers are nucleic acid-based high affinity ligands that are able to capture their corresponding target through molecular recognition. In this study, several DNA aptamers with high affinity and specificity for β-glucuronidases (PGUS-E) were obtained by our modified SELEX method. Among them, Apt5 and Apt9 were selected as representatives and covalently linked to magnetic beads, respectively. The aptamer-modified magnetic beads were characterized and successfully applied to one-step purification and immobilization of PGUS-E from the complex cell lysates. By conveniently adjusting the pH and ion strength, the PGUS-E purities reached 84% for Apt5-modified beads and 88% for Apt9-modified beads. Moreover, the maximum PGUS-E capturing capacity of the Apt5 and Apt9 modified magnetic beads were found to be 31.75μg/mg and 32.95μg/mg, respectively. The immobilized PGUS-E on aptamer-based magnetic beads showed good reusability, and the conversion of glycyrrhizin still remained more than 70% after 7 cycles. In addition, the aptamer-modified beads support can be easily regenerated, and the conversion rate of glycyrrhizin (GL) was still 62% after the 7th cycle of regeneration. This investigation can be easily extended to other enzyme systems and may help open a generic route to develop a novel enzyme immobilization technology for biocatalysis based on aptamer. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Capillary zone electrophoresis-multiple reaction monitoring from 100 pg of RAW 264.7 cell lysate digest

    PubMed Central

    Sun, Liangliang; Li, Yihan; Champion, Matthew M.; Zhu, Guijie; Wojcik, Roza; Dovichi, Norman J.

    2013-01-01

    Capillary zone electrophoresis-multiple/single reaction monitoring (CZE-MRM/SRM), which employed an electrokinetically driven sheath-flow electrospray interface, was used for the rapid and highly sensitive detection of protein analytes in complex tryptic digests. MRM channels were developed against a commercial exponential mixture of bovine proteins. Five proteins spanning four orders of magnitude concentration range were confidently detected from only 2.5 ng of the digest mixture; the mass detection limits (S/N=3) of two detected proteins, alpha-casein and glutamate dehydrogenasewere about 600 zmole and 30 amole, respectively. This technique was then applied to a RAW 264.7 cell lysate digest. Three proteins were confidently and reproducibly detected from 100 pg of this digest. The sample amount corresponds to the approximate protein content from a single cell, which suggests that CZE-MRM may be a useful analytical tool in chemical cytometry. In addition to providing highly sensitive detection of proteins in complex mixtures, this system is highly rapid; migration time of the protein digests was less than 10 min. PMID:23591184

  18. OrthoMaM v8: a database of orthologous exons and coding sequences for comparative genomics in mammals.

    PubMed

    Douzery, Emmanuel J P; Scornavacca, Celine; Romiguier, Jonathan; Belkhir, Khalid; Galtier, Nicolas; Delsuc, Frédéric; Ranwez, Vincent

    2014-07-01

    Comparative genomic studies extensively rely on alignments of orthologous sequences. Yet, selecting, gathering, and aligning orthologous exons and protein-coding sequences (CDS) that are relevant for a given evolutionary analysis can be a difficult and time-consuming task. In this context, we developed OrthoMaM, a database of ORTHOlogous MAmmalian Markers describing the evolutionary dynamics of orthologous genes in mammalian genomes using a phylogenetic framework. Since its first release in 2007, OrthoMaM has regularly evolved, not only to include newly available genomes but also to incorporate up-to-date software in its analytic pipeline. This eighth release integrates the 40 complete mammalian genomes available in Ensembl v73 and provides alignments, phylogenies, evolutionary descriptor information, and functional annotations for 13,404 single-copy orthologous CDS and 6,953 long exons. The graphical interface allows to easily explore OrthoMaM to identify markers with specific characteristics (e.g., taxa availability, alignment size, %G+C, evolutionary rate, chromosome location). It hence provides an efficient solution to sample preprocessed markers adapted to user-specific needs. OrthoMaM has proven to be a valuable resource for researchers interested in mammalian phylogenomics, evolutionary genomics, and has served as a source of benchmark empirical data sets in several methodological studies. OrthoMaM is available for browsing, query and complete or filtered downloads at http://www.orthomam.univ-montp2.fr/.

  19. Determination of the ortho to para ratio of H2Cl+ and H2O+ from submillimeter observations.

    PubMed

    Gerin, Maryvonne; de Luca, Massimo; Lis, Dariusz C; Kramer, Carsten; Navarro, Santiago; Neufeld, David; Indriolo, Nick; Godard, Benjamin; Le Petit, Franck; Peng, Ruisheng; Phillips, Thomas G; Roueff, Evelyne

    2013-10-03

    The opening of the submillimeter sky with the Herschel Space Observatory has led to the detection of new interstellar molecular ions, H2O(+), H2Cl(+), and HCl(+), which are important intermediates in the synthesis of water vapor and hydrogen chloride. In this paper, we report new observations of H2O(+) and H2Cl(+) performed with both Herschel and ground-based telescopes, to determine the abundances of their ortho and para forms separately and derive the ortho-to-para ratio. At the achieved signal-to-noise ratio, the observations are consistent with an ortho-to-para ratios of 3 for both H2O(+) and H2Cl(+), in all velocity components detected along the lines-of-sight to the massive star-forming regions W31C and W49N. We discuss the mechanisms that contribute to establishing the observed ortho-to-para ratio and point to the need for a better understanding of chemical reactions, which are important for establishing the H2O(+) and H2Cl(+) ortho-to-para ratios.

  20. Greater preexisting interferon γ responses to mycobacterial antigens and lower bacillary load during HIV-associated tuberculosis.

    PubMed

    Lahey, Timothy; Czechura, Tom; Crabtree, Scott; Arbeit, Robert D; Matee, Mecky; Horsburgh, C Robert; MacKenzie, Todd; Bakari, Muhammad; Pallangyo, Kisali; von Reyn, C Fordham

    2013-11-15

    The role of preexisting interferon (IFN) γ responses in controlling bacillary burden in human immunodeficiency virus (HIV)-associated tuberculosis is not known. Among BCG-immunized HIV-infected adults who developed tuberculosis in a phase III trial of an investigational tuberculosis vaccine, greater baseline IFN-γ responses to early secretory antigenic target 6 and Mycobacterium tuberculosis whole-cell lysate were associated with reduced bacillary burden on sputum smear grade, days to culture positivity on agar, and sputum culture grade during subsequent tuberculosis. This association was most consistent among recipients of the investigational vaccine. When HIV-associated tuberculosis develops, greater preexisting IFN-γ responses to mycobacterial antigens are associated with reduced tuberculosis bacillary burden. ClinicalTrials.gov Identifier. NCT0052195.

