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Sample records for purified protein derivative

  1. Genetic analysis of Mycobacterium avium complex strains used for producing purified protein derivatives

    NARCIS (Netherlands)

    Semret, M.; Bakker, D.; Smart, N.; Olsen, I.; Haslov, K.; Behr, M.A.

    2006-01-01

    For over a century, purified protein derivatives (PPD) have been used to detect mycobacterial infections in humans and livestock. Among these, reagents to detect infections by Mycobacterium avium complex organisms have been produced, but the utility of these reagents has not been clearly established

  2. Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

    Science.gov (United States)

    Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

    2012-11-01

    Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

  3. Prevalence of purified protein derivative positivity in human immunodeficiency virus infected individuals in Hong Kong

    Institute of Scientific and Technical Information of China (English)

    陈志伟; 邓伟祺; 黄加庆

    2002-01-01

    Objective To study the prevalence of skin positivity to purified protein derivative (PPD) in human immunodeficiency virus (HIV)-infected patients in Hong Kong.Methods Consecutive clients of an HIV clinic were administered the PPD test and 2 units of PPD-RT23 were used. The area of induration was then measured in 48 to 72 h. Results were related to patient characteristics and HIV-related parameters.Results Eight (17.0%) out of 47 clients tested positive to the administration of 2 units of PPD-RT23. If the cutoff were raised to 10 mm according to current practice, only two (4.3%) would test positive.Conclusion The prevalence of PPD positivity in HIV-infected patients in Hong Kong is 17%, when a cutoff of 5 mm is used. This figure may form the basis for further studies on the utility of isoniazid preventive therapy in this group of patients.

  4. Production and proteomic characterisation of purified protein derivative from Mycobacterium avium subsp. paratuberculosis

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    Wynne James W

    2012-03-01

    Full Text Available Abstract Background Effective diagnosis of Johne's disease (JD, particularly at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide. The IFN-γ test of cell mediated immunity is currently one of the most suitable diagnostics for subclinical infections, however a major limitation of this test is the lack of a standardised purified protein derivative (PPD antigen (also referred to as Johnin PPD or PPDj. While attempting to replace PPDj with more specific individual antigens is an attractive proposition, bacterial culture derived PPDj remains the most effective antigen preparation for the diagnosis of subclinical JD. It may be possible to increase the reproducibility and specificity of PPDj preparations by further characterising and standardising the PPDj production. Results Using a standardised protocol, five in-house preparations of PPDj were prepared from cultures of Mycobacterium avium subsp. paratuberculosis (MAP. Compared to PPDs obtained from other institutes/laboratories, these preparations appeared to perform similarly well in the IFN-γ test. Although the broad proteomic composition of all PPDj preparations was remarkably similar, the absolute abundance of individual proteins varied markedly between preparations. All PPDj preparations contained common immunogenic proteins which were also observed in PPD preparations from Mycobacterium avium subsp. avium (PPDa and Mycobacterium bovis (PPDb. Temporal difference in protein secretion of in vitro cultured MAP was observed between 20 and 34 weeks suggesting that the age of MAP culture used for PPDj preparations may markedly influence PPDj composition. Conclusions This study describes a protocol for the production of PPDj and its subsequent proteomic characterisation. The broad proteomic composition of different preparations of PPDj was, for the most part, highly similar. Compositional differences between PPDj preparations were found

  5. The ID93 tuberculosis vaccine candidate does not induce sensitivity to purified protein derivative.

    Science.gov (United States)

    Baldwin, Susan L; Reese, Valerie; Granger, Brian; Orr, Mark T; Ireton, Gregory C; Coler, Rhea N; Reed, Steven G

    2014-09-01

    The tuberculin skin test (TST) is a simple and inexpensive test to determine whether individuals have been exposed to Mycobacterium tuberculosis. This test is not always reliable, however, in people previously immunized with BCG and/or who have been exposed to environmental mycobacterial species due to a reaction to purified protein derivative (PPD) used in the skin test. An issue with BCG, therefore, is that the resulting sensitization to PPD in some individuals compromises the diagnostic use of the skin test. The ability to induce protective immune responses without sensitizing to the tuberculin skin test will be important properties of next-generation tuberculosis (TB) vaccine candidates. We show here that guinea pigs immunized with the candidate TB vaccine ID93/GLA-SE, currently in clinical trials, do not react to intradermal PPD administration. In contrast, positive DTH responses to both ID93 and components thereof were induced in ID93/GLA-SE-immunized animals, indicating robust but specific cellular responses were present in the immunized animals. Noninterference with the TST is an important factor for consideration in the development of a vaccine against M. tuberculosis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Immunotherapy using purified protein derivative in the treatment of warts: An open uncontrolled trial

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    Vikrant Saoji

    2016-01-01

    Full Text Available Background: Warts are known to clear spontaneously with the development of cell-mediated immunity (CMI to the virus. Purified protein derivative (PPD of tuberculin bacilli has been used as a non-specific stimulant of CMI to achieve this outcome. Aim: To study the effect of PPD in the treatment of warts. Methods: Patients with difficult-to-treat warts were selected for immunotherapy. Each patient received 2.5 TU of PPD intralesionally in a few warts. A total of four sessions were given at 2 weekly intervals and patients were followed up for 6 months after the last dose. Results: Sixty-one patients were recruited of which 55 completed 6 months follow up and were available for analysis. Of these, 25 had verruca vulgaris, 18 had verruca plana and 12 had plantar warts. Forty two (76% patients showed complete clearance after four sessions while the remaining 13 (24% patients were non-responders. One patient developed a recurrence after total clearance during the follow-up period. Adverse effects were erythema, edema and pain at the site of injections. Limitations: As this was an uncontrolled trial, there is no comparison with a non-intervention group. Also, a Mantoux test was not done due to practical difficulties. Conclusion: Immunotherapy with PPD is helpful in the treatment of cutaneous warts.