  1. Induction of antibody response in the oral cavity of dogs following intraocular (eye drop) immunization with Porphyromonas gingivalis cell lysate incorporated in pH-sensitive fusogenic polymer-modified liposomes

    PubMed Central

    SHIMIZU, Yosuke; IWASAKI, Tadashi; TAJIMA, Tomoko; YUBA, Eiji; KONO, Kenji; WATARAI, Shinobu

    2016-01-01

    Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associated cell lysate. Dogs immunized with P. gingivalis cell lysate-containing MGluPG-modified liposomes by intraocular (eye drop) route displayed significant levels of P. gingivalis cell lysate-specific serum IgG and IgA as well as mucosal IgA antibodies in saliva secretion. Serum and salivary antibodies generated by intraocularly immunized with MGluPG-modified liposome-associated P. gingivalis cell lysate revealed a significant aggregation activity against P. gingivalis, whereas serum and saliva from dogs receiving MGluPG-modified liposomes unentrapping P. gingivalis cell lysate did not show the aggregation activity against P. gingivalis. Furthermore, P. gingivalis-specific antibodies in saliva of immunized dogs inhibited the adherence of P. gingivalis to cultured HeLa cells. More importantly, salivary antibodies induced by intraocular immunization with P. gingivalis cell lysate-containing MGluPG-modified liposomes significantly inhibited the coaggregation of P. gingivalis with Actinomyces naeslundii and the cell damage activity of P. gingivalis against FaDu cells, an oral epithelial cell. These results suggest that intraocularly administered P. gingivalis cell lysate-containing MGluPG-modified liposomes should be an effective mucosal vaccine against P. gingivalis infection in dogs and may be an important tool for the prevention of periodontitis. PMID:27916762

  2. Induction of antibody response in the oral cavity of dogs following intraocular (eye drop) immunization with Porphyromonas gingivalis cell lysate incorporated in pH-sensitive fusogenic polymer-modified liposomes.

    PubMed

    Shimizu, Yosuke; Iwasaki, Tadashi; Tajima, Tomoko; Yuba, Eiji; Kono, Kenji; Watarai, Shinobu

    2017-02-14

    Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associated cell lysate. Dogs immunized with P. gingivalis cell lysate-containing MGluPG-modified liposomes by intraocular (eye drop) route displayed significant levels of P. gingivalis cell lysate-specific serum IgG and IgA as well as mucosal IgA antibodies in saliva secretion. Serum and salivary antibodies generated by intraocularly immunized with MGluPG-modified liposome-associated P. gingivalis cell lysate revealed a significant aggregation activity against P. gingivalis, whereas serum and saliva from dogs receiving MGluPG-modified liposomes unentrapping P. gingivalis cell lysate did not show the aggregation activity against P. gingivalis. Furthermore, P. gingivalis-specific antibodies in saliva of immunized dogs inhibited the adherence of P. gingivalis to cultured HeLa cells. More importantly, salivary antibodies induced by intraocular immunization with P. gingivalis cell lysate-containing MGluPG-modified liposomes significantly inhibited the coaggregation of P. gingivalis with Actinomyces naeslundii and the cell damage activity of P. gingivalis against FaDu cells, an oral epithelial cell. These results suggest that intraocularly administered P. gingivalis cell lysate-containing MGluPG-modified liposomes should be an effective mucosal vaccine against P. gingivalis infection in dogs and may be an important tool for the prevention of periodontitis.

  3. Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat-derived platelet concentrates.

    PubMed

    Glovinski, Peter V; Herly, Mikkel; Mathiasen, Anders B; Svalgaard, Jesper D; Borup, Rehannah; Talman, Maj-Lis M; Elberg, Jens J; Kølle, Stig-Frederik T; Drzewiecki, Krzysztof T; Fischer-Nielsen, Anne

    2017-02-01

    Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). Of the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. PLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights

  4. Construction of Escherichia coli Strains for Conversion of Nitroacetophenones to ortho-Aminophenols

    PubMed Central

    Kadiyala, Venkateswarlu; Nadeau, Lloyd J.; Spain, Jim C.

    2003-01-01

    The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds.   PMID:14602609

  5. Diphenylcarbene Protected by Four ortho-Iodine Groups: An Unusually Persistent Triplet Carbene.

    PubMed

    Hirai, Katsuyuki; Bessho, Kana; Tsujita, Kosaku; Kitagawa, Toshikazu

    2016-11-15

    Diphenyldiazomethane with four iodine groups at the ortho positions and two tert-butyl groups at the para positions, i.e., bis(4-tert-butyl-2,6-diiodophenyl)diazomethane (1a-N₂), was synthesized as a sterically hindered triplet carbene precursor. Irradiation of 1a-N₂ in solution effectively generated the corresponding triplet diphenylcarbene ³1a, which was characterized by UV-vis spectroscopy at low temperature, along with laser flash photolysis techniques at room temperature. The UV-vis spectrum of ³1a was obtained by irradiating 1a-N₂ in a 2-methyltetrahydrofuran matrix at 77 K. The ESR spectrum showed no triplet carbene signals, while a radical species was observed at the anticipated temperature of the decomposition of triplet carbene ³1a. Transient absorption bands ascribable to ³1a were observed by laser flash photolysis of 1a-N₂ in a degassed benzene solution and decayed very slowly with a second-order rate constant (2k/εl) of 5.5 × 10(-)³·s(-)¹. Steady-state irradiation of 1a-N₂ in degassed benzene afforded 9,10-diarylphenanthrene derivative 2a in a 31% yield. Triplet carbene ³1a was also trapped by either oxygen (kO2 = 6.5 × 10⁵ M(-)¹·s(-)¹) or 1,4-cyclohexadiene (kCHD = 1.5 M(-)¹·s(-)¹) to afford the corresponding ketone 1a-O or the diarylmethane 1a-H₂. The carbene was shown to be much less reactive than the triplet diphenylcarbene that is protected by two ortho-iodo and two ortho-bromo groups, ³1b.

  6. Poly(ortho ester amides): Acid-labile Temperature-responsive Copolymers for Potential Biomedical Applications

    PubMed Central

    Tang, Rupei; Palumbo, R. Noelle; Ji, Weihang; Wang, Chun

    2009-01-01

    A new, convenient pathway is developed to synthesize highly hydrolytically labile poly(ortho ester amide) (POEA) copolymers that overcomes some of the major weaknesses of the traditional methods of synthesizing poly(ortho esters) and their derivatives. A diamine monomer containing a built-in, stabilized ortho ester group was synthesized and was used for polycondensation with diacid esters, giving rise to a series of POEA copolymers with unique stimuli-responsive properties. The POEA undergoes temperature-responsive, reversible sol-gel phase transition in water. Phase diagrams of the POEA/H2O mixture reveal the concentration-dependent existence of different phases, including hydrogel and opaque or clear solution. Such behavior may be attributed to the temperature-dependent hydrogen-bonding involving the amide groups in the POEA backbone and hydrophobic interactions between POEA chains, and it is tunable by selecting diacid monomers with different chemical structures. The kinetics of POEA mass loss in physiological aqueous buffers and release of a model macromolecular drug, fluorescently labeled dextran, are nearly zero-order, suggesting predominantly surface-restricted polymer erosion. The rates of polymer erosion and drug release are much faster at pH 5.0 than pH 7.4. No cytotoxicity was found for the polymer extracts and the polymer degradation products at concentrations as high as 1 mg/ml. The normal morphology of fibroblasts cultured directly in contact with POEA films was not altered. These novel acid-labile temperature-responsive POEA copolymers may be potentially useful for a wide range of biomedical applications such as minimal invasive delivery of controlled-release drug formulations that respond to biological temperature and acidic-pH environments in cells and tissues. PMID:19281150

  7. Supported transition metal catalysts for para- to ortho-hydrogen conversion

    NASA Technical Reports Server (NTRS)

    Brooks, Christopher J.; Wang, Wei; Eyman, Darrell P.

    1994-01-01

    The main goal of this study was to develop and improve on existing catalysts for the conversion of ortho- to para-hydrogen. Starting with a commercially available Air Products nickel silicate, which had a beta value of 20, we were trying to synthesize catalysts that would be an improvement to AP. This was accomplished by preparing silicates with various metals as well as different preparation methods. We also prepared supported ruthenium catalysts by various techniques using several metal precursors to improve present technology. What was also found was that the activation conditions prior to catalytic testing was highly important for both the silicates and the supported ruthenium catalysts. While not the initial focus of the research, we made some interesting observations into the adsorption of H2 on ruthenium. This helped us to get a better understanding of how ortho- to para-H2 conversion takes place, and what features in a catalyst are important to optimize activity. Reactor design was the final area in which some interesting conclusions were drawn. As discussed earlier, the reactor catalyst bed must be constructed using straight 1/8 feet OD stainless steel tubing. It was determined that the use of 1/4 feet OD tubing caused two problems. First, the radius from the center of the bed to the wall was too great for thermal equilibrium. Since the reaction of ortho- to para-H2 is exothermic, the catalyst bed center was warmer than the edges. Second, the catalyst bed was too shallow using a 1/4 feet tube. This caused reactant blow-by which was thought to decrease the measured activity when the flow rate was increased. The 1/8 feet tube corrected both of these concerns.

  8. Anion photoelectron spectroscopy of deprotonated ortho-, meta-, and para-methylphenol

    NASA Astrophysics Data System (ADS)

    Nelson, Daniel J.; Gichuhi, Wilson K.; Miller, Elisa M.; Lehman, Julia H.; Lineberger, W. Carl

    2017-02-01

    The anion photoelectron spectra of ortho-, meta-, and para-methylphenoxide, as well as methyl deprotonated meta-methylphenol, were measured. Using the Slow Electron Velocity Map Imaging technique, the Electron Affinities (EAs) of the o-, m-, and p-methylphenoxyl radicals were measured as follows: 2.1991±0.0014, 2.2177±0.0014, and 2.1199±0.0014 eV, respectively. The EA of m-methylenephenol was also obtained, 1.024±0.008 eV. In all four cases, the dominant vibrational progressions observed are due to several ring distortion vibrational normal modes that were activated upon photodetachment, leading to vibrational progressions spaced by ˜500 cm-1. Using the methylphenol O-H bond dissociation energies reported by King et al. and revised by Karsili et al., a thermodynamic cycle was constructed and the acidities of the methylphenol isomers were determined as follows: Δa c i dH298K 0=348.39 ±0.25 , 348.82±0.25, 350.08±0.25, and 349.60±0.25 kcal/mol for cis-ortho-, trans-ortho-, m-, and p-methylphenol, respectively. The excitation energies for the ground doublet state to the lowest excited doublet state electronic transition in o-, m-, and p-methylphenoxyl were also measured as follows: 1.029±0.009, 0.962±0.002, and 1.029±0.009 eV, respectively. In the photoelectron spectra of the neutral excited states, C-O stretching modes were excited in addition to ring distortion modes. Electron autodetachment was observed in the cases of both m- and p-methylphenoxide, with the para isomer showing a lower photon energy onset for this phenomenon.

  9. Poly(ortho ester amides): acid-labile temperature-responsive copolymers for potential biomedical applications.

    PubMed

    Tang, Rupei; Palumbo, R Noelle; Ji, Weihang; Wang, Chun

    2009-04-13

    A new, convenient pathway is developed to synthesize highly hydrolytically labile poly(ortho ester amide) (POEA) copolymers that overcomes some of the major weaknesses of the traditional methods of synthesizing poly(ortho esters) and their derivatives. A diamine monomer containing a built-in, stabilized ortho ester group was synthesized and was used for polycondensation with diacid esters, giving rise to a series of POEA copolymers with unique stimuli-responsive properties. The POEA undergoes temperature-responsive, reversible sol-gel phase transition in water. Phase diagrams of the POEA/H(2)O mixture reveal the concentration-dependent existence of different phases, including hydrogel and opaque or clear solution. Such behavior may be attributed to the temperature-dependent hydrogen-bonding involving the amide groups in the POEA backbone and hydrophobic interactions between POEA chains, and it is tunable by selecting diacid monomers with different chemical structures. The kinetics of POEA mass loss in physiological aqueous buffers and release of a model macromolecular drug, fluorescently labeled dextran, are nearly zero-order, suggesting predominantly surface-restricted polymer erosion. The rates of polymer erosion and drug release are much faster at pH 5.0 than pH 7.4. No cytotoxicity was found for the polymer extracts and the polymer degradation products at concentrations as high as 1 mg/mL. The normal morphology of fibroblasts cultured directly in contact with POEA films was not altered. These novel acid-labile temperature-responsive POEA copolymers may be potentially useful for a wide range of biomedical applications such as minimal invasive delivery of controlled-release drug formulations that respond to biological temperature and acidic-pH environments in cells and tissues.