  7. Purified protein derivative skin testing on HIV/AIDS patients and logistic regression analysis of its risk factors

    Institute of Scientific and Technical Information of China (English)

    Fengren Liu; Jianjun Ye; Changfu Xiong; Jiguo Yin; Weihua He; Gaobo Li; Dingyuan Zhao; Linxiang Ye

    2007-01-01

    Objective: To understand the reactivity of purified protein derivative skin test(PPD test) in HIV-infected persons and to determine the influential factors associated with PPD. Methods: 174 HIV/AIDS patients registered in the local center for disease control and prevention(CDC) participated this study from April to June in 2006. Questionnaire, CD4 count and thoracic roentgenogram were performed for all participants. Results: In this study, response rate of questionnaires was 83.65%. The majority of these participants had a different degree of immunodeficiency that accounted for 93.64%. Female patients had a higher CD4 count than that of males. The total positive rate of PPD was 38.15%. Analysis of single factor in our study indicated that CD4 count, previous tuberculosis history, tuberculosis contact history and thoracic roentgenogram manifestation of patients were related to their PPD diameters. Further analysis of multiple factors also supports the previous conclusion that CD4 count and previous tuberculosis history of patients were risk factors in the PPD test. Conclusion: The PPD test of HIV/AIDS patients could be affected by several factors. For persons infected with HIV, the confirmation of latent tuberculosis infection (LTBI) should be considered the combination effect of previous MTB infection and body cellular immune function.

  8. Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1996-01-01

    with no history of previous or active mycobacterial infection. Antigen-specific killing of macrophages presenting mycobacterial antigens (purified protein derivative or M. avium culture filtrate) was conducted. The phenotype of the killer cells was determined by a fluorescence-activated cell sorter after antigen...

  9. Immunotherapy in viral warts with intradermal Bacillus Calmette–Guerin vaccine versus intradermal tuberculin purified protein derivative: A double-blind, randomized controlled trial comparing effectiveness and safety in a tertiary care center in Eastern India

    Directory of Open Access Journals (Sweden)

    Indrashis Podder

    2017-01-01

    Conclusion: Both intradermal Bacillus Calmette–Guerin and tuberculin purified protein derivative hold promise in the treatment of viral warts. Bacillus Calmette–Guerin may be more effective, though it had more adverse events in our study.

  10. Short-term stimulation with interleukin (IL-4 enhances purified protein derivative-induced production of an eosinophil chemotactic lymphokine, but suppresses IL-5 production

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    Takehiko Nishiyama

    1998-01-01

    Full Text Available The effect of interleukin (IL-4 on eosinophil chemotactic lymphokine (ECL production from peripheral blood mononuclear cells (PBMC stimulated with purified protein derivative (PPD was examined. The PBMC stimulated with PPD in the absence of IL-4 failed to produce evident ECL. However, PPD-induced eosinophil chemotactic activity (ECA production was markedly enhanced in a dose-dependent manner by pretreatment of PBMC with IL-4. The most potent enhancement was induced by IL-4 at a concentration of 30 U in tuberculin-sensitive PBMC. Short-term pretreatment (30 min to 3 h was sufficient for the enhancement, whereas longer-term treatment was less effective. Eosinophil chemotactic lymphokine was found to be a CD4+ T cell-derived factor with an isoelectric point of approximately pH 7.0 and without heparin affinity, unlike chemokines such as RANTES and eotaxin. The effect of IL-4 on the production of other cytokines, such as interferon (IFN-γ, IL-5, RANTES (regulated on activation, normal T expressed and secreted, and granulocyte-macrophage colony stimulating factor (GM-CSF was also examined. Peripheral blood mononuclear cells produced all these cytokines when they were treated with PPD, even in the absence of IL-4. When PBMC were pretreated with IL-4, interestingly not only IFNy but also IL-5 production was suppressed by pretreatment with IL-4, although ECL production was enhanced by the pretreatment. In the case of RANTES and GM-CSF, significant amounts of these cytokines were produced, even without antigenic stimulation, and IL-4 pretreatment did not result in an enhancement of their production. It is thus suggested that IL-4, existing in allergic lesions, plays a crucial role in eosinophil accumulation mediated by the T cell-derived ECL.

  11. Effect of the inoculation site of bovine purified protein derivative (PPD) on the skin fold thickness increase in cattle from officially tuberculosis free and tuberculosis-infected herds.

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    Casal, Carmen; Alvarez, Julio; Bezos, Javier; Quick, Harrison; Díez-Guerrier, Alberto; Romero, Beatriz; Saez, Jose L; Liandris, Emmanouil; Navarro, Alejandro; Perez, Andrés; Domínguez, Lucas; de Juan, Lucía

    2015-09-01

    The official technique for diagnosis of bovine tuberculosis (bTB) worldwide is the tuberculin skin test, based on the evaluation of the skin thickness increase after the intradermal inoculation of a purified protein derivative (PPD) in cattle. A number of studies performed on experimentally infected or sensitized cattle have suggested that the relative sensitivity of the cervical test (performed in the neck) may vary depending on the exact location in which the PPD is injected. However, quantitative evidence on the variation of the test accuracy associated to changes in the site of inoculation in naturally infected animals (the population in which performance of the test is most critical for disease eradication) is lacking. Here, the probability of obtaining a positive reaction (>2 or 4 millimeters and/or presence of local clinical signs) after multiple inoculations of bovine PPD in different cervical and scapular locations was assessed in animals from five bTB-infected herds (818 cattle receiving eight inoculations) using a hierarchical Bayesian logistic regression model and adjusting for the potential effect of age and sex. The effect of the inoculation site was also assessed qualitatively in animals from four officially tuberculosis free (OTF) herds (two inoculations in 210 animals and eight inoculations in 38 cattle). Although no differences in the qualitative outcome of the test were observed in cattle from OTF herds, a statistically important association between the test outcome and the inoculation site in animals from infected herds was observed, with higher probabilities of positive results when the test was performed in the neck anterior area. Our results suggest that test sensitivity may be maximized by considering the area of the neck in which the test is applied, although lack of effect of the inoculation site in the specificity of the test should be confirmed in a larger sample.