  10. Anion photoelectron spectroscopy of deprotonated ortho-, meta-, and para-methylphenol.

    PubMed

    Nelson, Daniel J; Gichuhi, Wilson K; Miller, Elisa M; Lehman, Julia H; Lineberger, W Carl

    2017-02-21

    The anion photoelectron spectra of ortho-, meta-, and para-methylphenoxide, as well as methyl deprotonated meta-methylphenol, were measured. Using the Slow Electron Velocity Map Imaging technique, the Electron Affinities (EAs) of the o-, m-, and p-methylphenoxyl radicals were measured as follows: 2.1991±0.0014, 2.2177±0.0014, and 2.1199±0.0014 eV, respectively. The EA of m-methylenephenol was also obtained, 1.024±0.008 eV. In all four cases, the dominant vibrational progressions observed are due to several ring distortion vibrational normal modes that were activated upon photodetachment, leading to vibrational progressions spaced by ∼500 cm(-1). Using the methylphenol O-H bond dissociation energies reported by King et al. and revised by Karsili et al., a thermodynamic cycle was constructed and the acidities of the methylphenol isomers were determined as follows: ΔacidH298K(0)=348.39±0.25, 348.82±0.25, 350.08±0.25, and 349.60±0.25 kcal/mol for cis-ortho-, trans-ortho-, m-, and p-methylphenol, respectively. The excitation energies for the ground doublet state to the lowest excited doublet state electronic transition in o-, m-, and p-methylphenoxyl were also measured as follows: 1.029±0.009, 0.962±0.002, and 1.029±0.009 eV, respectively. In the photoelectron spectra of the neutral excited states, C-O stretching modes were excited in addition to ring distortion modes. Electron autodetachment was observed in the cases of both m- and p-methylphenoxide, with the para isomer showing a lower photon energy onset for this phenomenon.

  11. Kinetics of proton transfer between ortho substituted benzoic acids and the carbinol base of crystal violet in toluene. Ortho effect on the reactivity of benzoic acids in apolar aprotic solvents.

    PubMed

    Sen Gupta, Susanta K; Mishra, Sangeeta

    2011-05-12

    Apolar aprotic solvents are particularly advantageous for investigating the intrinsic ortho effect free from complications of specific solvent effects. A kinetic study for toluene-phase proton transfers between ortho F, Cl, Br, I, OMe, OEt, OPh, OAc, Me, NO(2), COMe, COPh, OH, NH(2), and H benzoic acids and crystal violet carbinol base has shown the forward rate constant (log k(+1)) is the most appropriate reactivity parameter in toluene. log k(+1) (toluene) as compared to other reported reactivity parameters in benzene, toluene, or chlorobenzene has been found more sensitive to the ortho substituent effect. The regression results of the correlation of log k(+1) (toluene) of the acids (except OH and NH(2) substituted ones) according to seven ortho effect models are all very significant, and the best result is given by Fujita-Nishioka's model. The overall analysis reveals that a substituent's ortho effect pattern is a 58:24:18 ratio of its ordinary electrical, proximity electrical, and steric effects and that the proximity electrical effect is the major component to account for the peculiarity of the substituent's ortho effect. The results further favor the transmission of this effect mainly through the molecular cavity. The effect may, however, be outweighed by the steric component for bulky enough substituents, e.g., Me. The enhanced strength exhibited by salicylic acid in toluene has been quantitatively described using Pytela-Liška's σ(HB)(i) parameter. The abnormally high log k(+1) observed for anthranilic acid in toluene has been ascribed to a very extensive homoconjugation in its acid-acid anion complex induced by the acid's three hydrogen bond donors.

  12. Immune recognition of protein antigens

    SciTech Connect

    Laver, W.G.; Air, G.M.

    1985-01-01

    This book contains 33 papers. Some of the titles are: Antigenic Structure of Influenze Virus Hemagglutinin; Germ-line and Somatic Diversity in the Antibody Response to the Influenza Virus A/PR/8/34 Hemagglutinin; Recognition of Cloned Influenza A Virus Gene Products by Cytotoxic T Lymphocytes; Antigenic Structure of the Influenza Virus N2 Neuraminidase; and The Molecular and Genetic Basis of Antigenic Variation in Gonococcal Pillin.

  13. Purification and Low Temperature (15 MK-10 k) NMR of Ultra Pure Ortho-Deuterium

    NASA Astrophysics Data System (ADS)

    Yucel, Sermet

    1988-06-01

    Pure solid ortho-deuterium, whose spin-lattice relaxation time in the liquid helium region is expected to be the order of days, is a promising candidate for nuclear spin polarized fusion fuels and targets for high energy physics applications. For purification of ortho-deuterium by conversion of para-deuterium, we present here a new catalytic technique in which conversion takes place in the physisorbed phase on copper powder or on grafoil at temperatures between 12 and 8 K. This catalysis has yielded an ortho-D_2 purity in which para -D_2 concentration is reduced to 1.1 times 10^{ -3}, better by more than an order of magnitude than in any previous work. By subsequent aging of the purified samples at 4.2 K for about 9 months, o-D _2 with residual p-D_2 concentration of 5 +/- 2 times 10^{-5} was attained, whose spin-lattice relaxation time at 1.2 K in a 0.3 T magnetic field is 11 hours. A theory of ortho -para conversion on clean metal surfaces due to interaction with conduction electrons is given. In the case of grafoil, the conversion rates in 2-D solid phases of physisorbed D_2 by emission of one and two photons (of the D_2 solid) are calculated. A temperature independent conversion rate is predicted and is consistent with our measurements in the 2-D solid regime. A sample of moderately pure o-D_2, (p-D_2 concentration of 0.004), chosen for acceptable heat of conversion as well as relatively short spin-lattice relaxation time even in the mK region, was cooled in our dilution refrigerator to 15 mK, a record low temperature for solid hydrogens. The special NMR probe and sample chamber designed and constructed for this purpose is described. With our present apparatus (magnetic field of 13 Tesla), protein polarizations of about 70% are attainable, from which deuteron polarizations in excess of 50% can be driven by dynamic polarization techniques in an o-D _2-HD mixture.