  12. Biological characterization of purified macrophage-derived neutrophil chemotactic factor

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    M. Dias-Baruffi

    1995-01-01

    Full Text Available We have recently described the purification of a 54 kDa acidic protein, identified as macrophage-derived neutrophil chemotactic factor (MNCF. This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. This in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. MNCF is released in the supernatant by macrophage monolayers stimulated with lipopolysaccharide (LPS. In the present study, we describe some biological characteristics of homogenous purified MNCF. When assayed in vitro, MNCF gave a bell-shaped dose–response curve. This in vitro activity was shown to be caused by haptotaxis. Unlike N-formyl-methionylleucyl- phenylalanine (FMLP or interleukin 8 (IL-8, the chemotactic activity of MNCF in vivo and in vitro, was inhibited by preincubation with D-galactose but not with D-mannose. In contrast with IL-8, MNCF did not bind to heparin and antiserum against IL-8 was ineffective in inhibiting its chemotactic activity. These data indicate that MNCF induces neutrophil migration through a carbohydrate recognition property, but by a mechanism different from that of the known chemokines. It is suggested that MNCF may be an important mediator in the recruitment of neutrophils via the formation of a substrate bound chemotactic gradient (haptotaxis in the inflamed tissues.

  13. Tuberculin-purified protein derivative-, MPT-64-, and ESAT-6-stimulated gamma interferon responses in medical students before and after Mycobacterium bovis BCG vaccination and in patients with tuberculosis

    DEFF Research Database (Denmark)

    Johnson, P D; Stuart, R L; Grayson, M L

    1999-01-01

    of QIFN in medical students before and after BCG immunization was assessed, and sensitivity in patients with tuberculosis was assessed. Antigens were PPD derived from M. tuberculosis and two M. tuberculosis-specific proteins, ESAT-6 and MPT-64. Of 60 medical students, all of whom had 0-mm tuberculin skin......QuantiFERON-TB (QIFN) (CSL Limited) is a whole-blood assay for the recognition of infection with Mycobacterium tuberculosis. QIFN measures gamma interferon (IFN-gamma) production when purified protein derivatives (PPDs) of mycobacteria are incubated with venous blood samples. The specificity...... tests (TSTs) at study entry, 58 (97%) were initially classified as negative for M. tuberculosis infection by PPD QIFN. Five months after BCG immunization, 7 of 54 students (13%) had a TST result of >/=10 mm and 11 of 54 students (20%) tested positive by PPD QIFN. ESAT-6- and MPT-64-stimulated IFN...

  14. Tea derived galloylated polyphenols cross-link purified gastrointestinal mucins.

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    Pantelis Georgiades

    Full Text Available Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria.

  15. Venom neutralization by purified bioactive molecules: Synthetic peptide derivatives of the endogenous PLA(2) inhibitory protein PIP (a mini-review).

    Science.gov (United States)

    Thwin, Maung-Maung; Samy, Ramar Perumal; Satyanarayanajois, Seetharama D; Gopalakrishnakone, Ponnampalam

    2010-12-15

    Envenomation due to snakebite constitutes a significant public health problem in tropical and subtropical countries. Antivenom therapy is still the mainstay of treatment for snake envenomation, and yet despite recent research focused on the prospects of using antivenom adjuncts to aid in serotherapy, no new products have emerged so far for therapeutic use. Various methodologies including molecular biology, crystallography, functional and morphological approaches, etc., are employed in the search for such inhibitors with a view to generate molecules that can stop partially or completely the activities of toxic phospholipase A(2) (PLA(2)) and snake venom metalloproteinase (SvMPs) enzymes at the molecular level. Herein, both natural and synthetic inhibitors derived from a variety of sources including medicinal plants, mammals, marine animals, fungi, bacteria, and from the venom and blood of snakes have been briefly reviewed. Attention has been focused on the snake serum-based phospholipase A(2) inhibitors (PLIs), particularly on the PLI derived from python snake serum (PIP), highlighting the potential of the natural product, PIP, or possible derivatives of it, as a complementary treatment to serotherapy against the inflammation and/or muscle-damaging activity of snake venoms. The data indicate a more efficient pathway for inhibition and blocking the activity of PLA(2)s and matrix metalloproteinases (MMPs), thus representing a feasible complementary treatment for snakebites. Such information may be helpful for interfering on the biological processes that these molecules are involved in human inflammatory-related diseases, and also for the development of new drugs for treatment of snake envenomation.

  16. Purified protein derivative anergy in Kawasaki disease.

    Science.gov (United States)

    Kollmann, T R; Klein, E J; Stefanelli, C B; Marcuse, E K

    2001-01-01

    It was previously reported from Italy that all patients with Kawasaki disease had a positive tuberculin intradermal test. In this study from Seattle, WA, nine patients with Kawasaki disease showed no reaction to intradermal tuberculin. The difference in results might be caused by the different tuberculin products.

  17. Using ion exchange chromatography to purify a recombinantly expressed protein.

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    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

  18. Purified umbilical cord derived mesenchymal stem cell treatment in a case of systemic lupus erythematosus.

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    Phillips, Christopher D; Wongsaisri, Pornpatcharin; Htut, Thein; Grossman, Terry

    2017-12-01

    Systemic lupus erythematosus (SLE) is a multiple organ system autoimmune disorder for which there is no known cure. We report a case of a young adult lady with SLE and Sjogren's with diagnostic and clinical resolution following purified umbilical cord derived mesenchymal stem cell (MSC) and globulin component protein macrophage activating factor (GcMAF) therapy in a combined multidisciplinary integrative medicine protocol. Our patient had complete reversal of all clinical and laboratory markers. We recommend a prospective randomized double blind study to assess the sustained efficacy of MSC and GcMAF in the treatment of autoimmune connective tissue diseases such as systemic lupus erythematosus.