  14. Ortho/para ratio of H2O+ toward Sagittarius B2(M) revisited.

    PubMed

    Schilke, Peter; Lis, Dariusz C; Bergin, Edwin A; Higgins, Ronan; Comito, Claudia

    2013-10-03

    The HIFI instrument aboard the Herschel satellite has allowed the observation and characterization of light hydrides, the building blocks of interstellar chemistry. In this article, we revisit the ortho/para ratio for H2O(+) toward the Sgr B2(M) cloud core. The line of sight toward this star forming region passes through several spiral arms and the gas in the Bar potential in the inner Galaxy. In contrast to earlier findings, which used fewer lines to constrain the ratio, we find a ratio of 3, which is uniformly consistent with high-temperature formation of the species. In view of the reactivity of this ion, this matches the expectations.

  15. Short and Efficient Synthesis of Alkyl- and Aryl-Ortho-Hydroxy-Anilides and their Antibiotic Activity

    PubMed Central

    Krauß, Jürgen; Plesch, Eva; Clausen, Sabine; Bracher, Franz

    2014-01-01

    Abstract Ortho-hydroxy-anilides are part of natural products like the new antibiotics platencin (A) and platensimycin (B). An important step in the total synthesis of these antibiotics or their derivatives is the preparation of the o-hydroxy-anilide partial structure. The presented method allows the preparation of o-hydroxy-anilides and o-dihydroxy-anilides from 2-nitrophenol esters in a one-step synthesis without protecting the hydroxy group. Aryl- and alkyl-anilides were prepared following this method as simple analogues of platensimycin (A). The resulting compounds were tested in an agar diffusion assay for their antibiotic potency. PMID:25853064

  16. Tetrabenzanthanthrenes by mitigation of rearrangements in the planarization of ortho-phenylene hexamers.

    PubMed

    He, Jian; Mathew, Sanyo; Kinney, Zacharias J; Warrell, Rachel M; Molina, James S; Hartley, C Scott

    2015-04-28

    In general, ortho-phenylene hexamers are not good substrates for oxidative planarization because of competing backbone rearrangements. However, by first planarizing the ends, a target tetrabenzanthanthrene has been obtained by oxidation in good yield. DFT calculations suggest that the larger polycyclic aromatic subunits of the preplanarized substrate increase the rate of planarization relative to that of rearrangement. By implication, it may be possible to prepare graphene structures that cannot be made directly from simple polyphenylenes by instead designing precursors with larger polycyclic aromatic moieties. The photophysical properties of the tetrabenzanthanthrene core indicate that it may have promise as a functional chromophore.

  17. Exchange of carbon-bound hydrogen atoms ortho to the hydroxyl group in tyrosine.

    PubMed

    Martin, R B; Morlino, V J

    1965-10-22

    The carbon-bound hydrogen atoms of tyrosine that exchange with solvent protons in strongly acid solutions at about 100 degrees C are not the methylene hydrogen atoms but a pair on the aromatic ring. Of the two pairs of protons on the aromatic ring, observed in the proton magnetic resonance spectra, the pair at higher field undergoes exchange in 2.4N DCI at 100 degrees C. Other hydrogen atoms, attached either to aliphatic or aromatic carbon atoms, exhibit no noticeable exchange under the same conditions. From a chemicalshift analysis the exchanging protons are assigned as those ortho to the hydroxyl group on the aromatic ring.

  18. Pd(II)-catalyzed ortho- or meta-C-H olefination of phenol derivatives.

    PubMed

    Dai, Hui-Xiong; Li, Gang; Zhang, Xing-Guo; Stepan, Antonia F; Yu, Jin-Quan

    2013-05-22

    A combination of weakly coordinating auxiliaries and ligand acceleration allows for the development of both ortho- and meta-selective C-H olefination of phenol derivatives. These reactions demonstrate the feasibility of directing C-H functionalizations when functional groups are distal to target C-H bonds. The meta-C-H functionalization of electron-rich phenol derivatives is unprecedented and orthogonal to previous electrophilic substitution of phenols in terms of regioselectivity. These methods are also applied to functionalize α-phenoxyacetic acids, a fibrate class of drug scaffolds.

  19. A nuclear magnetic resonance study of higher-order correlation functions in supercooled ortho-terphenyl

    NASA Astrophysics Data System (ADS)

    Böhmer, R.; Diezemann, G.; Hinze, G.; Sillescu, H.

    1998-01-01

    Using deuteron NMR techniques two-, effective three-, and various four-time correlation functions were recorded for supercooled ortho-terphenyl at 10-15 K above the calorimetric glass transition in order to characterize the heterogeneous nature of its primary response. The experimental results could successfully be described within various energy landscape models as well as via continuous time random walk simulations. These theoretical considerations provide a suitable basis for a definition of the term dynamic heterogeneity. We discuss the power but also some limitations of the present multidimensional NMR techniques when applied to amorphous materials.

  20. Microhydration effects on geometric properties and electronic absorption spectra of ortho-aminobenzoic acid.

    PubMed

    Olivier, Danilo da Silva; Ito, Amando Siuiti; Galembeck, Sergio Emanuel

    2015-08-05

    TD-DFT and a combination of polarized continuum model (PCM) and microhydration methods helped to simulate the optical electronic absorption spectrum of ortho-aminobenzoic acid (o-Abz). The microhydration method involved the use of different numbers, from 1 to 5, of first solvation layer water molecules. We examined how implicit and explicit water affected the energies of the HOMO-LUMO transition in the o-Abz/water systems. Adding until five water molecules, the theoretical spectrum becomes closer to the experimental data. Microhydration combined with the PCM method leads to agreement between the theoretical result for five water molecules and the experimentally measured absorption bands.

  1. Mechanism of trifluoromethylation of aryl halides with CuCF3 and the ortho effect.

    PubMed

    Konovalov, Andrey I; Lishchynskyi, Anton; Grushin, Vladimir V

    2014-09-24

    A combined experimental (radical clock, kinetic, Hammett) and computational (DFT, MM) study of the trifluoromethylation reaction of aryl halides with CuCF3 reveals a nonradical mechanism involving Ar-X oxidative addition to the Cu(I) center as the rate determining step. The reaction is second order, first order in each reactant with ΔG(⧧) ≈ 24 kcal/mol for PhI (computed ΔG(⧧) = 21.9 kcal/mol). An abrupt change in the gradient on the Hammett plot of log(kR/kH) versus σp for 11 p-RC6H4I substrates produces two correlations (ρ = +0.69 and +1.83), which is temptingly suggestive of two different reaction pathways. Only one mechanism is operational, however, as advocated by a single linear correlation with σp(-) (ρ = +0.91), analysis of the experimental ρ values, close similarity of the transition states varying in R and displaying clear signs of -M interactions, and excellent reproduction of the plot by DFT. The long-known yet previously uncomprehended ortho effect has been quantified, for the first time, using the reaction of CuCF3 with a series of o-RC6H4Br: R(kR/kH) = H (1) < Me (3.5) < MeO (4) < CN (20) < CHO (250) < CO2Me (850) < NO2 (4300) < Ac (7300) < CO2H (150 000). With minor contributions from electronic factors, the ortho effect is largely determined by (i) the stabilizing coordination of the o-substituent to Cu in the transition state with the Cu···O distance varying directly with the barrier and (ii) the steric bulk of the o-substituent that raises the ground state free energy of the haloarene (G(o)ortho - G(o)H or G(o)ortho - G(o)para) by inflicting molecular strain and consequently weakening the Ar-X bond.

  2. Increasing sensitivity of the Ortho analytical cytofluorograph by modifying the fluid system.

    PubMed

    Zucker, R M; Elstein, K H; Gershey, E L; Massaro, E J

    1990-01-01

    By adding Teflon tubing to the effluent line of the Ortho 50H analytical cytofluorograph, we were able to increase the sensitivity of fluorescence and scatter detection threefold without compromising resolution. By increasing sheath backpressure (resistance), the additional tubing increases particle residence time within the detection area and thereby increases the total photon emission density per particle. In addition, the longer, wider tubing dampens the stream-destabilizing effects of minor obstructions in or movement of the effluent line. This increase in sensitivity and stability is desirable in applications requiring detection of particles exhibiting low-level fluorescence.

  3. Michael Additions of Highly Basic Enolates to ortho-Quinone Methides.

    PubMed

    Lewis, Robert S; Garza, Christopher J; Dang, Ann T; Pedro, Te Kie A; Chain, William J

    2015-05-01

    A protocol by which ketone or ester enolates and ortho-quinone methides (o-QMs) are generated in situ in a single reaction flask from silylated precursors under the action of anhydrous fluoride is reported. The reaction partners are joined to give a variety of β-(2-hydroxyphenyl)-carbonyl compounds in 32-94% yield in a single laboratory operation. The intermediacy of o-QMs is supported by control experiments utilizing enolate precursors and conventional alkyl halides as competitive alkylating agents and the isolation of 1,5-dicarbonyl products resulting from conjugate additions that do not restore the aromatic system.

  4. Michael Additions of Highly Basic Enolates to ortho-Quinone Methides

    PubMed Central

    Lewis, Robert S.; Garza, Christopher J.; Dang, Ann T.; Pedro, Te Kie A.; Chain, William J.

    2015-01-01

    A protocol by which ketone or ester enolates and ortho-quinone methides (o-QMs) are generated in situ in a single reaction flask from silylated precursors under the action of anhydrous fluoride is reported. The reaction partners are joined to give a variety of β-(2-hydroxyphenyl)-carbonyl compounds in 32–94% yield in a single laboratory operation. The intermediacy of o-QMs is supported by control experiments utilizing enolate precursors and conventional alkyl halides as competitive alkylating agents and the isolation of 1,5-dicarbonyl products resulting from conjugate additions that do not restore the aromatic system. PMID:25906358

  5. Another side of the oxazaphospholidine oxide chiral ortho-directing group.

    PubMed

    Martins, Nelson; Mateus, Nuno; Vinci, Daniele; Saidi, Ourida; Brigas, Amadeu; Bacsa, John; Xiao, Jianliang

    2012-05-28

    A new ferrocenyl oxazaphospholidine oxide 3 was synthesized together with its P-epimer 2 in the reaction of ferrocene lithium with phosphoramidite chloride 1. 3 was successfully derivatized into planar chiral 1,2-ferrocenes, including phosphine ligands, via highly diastereoselective ortho-lithiation and subsequent functionalization; these compounds display opposite planar chirality to those obtained from 2. Some of these 1,2-ferrocenes were further lithiated, allowing for the introduction of a free phosphine group at the oxazaphospholidine ring. The X-ray structures of the compounds 2 and 3 as well as those of the new 1,2-ferrocenes 4 and 7 have been determined.

  6. Ortho effects on the change in electronic absorption spectrum of pyridinium salts of saturated bromohydrocarbon.

    PubMed

    Song, Jin-Ling; Gong, Li-Ming; Feng, Shou-Ai; Zhao, Jiang-Hong; Zheng, Jian-Feng; Zhu, Zhen-Ping

    2009-12-01

    The quaterisation process of 1,2-dibromoethane and pyridine is in situ traced by electronic absorption spectrum. Two absorption peaks, induced by mono- and bis-pyridinium salt of 1,2-dibromoethane, appear at 429 nm and 313 nm, respectively. To explain the phenomena, several kinds of alkyl bromides with special structures were selected and compared by experimental measurement and theoretical calculation. The results indicate that for mono-pyridinium salt of 1,2-dibromoethane, the electron donor property of ortho-bromine group increases the electron cloud density of the carbon atom associated with pyridinium cation, which induces red-shift of absorption wavelength.

  7. 77 FR 49793 - Ortho-Phthalaldehyde; Receipt of Application for Emergency Exemption, Solicitation of Public Comment

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-17

    ...EPA has received a specific exemption request from the National Aeronautics and Space Administration (NASA) to use the pesticide ortho-phthalaldehyde (OPA) (CAS No. 643-79-8) to treat the International Space Station internal active thermal control system (IATCS) coolant to control Cupriavidus metallidurans, Variovorax paradoxus, Acidovorax sp., Sphingomonas parapaucimobilis, Stenotrophomonas maltophilia, Methylobacterium extorquens, and unidentified gram negative rods. This emergency exemption involves the use of a chemical which has not been registered by the EPA. EPA is soliciting public comment on the exemption.