  19. Replication of adenovirus DNA-protein complex with purified proteins.

    OpenAIRE

    Ikeda, J E; Enomoto, T.; Hurwitz, J

    1981-01-01

    A protein fraction isolated from the cytosol of adenovirus-infected HeLa cells, which contained DNA polymerase alpha, catalyzed adenoviral DNA replication in the presence of adenovirus DNA binding protein, eukaryotic DNA polymerase beta, ATP, all four dNTPs, and MgCl2. DNA replication started at either end of exogenously added adenoviral DNA and was totally dependent on the presence of terminal 55,000-dalton proteins on the DNA template. The replicaton of adenovirus DNA in the system was sens...

  20. 纯化的结核菌素衍生蛋白免疫刺激后小鼠脑内的Fos表达%Fos expression in brain of purified protein derivative of tuberculin immunized mice

    Institute of Scientific and Technical Information of China (English)

    王凌; 汉江; 邝芳; 王百忍; 鞠躬

    2001-01-01

    AIM To further identify the response of brain areas to the immune stimulus. METHODS Using immunohistochemistry method, we observed the Fos expression in the brain of purified protein derivative of tuberculin (PPD) immunized mice at different time points. RESULTS At 3, 9, 14, 18, 21 d following the primary immunization and 7, 14 d following the second immunization, strong Fos immunostaining was observed in many brain areas such as the ventrolater-al medulla, central gray, hippocampus and neocortex. But the patterns at different time were not completely the same, especially in the cingulate cortex. Fos expression in the right side is stronger than that in the left side after second immunization with PPD. CONCLUSION It is suggested that many brain areas participate in the interaction of immune and central nervous system, and that brain asymmetry might be associated with the peripheral immune response.%目的进一步阐明中枢神经系统参与免疫调节的部位. 方法用免疫组织化学方法观察纯化的结核菌素衍生蛋白(PPD)免疫刺激后不同时间点小鼠脑内的Fos蛋白表达. 结果 PPD初次免疫后3, 9, 14, 18和21 d时再追加免疫后7, 14 d的动物,孤束核、延髓腹外侧区、中央灰质、海马、新皮质等多数脑区在各个时间点均可见到较强的Fos表达. 但不同时间点的Fos表达不完全相同,尤为明显的是,追加免疫后扣带回出现明显的Fos表达,且左侧较右侧强. 结论免疫应答过程中有多个脑区参与对免疫信息的感知和调控,皮质在对免疫反应的调节中有侧别差异.

  1. Synaptosomal protein synthesis in P2 and Ficoll purified fractions.

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    Eyman, Maria; Cefaliello, Carolina; Bruno, Anna Paola; Bruno, Annapaola; Crispino, Marianna; Giuditta, Antonio

    2012-01-30

    Cytoplasmic protein synthesis of brain synaptosomes has generally been determined in the Ficoll purified fraction which contains fewer contaminating mitochondria, microsomes and myelin fragments than the parent P2 fraction. Using a highly selective assay of this activity we have compared the total translation activity and the specific activity of the proteins synthesized by either fraction in control rats and in rats trained for a two-way active avoidance task. In control rats the specific activity remained essentially the same in both fractions but in trained rats the value of the Ficoll fraction was markedly lower (38.5%) than in the P2 fraction. Furthermore, the total translation activity of the Ficoll fraction was 30% lower than in the P2 fraction in control rats and 62% lower in trained rats. These decrements indicate that a large proportion of active synaptosomes present in the P2 fraction is not recovered in the Ficoll fraction, notably in rats undergoing plastic brain changes. We conclude that cytoplasmic protein synthesis of brain synaptosomes is better preserved in the P2 fraction.

  2. Tuberculin-purified protein derivative-, MPT-64-, and ESAT-6-stimulated gamma interferon responses in medical students before and after Mycobacterium bovis BCG vaccination and in patients with tuberculosis

    DEFF Research Database (Denmark)

    Johnson, P D; Stuart, R L; Grayson, M L

    1999-01-01

    of QIFN in medical students before and after BCG immunization was assessed, and sensitivity in patients with tuberculosis was assessed. Antigens were PPD derived from M. tuberculosis and two M. tuberculosis-specific proteins, ESAT-6 and MPT-64. Of 60 medical students, all of whom had 0-mm tuberculin skin...... tests (TSTs) at study entry, 58 (97%) were initially classified as negative for M. tuberculosis infection by PPD QIFN. Five months after BCG immunization, 7 of 54 students (13%) had a TST result of >/=10 mm and 11 of 54 students (20%) tested positive by PPD QIFN. ESAT-6- and MPT-64-stimulated IFN...

  3. Identification of a Protein that Purifies with the Scrapie Prion

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    Bolton, David C.; McKinley, Michael P.; Prusiner, Stanley B.

    1982-12-01

    Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain. The protein migrated with an apparent molecular size of 27,000 to 30,000 daltons in sodium dodecyl sulfate polyacrylamide gels. The resistance of this protein to digestion by proteinase K distinguished it from proteins of similar molecular weight found in normal hamster brain. Initial results suggest that the amount of this protein correlates with the titer of the agent.

  4. [An easy way to purify the inclusion body protein with high purity from prokaryotic expression cells].

    Science.gov (United States)

    Liu, Rong; Zhong, Qin-Ping; Jiang, Ming-Sen; Dong, Hui-Fen

    2011-10-01

    To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a(+) plasmids, and then to transform them into the competent cells E. coli BL21 (DE3), finally a positive clone was used to be induced by IPTG. The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni(2+)-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction. The purified protein was used to immune rabbits and make antiserum against the SjBMP, and the antiserum were then used to identify the rSjBMP by Western blotting. The target protein obtained by Ni(2+)-NTA Agarose affinity purification was not pure with unspecific proteins, but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure, and the recovery rate was more than 11.0%. Meanwhile, Western blotting was used to identify the recombinant SjBMP protein by antiserum, only a specific single strip appeared, which suggested the protein purified by this method kept its antigenicity, and could be used for common immunological studies. Therefore, the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.

  5. Soybean plant growth study conducted using purified protein hydrolysate-based fertilizer made from chrome-tanned leather waste.