  8. 63Cu-NMR study of oxygen disorder in ortho-II YBa2Cu3Oy

    NASA Astrophysics Data System (ADS)

    Wu, T.; Zhou, R.; Hirata, M.; Vinograd, I.; Mayaffre, H.; Liang, R.; Hardy, W. N.; Bonn, D. A.; Loew, T.; Porras, J.; Haug, D.; Lin, C. T.; Hinkov, V.; Keimer, B.; Julien, M.-H.

    2016-04-01

    We show that 63Cu-NMR spectra place strong constraints on both the nature and the concentration of oxygen defects in ortho-II YBa2Cu3Oy . Systematic deviation from ideal ortho-II order is revealed by the presence of inequivalent Cu sites in either full or empty chains. The results can be explained by two kinds of defects: oxygen clustering into additional chains, or fragments thereof, most likely present at all concentrations (6.4 ortho-II order (y ≃6.55 ) shows that chain-oxygen disorder, known to limit electronic coherence, is ineluctable because it is inherent to these compounds.

  9. Amine vs. carboxylic acid protonation in ortho-, meta-, and para-aminobenzoic acid: An IRMPD spectroscopy study

    NASA Astrophysics Data System (ADS)

    Cismesia, Adam P.; Nicholls, Georgina R.; Polfer, Nicolas C.

    2017-02-01

    Infrared multiple photon dissociation (IRMPD) spectroscopy and computational chemistry are applied to the ortho-, meta-, and para- positional isomers of aminobenzoic acid to investigate whether the amine or the carboxylic acid are the favored sites of proton attachment in the gas phase. The NH and OH stretching modes yield distinct patterns that establish the carboxylic acid as the site of protonation in para-aminobenzoic acid, as opposed to the amine group in ortho- and meta-aminobenzoic acid, in agreement with computed thermochemistries. The trends for para- and meta-substitutions can be rationalized simplistically by inductive effects and resonant stabilization, and will be discussed in light of computed charge distributions based from electrostatic potentials. In ortho-aminobenzoic acid, the close proximity of the amine and acid groups allow a simultaneous interaction of the proton with both groups, thus stabilizing and delocalizing the charge more effectively, and compensating for some of the resonance stabilization effects.

  10. Lewis Acid-Base Interaction-Controlled ortho-Selective C-H Borylation of Aryl Sulfides.

    PubMed

    Li, Hong Liang; Kuninobu, Yoichiro; Kanai, Motomu

    2017-02-01

    An iridium/bipyridine-catalyzed ortho-selective C-H borylation of aryl sulfides was developed. High ortho-selectivity was achieved by a Lewis acid-base interaction between a boryl group of the ligand and a sulfur atom of the substrate. This is the first example of a catalytic and regioselective C-H transformation controlled by a Lewis acid-base interaction between a ligand and a substrate. The C-H borylation reaction could be conducted on a gram scale, and with a bioactive molecule as a substrate, demonstrating its applicability to late-stage regioselective C-H borylation. A bioactive molecule was synthesized from an ortho-borylated product by converting the boryl and methylthio groups of the product.

  11. Molecular hydrogen in the vicinity of NGC 7538 IRS 1 and IRS 2 - Temperature and ortho-to-para ratio

    NASA Technical Reports Server (NTRS)

    Hoban, Susan; Reuter, Dennis C.; Mumma, Michael J.; Storrs, Alex D.

    1991-01-01

    Near-infrared spectroscopic observations of the active star-forming region near NGC 7538 IRS 1 and IRS 2 were made. The relative intensities of the v = 1-0 Q(1), Q(3), and Q(5) lines of molecular hydrogen are used to calculate a rotational excitation temperature. Comparison of the measured intensity of the Q(2) transition relative to the intensity of Q(1) and Q(3) permitted the retrieval of the ratio of ortho-to-para hydrogen. It is found that an ortho-to-para ratio of between 1.6 and 2.35 is needed to explain the Q-branch line intensity ratios, depending on the excitation model used. This range in ortho-to-para ratios implies a range of molecular hydrogen formation temperature of approximately 105 K to 140 K.

  12. Ortho-Functionalized Aryltetrazines by Direct Palladium-Catalyzed C-H Halogenation: Application to Fast Electrophilic Fluorination Reactions.

    PubMed

    Testa, Christelle; Gigot, Élodie; Genc, Semra; Decréau, Richard; Roger, Julien; Hierso, Jean-Cyrille

    2016-04-25

    A general catalyzed direct C-H functionalization of s-tetrazines is reported. Under mild reaction conditions, N-directed ortho-C-H activation of tetrazines allows the introduction of various functional groups, thus forming carbon-heteroatom bonds: C-X (X=I, Br, Cl) and C-O. Based on this methodology, we developed electrophilic mono- and poly-ortho-fluorination of tetrazines. Microwave irradiation was optimized to afford fluorinated s-aryltetrazines, with satisfactory selectivity, within only ten minutes. This work provides an efficient and practical entry for further accessing highly substituted tetrazine derivatives (iodo, bromo, chloro, fluoro, and acetate precursors). It gives access to ortho-functionalized aryltetrazines which are difficult to obtain by classical Pinner-like syntheses.

  13. Identification of proteolytic activities in ROS 17/2.8 cell lysates which cleave peptide substrates for protein kinase C-mediated phosphorylation.

    PubMed

    Guidon, P T; Harrison, P

    1996-04-01

    We have observed two proteolytic activities in cell lysates from the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation, and other peptides containing similar sequences. Both activities are inhibited by Pefabloc, a serine protease inhibitor, while one of the activities is inhibited by either EDTA or aprotinin. The protease inhibitors pepstatin, bestatin, E-64, leupeptin and phosphoramidon do not block either of these proteolytic activities.

  14. Effects of nonpathogenic gram-negative bacterium Vitreoscilla filiformis lysate on atopic dermatitis: a prospective, randomized, double-blind, placebo-controlled clinical study.