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    Pati, Anupama; Chaudhary, Rubina

    2015-12-01

    Leather processing discharges enormous amount of chrome containing leather solid waste which creates a major disposal problem. Chrome-tanned leather solid waste is a complex of collagen and chromium. The presence of chromium limits protein application in fertilizer industry. The purified protein hydrolysate with zero chromium could be used as a nitrogen source for fertilizer formulation. In this study, an attempt has been made to employ purified protein hydrolysate derived from chrome-tanned leather shavings (CTLS) in formulation of fertilizer. The formulated fertilizer (1–3 t ha(-1)) is employed as nitrogen source in production of soybean. Plant growth study demonstrates that formulated fertilizer dosage 3 t ha(-1) produced similar effects of commercial fertilizer-treated plants. Application of formulated fertilizer yielded higher seed in plant than commercial fertilizer.

  6. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells.

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    Longmire, Tyler A; Ikonomou, Laertis; Hawkins, Finn; Christodoulou, Constantina; Cao, Yuxia; Jean, J C; Kwok, Letty W; Mou, Hongmei; Rajagopal, Jayaraj; Shen, Steven S; Dowton, Anne A; Serra, Maria; Weiss, Daniel J; Green, Michael D; Snoeck, Hans-Willem; Ramirez, Maria I; Kotton, Darrell N

    2012-04-06

    Two populations of Nkx2-1(+) progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1(GFP) knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.

  7. Protease Activities of Semi-purified Pseudomonas fluorescensin Protein Degradation of Pasteurized Milk at Storage

    Directory of Open Access Journals (Sweden)

    Tatik Khusniati

    2012-10-01

    Full Text Available Protein on stored milks spoiled due to protease activities of Pseudomonas fluorescens. To know protease effect on milks, protease activities of semi-purified P. fluorescens on protein degradation in stored pasteurized milks were detected. Protease was semi-purified by ethanol 70%. Protease activities were detected by modified Lowry method, protein degradation by formol titration, and protein content by Kjeldahl method. Milk storage' times were conducted on 0 day (4 days before expired date up to 12 days (8 days after expired date. The results show that the longer the storage times the higher protease activities and protein degradation of milks. At storage 12 days, protease activities on control were 0.2556 Unit/mL (skim and 0.2116 Unit/mL (whole, and on inoculated milk (crude was 2.2044 Unit/mL (skim and 1.5378 Unit/mL (whole; while on inoculated milk (semi-purified was 3.5355 Unit/mL (skim and 1.6778 Unit/mL (whole, respectively. The decrease of inoculated milk' homogeneous were faster than that of control. Protein degradation on control, inoculated skim milks (crude and semi-purified on 12 days were 4.48%, 7.28% and 7.62%, while that on whole milks were 3.81%, 7.28% and 6.04%, respectively. Based on protease, protein degradation and homogeneous, it can be concluded that skim milk spoiled faster than whole milk.

  8. Immunogenicity of a purified fragment of 17D yellow fever envelope protein.

    Science.gov (United States)

    Brandriss, M W; Schlesinger, J J; Walsh, E E

    1990-06-01

    Information on the immunogenic properties of purified flavivirus proteins may be useful in the development of recombinant or synthetic peptide vaccines. Using a monoclonal antibody, an attempt was made to purify the envelope (E) protein of 17D yellow fever virus (17D YF) by affinity chromatography. The purified material could not be identified as intact E protein but it did bear antigenic determinants of E as determined by selective reactivity with anti-E monoclonal antibodies. Rabbits immunized with this material produced antibodies that neutralized 17D YF and dengue-2 viruses in comparable titers, indicating that cross-reactive antigenic determinants were preserved. Immunization of mice resulted in protection against intracerebral challenge with 17D YF.

  9. Effect of Purified Mushroom Tyrosinase on Melanin Content and Melanogenic Protein Expression

    Directory of Open Access Journals (Sweden)

    Kamal Uddin Zaidi

    2016-01-01

    Full Text Available In mammalian melanocytes, melanosome is a highly specialized organelle where melanin is synthesized. Melanin synthesis is controlled by tyrosinase, the vital enzyme in melanogenic pathway. The present investigation is based on an effect of purified mushroom tyrosinase of Agaricus bisporus on B16F10 melanocytes for the melanin production via blocking pigment cell machinery. Using B16F10 melanocytes showed that the stimulation of melanogenesis by purified tyrosinase is due to increased tyrosinase absorption. Cellular tyrosinase activity and melanin content in B16F10 melanocytes were increased by purified tyrosinase in a dose-dependent manner. Western blot analysis revealed that cellular tyrosinase levels were enhanced after treatment with purified tyrosinase for 48 hours. Furthermore, tyrosinase induced phosphorylation of cyclic adenosine monophosphate (cAMP response element-binding protein (CREB in a dose-dependent manner. The purified tyrosinase-mediated increase of tyrosinase activity was significantly attenuated by H89, LY294002, Ro-32-0432, and PD98059, cAMP-dependent protein kinase inhibitors. The results indicate that purified tyrosinase can be used as contestant for the treatment of vitiligous skin conditions.

  10. Effect of Purified Mushroom Tyrosinase on Melanin Content and Melanogenic Protein Expression

    Science.gov (United States)

    Ali, Ayesha S.

    2016-01-01

    In mammalian melanocytes, melanosome is a highly specialized organelle where melanin is synthesized. Melanin synthesis is controlled by tyrosinase, the vital enzyme in melanogenic pathway. The present investigation is based on an effect of purified mushroom tyrosinase of Agaricus bisporus on B16F10 melanocytes for the melanin production via blocking pigment cell machinery. Using B16F10 melanocytes showed that the stimulation of melanogenesis by purified tyrosinase is due to increased tyrosinase absorption. Cellular tyrosinase activity and melanin content in B16F10 melanocytes were increased by purified tyrosinase in a dose-dependent manner. Western blot analysis revealed that cellular tyrosinase levels were enhanced after treatment with purified tyrosinase for 48 hours. Furthermore, tyrosinase induced phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in a dose-dependent manner. The purified tyrosinase-mediated increase of tyrosinase activity was significantly attenuated by H89, LY294002, Ro-32-0432, and PD98059, cAMP-dependent protein kinase inhibitors. The results indicate that purified tyrosinase can be used as contestant for the treatment of vitiligous skin conditions. PMID:27699070

  11. Cutin-derived CuO reaction products from purified cuticles and tree leaves

    Science.gov (United States)

    Goñi, Miguel A.; Hedges, John I.

    1990-11-01

    Long chain (C 16-C 18) hydroxy fatty acids are obtained among the nonlignin-derived reaction products from the CuO oxidation of a variety of geochemical samples. In order to investigate the origin of these acids, the CuO reaction products of isolated cuticles and whole leaves were investigated. The reaction products from the CuO oxidation of purified apple ( Malus pumila) cuticle include 16-hydroxy-hexadecanoic acid, 10,16-dihydroxyhexadecanoic acid, 9,10,18-trihydroxyoctadec-12-enoic acid, and 9,10,18-trihydroxyoctadecanoic acid as major components. The distribution of these cutin-derived CuO reaction products is similar to the monomer compositions deduced from traditional methods of cutin analysis. Oxidation of whole English Holly ( Ilex aquifolium) leaves yields cutin-derived acidic reaction products (in addition to lignin-derived phenols) similar to those obtained from oxidation of the corresponding isolated cuticles, indicating that CuO oxidation of bulk plant tissue is a viable procedure of cutin analysis in geochemical applications.

  12. Ni2+-PAA Adsorbent for Purifying 6His-OmpTS Recombinant Protein

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Functional Ni2+-polyacrylic acid (Ni2+-PAA) adsorbent has been prepared for metal chelate affinity chromatography. DNA elements coding for adjacent histidines were fused to the Aeromonas hydrophila ompTS gene. Subsequent expression in E. coli resulted in the production of hybrid protein 6His-OmpTS that could be purified by Ni2+-PAA affinity chromatography.

  13. Identification and removal of proteins that co-purify with infectious prion protein improves the analysis of its secondary structure.

    Science.gov (United States)

    Moore, Roger A; Timmes, Andrew G; Wilmarth, Phillip A; Safronetz, David; Priola, Suzette A

    2011-10-01

    Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrP(Sc) ) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of α-helix, β-sheet and other secondary structures have not always been consistent between studies, suggesting that other proteins might be confounding the analysis of PrP(Sc) secondary structure. We have used FTIR and LC-MS/MS to analyze enriched PrP(Sc) from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrP(Sc) . Our data show that non-PrP proteins do contribute to absorbances that have been associated with α-helical, loop, turn and β-sheet structures attributed to PrP(Sc) . The major contaminant, the α-helical protein ferritin, absorbs strongly at 1652 cm(-1) in the FTIR spectrum associated with PrP(Sc) . However, even the removal of more than 99% of the ferritin from PrP(Sc) did not completely abolish absorbance at 1652 cm(-1) . Our results show that contaminating proteins alter the FTIR spectrum attributed to PrP(Sc) and suggest that the α-helical, loop/turn and β-sheet secondary structure that remains following their removal are derived from PrP(Sc) itself.

  14. Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

    Science.gov (United States)

    Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

    2013-07-19

    Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

  15. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Directory of Open Access Journals (Sweden)

    Hua-zhen Wang

    Full Text Available Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP could greatly increase the soluble expression level of Glucokinase (GlcK, α-Amylase (Amy and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  16. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Science.gov (United States)

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  17. The 73 kilodalton heat shock cognate protein purified from rat brain contains nonesterified palmitic and stearic acids.

    Science.gov (United States)

    Guidon, P T; Hightower, L E

    1986-08-01

    A protein related to the 71 kilodalton inducible rat heat shock protein was purified to electrophoretic homogeneity in milligram amounts from brain tissue of nonheat-stressed rats. The protein has been designated as a stress cognate protein based on previous studies and data presented herein that this protein cross-reacted with a monoclonal antibody originally raised against the Drosophila 70 kilodalton heat shock protein. The purified protein had an apparent molecular mass of 73 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent mass of 150 kilodaltons as determined by nondissociative gel chromatography, suggesting that the purified protein is a homodimer. The purified protein had isoelectric points of 5.0 under nondissociative conditions and 5.6 when exposed to protein denaturants, suggesting loss of bound anionic molecules and/or net exposure of basic residues upon denaturation. Chloroform/methanol extraction of the purified protein and subsequent analyses by thin layer and gas-liquid chromatography resulted in the identification of palmitic and stearic acids noncovalently bound to the protein. Approximately four molecules of fatty acids were bound per dimer with palmitic and stearic acids present in a one-to-one ratio. The purified protein did not bind exogenously added radioactive palmitate, indicating that the fatty acid-binding sites of the cognate protein were fully occupied and that the associated fatty acids were too tightly bound to exchange readily. The possible significance of the fatty acids associated with the 73 kilodalton stress cognate protein is discussed.

  18. Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

    Energy Technology Data Exchange (ETDEWEB)

    Radulescu, Razvan T., E-mail: ratura@gmx.net [Molecular Concepts Research (MCR), Muenster (Germany); Duckworth, William C. [Department of Medicine, Phoenix VA Health Care System, Phoenix, AZ (United States); Levy, Jennifer L. [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States); Fawcett, Janet, E-mail: janet.fawcett@va.gov [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States)

    2010-04-30

    Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

  19. In vitro DNA binding of purified CcpA protein from Lactococcus lactis IL1403.

    Science.gov (United States)

    Kowalczyk, Magdalena; Borcz, Barbara; Płochocka, Danuta; Bardowski, Jacek

    2007-01-01

    During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites. However, purified HPr-Ser-P protein from Bacillus subtilis was shown to slightly increase the DNA-binding capacity of the CcpA protein. It was also observed that CcpA bound to the cre box forms an apparently more stable complex than that resulting from unspecific binding. Competition gel retardation assay performed on DNA sequences from two PEP:PTS regions demonstrated that the ybhE, bglS, rheB, yebE, ptcB and yecA genes situated in these regions are most probably directly regulated by CcpA.