    PubMed

    Gueniche, A; Knaudt, B; Schuck, E; Volz, T; Bastien, P; Martin, R; Röcken, M; Breton, L; Biedermann, T

    2008-12-01

    Atopic dermatitis (AD) is associated with elevated IgE levels and Th2 responses. The oral administration of nonpathogenic bacteria such as probiotics may improve the course of atopic diseases. It is believed that nonpathogenic bacteria prevent the development of allergic diseases by modulating intestinal immune responses. However, the effects of oral probiotics on AD could not be reproduced in all studies and the direct immunomodulation of the skin-associated immune response by nonpathogenic bacteria has not yet been investigated. We performed a prospective, double-blind, placebo-controlled clinical study with a cream containing a 5% lysate of the nonpathogenic bacteria Vitreoscilla filiformis. Seventy-five volunteers with AD (6-70 years of age) were randomized to receive either V. filiformis cream 5% or vehicle cream daily for 30 days. Efficacy was evaluated by the SCORe of Atopic Dermatitis (SCORAD), transepidermal water loss (TEWL), assessment of microflora, and the patient's assessment of itch and loss of sleep. Compared with placebo, V. filiformis lysate significantly decreased SCORAD levels (P=0.0044) and pruritus (P=0.0171). Active cream significantly decreased loss of sleep from day 0 to day 29 (P=0.0074). Qualitative and quantitative assessment of cutaneous microbial colonization revealed that V. filiformis lysate reduced Staphylococcus aureus colonization of the skin. The skin barrier as determined by TEWL also improved significantly with the cream alone. V. filiformis lysate significantly improved AD. This may be in part due to reduction of S. aureus, but seems to relate in most parts to a direct immunomodulatory effect on skin-associated immune responses.

  15. Protection against Naegleria fowleri infection in mice immunized with Cry1Ac plus amoebic lysates is dependent on the STAT6 Th2 response.

    PubMed

    Carrasco-Yepez, M; Rojas-Hernandez, S; Rodriguez-Monroy, M A; Terrazas, L I; Moreno-Fierros, L

    2010-01-01

    We previously reported that intranasal administration of Cry1Ac protoxin alone or in combination with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice. Those results suggested that both antibody responses and innate immune mechanisms may be participating in the protective effects observed. The present study was aimed to investigate whether the STAT6-induced Th2 immune response is essential for the resistance to N. fowleri infection, conferred by immunization with amoebic lysates plus Cry1Ac. STAT6-deficient (STAT6-/-) and wild-type (STAT6+/+) BALB/c mice were immunized by the intranasal route with a combination of N. fowleri lysates plus Cry1Ac, and subsequently challenged with lethal doses of N. fowleri trophozoites. STAT6+/+ mice displayed 100% protection, while no protection was observed in STAT6-/- mice. Significantly higher titres of Th2-associated IgG1 as well as interleukin-4 (IL-4) were found in STAT6+/+ mice, whereas in STAT6-/- mice significantly more IL-12 and IFN-gamma as well as significantly higher titres of Th1-associated IgG2a were detected. Thus, whereas protected STAT6+/+-immunized mice elicited a Th-2 type inclined immune response that produced predominantly humoral immunity, unprotected STAT6-/- mice exhibited a polarized Th1 type cellular response. These findings suggest that the STAT6-signalling pathway is critical for defence against N. fowleri infection.

  16. Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine-affinity chromatography: effects of spacer arms and ligand density.

    PubMed

    Bai, Jin-Shan; Bai, Shu; Shi, Qing-Hong; Sun, Yan

    2014-06-01

    Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine-affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10-atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two-stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine-affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

    PubMed Central

    Piras, Bryan A; Drury, Jason E; Morton, Christopher L; Spence, Yunyu; Lockey, Timothy D; Nathwani, Amit C; Davidoff, Andrew M; Meagher, Michael M

    2016-01-01

    With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development. PMID:27069949

  18. Mesenchymal Stromal Cells Implantation in Combination with Platelet Lysate Product Is Safe for Reconstruction of Human Long Bone Nonunion

    PubMed Central

    Fazeli, Roghayeh; Mohseni, Fatemeh; Hosseini, Seyedeh Esmat; Moghadasali, Reza; Mardpour, Soura; Azimian, Vajiheh; Ghorbani Liastani, Maede; Mirazimi Bafghi, Ali; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser

    2016-01-01

    Objective Nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell (MSC) implantation in combination with platelet lysate (PL) product for treating human long bone nonunion. Materials and Methods In this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells (20 million cells implanted once into the nonunion site using a fluoroscopic guide) in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects (local or systemic, serious or non-serious, related or unrelated) were observed during this time period. Results From a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control Xray evidence, bony union had occurred. Conclusion Results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179). PMID:27602311

  19. Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH.

    PubMed

    Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Yamada, Atsushi; Endo, Mika; Koike, Tohru

    2006-10-01

    While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.

  20. Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study

    PubMed Central

    2014-01-01

    Endotoxins are part of the cell wall of Gram-negative bacteria. They are potent immune stimulators and can lead to death if present in high concentrations. Feed additives, which bind endotoxins in the gastrointestinal tract of animals, could help to prevent their negative impact. The objective of our study was to determine the potential of a bentonite (Bentonite 1), a sodium bentonite (Bentonite 2), a chemically treated smectite (Organoclay 1) and a modified attapulgite (Organoclay 2) to bind endotoxins in vitro. Polymyxin B served as positive control. The kinetic chromogenic Limulus Amebocyte lysate test was adapted to measure endotoxin activity. Firstly, a single sorption experiment (10 endotoxin units/mL (EU/mL)) was performed. Polymyxin B and organoclays showed 100% binding efficiency. Secondly, the adsorption efficiency of sorbents in aqueous solution with increasing endotoxin concentrations (2,450 – 51,700 EU/mL) was investigated. Organoclay 1 (0.1%) showed a good binding efficiency in aqueous solution (average 81%), whereas Bentonite 1 (0.1%) obtained a lower binding efficiency (21-54%). The following absorbent capacities were calculated in highest endotoxin concentration: 5.59 mg/g (Organoclay 1) > 3.97 mg/g (Polymyxin B) > 2.58mg/g (Organoclay 2) > 1.55 mg/g (Bentonite 1) > 1.23 mg/g (Bentonite 2). Thirdly, a sorption experiment in artificial intestinal fluid was conducted. Especially for organoclays, which are known to be unspecific adsorbents, the endotoxin binding capacity was significantly reduced. In contrast, Bentonite 1 showed comparable results in artificial intestinal fluid and aqueous solution. Based on the results of this in vitro study, the effect of promising clay minerals will be investigated in in vivo trials. PMID:24383578