  20. Detection of D-amino acids in purified proteins synthesized in Escherichia coli.

    Science.gov (United States)

    Miyamoto, Tetsuya; Sekine, Masae; Ogawa, Tetsuhiro; Hidaka, Makoto; Homma, Hiroshi; Masaki, Haruhiko

    2010-05-01

    It has long been believed that amino acids comprising proteins of all living organisms are only of the L-configuration, except for Gly. However, peptidyl D-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of D-amino acids are naturally present in usual proteins. Thus we analyzed the D-amino acid contents of His-tag-purified beta-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110 degrees C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into D- and L-enantiomers. The contents of D-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index D/(D + L) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of D-amino acids were reproducibly detected, the D-amino acid profile being specific to an individual protein. This finding indicated the likelihood that D-amino acids are in fact present in the purified proteins. On the other hand, the D-amino acid contents of proteins were hardly influenced by the addition of D- or L-amino acids to the cultivation medium, whereas intracellular free D-amino acids sensitively varied according to the extracellular conditions. The origin of these D-amino acids detected in proteins was discussed.

  1. Use of sequential chemical extractions to purify nuclear membrane proteins for proteomics identification.

    Science.gov (United States)

    Korfali, Nadia; Fairley, Elizabeth A L; Swanson, Selene K; Florens, Laurence; Schirmer, Eric C

    2009-01-01

    The nuclear envelope (NE) is a double membrane system that is both a part of the endoplasmic reticulum and part of the nucleus. As its constituent proteins tend to be highly complexed with nuclear and cytoplasmic components, it is notoriously difficult to purify. Two methods can reduce this difficulty for the identification of nuclear membrane proteins: comparison to contaminating membranes and chemical extractions to enrich for certain groups of proteins. The purification of nuclear envelopes and contaminating microsomal membranes is described here along with procedures for chemical extraction using salt and detergent, chaotropes, or alkaline solutions. Each extraction method enriches for different combinations of nuclear envelope proteins. Finally, we describe the analysis of these fractions with MudPIT, a proteomics methodology that avoids gel extraction of bands to facilitate identification of minor proteins and membrane proteins that do not resolve well on gels. Together these three approaches can significantly increase the output of proteomics studies aimed at identifying the protein complement of subcellular membrane systems.

  2. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

    Energy Technology Data Exchange (ETDEWEB)

    Dick, Robert A.; Datta, Siddhartha A.K.; Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M. (NCI); (Cornell); (CM); (NIST)

    2016-05-06

    Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

    Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our

  3. Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells.

    Science.gov (United States)

    Kremers, Gert-Jan; Piston, David

    2010-06-26

    Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles. Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion. This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

  4. Antioxidant and ACE Inhibitory Bioactive Peptides Purified from Egg Yolk Proteins

    Directory of Open Access Journals (Sweden)

    Marwa Yousr

    2015-12-01

    Full Text Available Protein by-products from the extraction of lecithin from egg yolk can be converted into value-added products, such as bioactive hydrolysates and peptides that have potential health enhancing antioxidant, and antihypertensive properties. In this study, the antioxidant and angiotensin converting enzyme (ACE inhibitory activities of peptides isolated and purified from egg yolk protein were investigated. Defatted egg yolk was hydrolyzed using pepsin and pancreatin and sequentially fractionated by ultrafiltration, followed by gel filtration to produce egg yolk gel filtration fractions (EYGF. Of these, two fractions, EYGF-23 and EYGF-33, effectively inhibited the peroxides and thiobarbituric acid reactive substance (TBARS in an oxidizing linoleic acid model system. The antioxidant mechanism involved superoxide anion and hydroxyl radicals scavenging and ferrous chelation. The presence of hydrophobic amino acids such as tyrosine (Y and tryptophan (W, in sequences identified by LC-MS as WYGPD (EYGF-23 and KLSDW (EYGF-33, contributed to the antioxidant activity and were not significantly different from the synthetic BHA antioxidant. A third fraction (EYGF-56 was also purified from egg yolk protein by gel filtration and exhibited high ACE inhibitory activity (69% and IC50 value (3.35 mg/mL. The SDNRNQGY peptide (10 mg/mL had ACE inhibitory activity, which was not significantly different from that of the positive control captopril (0.5 mg/mL. In addition, YPSPV in (EYGF-33 (10 mg/mL had higher ACE inhibitory activity compared with captopril. These findings indicated a substantial potential for producing valuable peptides with antioxidant and ACE inhibitory activity from egg yolk.

  5. An evaluation of purified Salmonella Typhi protein antigens for the serological diagnosis of acute typhoid fever.

    Science.gov (United States)

    Tran Vu Thieu, Nga; Trinh Van, Tan; Tran Tuan, Anh; Klemm, Elizabeth J; Nguyen Ngoc Minh, Chau; Voong Vinh, Phat; Pham Thanh, Duy; Ho Ngoc Dan, Thanh; Pham Duc, Trung; Langat, Pinky; Martin, Laura B; Galan, Jorge; Liang, Li; Felgner, Philip L; Davies, D Huw; de Jong, Hanna K; Maude, Rapeephan R; Fukushima, Masako; Wijedoru, Lalith; Ghose, Aniruddha; Samad, Rasheda; Dondorp, Arjen M; Faiz, Abul; Darton, Thomas C; Pollard, Andrew J; Thwaites, Guy E; Dougan, Gordon; Parry, Christopher M; Baker, Stephen

    2017-08-01

    The diagnosis of typhoid fever is a challenge. Aiming to develop a typhoid diagnostic we measured antibody responses against Salmonella Typhi (S. Typhi) protein antigens and the Vi polysaccharide in a cohort of Bangladeshi febrile patients. IgM against 12 purified antigens and the Vi polysaccharide was measured by ELISA in plasma from patients with confirmed typhoid fever (n = 32), other confirmed infections (n = 17), and healthy controls (n = 40). ELISAs with the most specific antigens were performed on plasma from 243 patients with undiagnosed febrile disease. IgM against the S. Typhi protein antigens correlated with each other (rho > 0.8), but not against Vi (rho Typhoid patients exhibited higher IgM against 11/12 protein antigens and Vi than healthy controls and those with other infections. Vi, PilL, and CdtB exhibited the greatest sensitivity and specificity. Specificity and sensitivity was improved when Vi was combined with a protein antigen, generating sensitivities and specificities of 0.80 and >0.85, respectively. Applying a dynamic cut-off to patients with undiagnosed febrile disease suggested that 34-58% had an IgM response indicative of typhoid. We evaluated the diagnostic potential of several S. Typhi antigens; our assays give good sensitivity and specificity, but require further assessment in differing patient populations. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  6. Strategies for purifying variants of human rhinovirus 14 2C protein.

    Science.gov (United States)

    Sára, Tomáš; Konrat, Robert; Skern, Tim

    2014-03-01

    The positive strand RNA genome of picornaviruses, including human rhinovirus (HRV), poliovirus (PV) and foot-and-mouth disease virus, is translated immediately into a polyprotein that is cleaved by virally encoded proteinases into 10-13 mature proteins. These include the four proteins required to assemble the viral particle as well as 3D(pol) (the viral RNA polymerase) and 2C, an ATPase and putative helicase. 2C is a protein which is responsible, together with 2B and 3A, for anchoring the replication complexes to membranous structures in the infected cell on which RNA replication takes place. Additionally, expression of 2C and its precursor 2BC in mammalian cells leads to vesicle formation observed in infected cells. 2C is encoded by all picornaviruses; nevertheless, its exact role in viral replication remains unclear. A contributing factor is the absence of structural data for this hydrophobic protein the generation of which has been hampered by an inability to produce soluble and stable material. Here, we compare 2C from several genera and show that the 2C protein has considerable heterogeneity. Using protein structure meta-analysis, we developed models of HRV14 2C that should be useful for mutational analysis. Based on these analyses, we expressed and purified two domains of HRV14 2C using three different protocols and examined the folding by thermal denaturation or (1)H NMR. Both domains were concentrated sufficiently to allow crystal screens or NMR pilot experiments to be performed. This work provides a platform to explore 2C proteins from all picornaviral genera to generate candidates for structural analysis.

  7. Use of a purified outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine in mice.

    OpenAIRE

    Gilleland, H. E.; Parker, M G; Matthews, J.M.; Berg, R D

    1984-01-01

    The outer membrane protein F (porin) from the PAO1 strain of Pseudomonas aeruginosa was purified by two different methods. One procedure involved separation by column chromatography of proteins extracted from isolated outer membranes, whereas the other involved extraction from gels after slab polyacrylamide gel electrophoresis of proteins extracted from cell envelopes. Both procedures yielded protein F preparations which successfully immunized mice from subsequent challenge with the PAO1 stra...

  8. Properties of Folate Binding Protein Purified from Cow’s Milk

    Directory of Open Access Journals (Sweden)

    SUBANDRATE

    2012-09-01

    Full Text Available Folic acid played an important role in the metabolism of the body. To measure the serum folic acid levels could use the folate binding protein (FBP from cow’s milk with a technique analogous to ELISA. The aims of this study were to identify characteristics of FBP from cow’s milk and binding capacity of FBP to folic acid and to purify FBP from other whey protein passed through DEAE-cellulose chromatography column. Each of DEAE-cellulose peaks was passed in affinity chromatography column. FBP was released from affinity column with sodium acetate buffer pH 3.5. The purity of obtained FBP was demonstrated by a single spot in SDS-PAGE analysis and the estimated molecular weight of FBP was around 31 kDa. Our study indicated that 1 mol FBP bound 1 mol folic acid. Alkylation with iodoacetic acid decreased the binding capacity of FBP which suggested the presence of a–SH or imidazol group in its active site. The importance of disulfide bridge was proven by decreasing of folate binding capacity of FBP after -mercaptoethanol treatment. In contrary, the folate binding didn need Ca2+ ion, as indicated by EDTA test which gave the same result as control.

  9. A novel method for purifying bluetongue virus with high purity by co-immunoprecipitation with agarose protein A

    Directory of Open Access Journals (Sweden)

    Ming Dai

    2010-06-01

    Full Text Available Abstract Background Bluetongue virus (BTV is an icosahedral non-enveloped virus within the genus Orbivirus of Reoviridae and exists as 24 distinct serotypes. BTV can infect all ruminant species and causes severe sickness in sheep. Recently, it was reported that BTV can infect some human cancer cells selectively. Because of the important oncolysis of this virus, we developed a novel purifying method for large-scale production. The purifying logic is simple, which is picking out all the components unwanted and the left is what we want. The process can be summarized in 4 steps: centrifugation, pulling down cell debrises and soluble proteins by co-immunoprecipitation with agarose Protein A, dialysis and filtration sterilization after concentration. Results The result of transmission electron microscope (TEM observation showed that the sample of purified virus has a very clear background and the virions still kept intact. The result of 50% tissue culture infective dose (TCID50 assay showed that the bioactivity of purified virus is relatively high. Conclusions This method can purify BTV-10 with high quality and high biological activity on large-scale production. It also can be used for purifying other BTV serotypes.

  10. Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.

    Science.gov (United States)

    Eckford, Paul D W; Li, Canhui; Bear, Christine E

    2015-03-09

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

  11. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells

    OpenAIRE

    Longmire, Tyler A.; Ikonomou, Laertis; Hawkins, Finn; Christodoulou, Constantina; Cao, Yuxia; Jean, JC; Kwok, Letty W.; Mou, Hongmei; Rajagopal, Jayaraj; Shen, Steven S.; Dowton, Anne A.; Serra, Maria; Weiss, Daniel J.; Green, Michael D.; Snoeck, Hans-Willem

    2012-01-01

    Two populations of Nkx2-1+ progenitors in the developing foregut endoderm give rise to the entire post-natal lung and thyroid epithelium, but little is known about these cells, as they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling can specify t...

  12. Preparation and physiological activities of carboxymethylated derivative purified from corn bran

    Science.gov (United States)

    Zhu, Linghui; Fang, Miaoli; Ma, Jianjun; Mo, Qing

    2017-06-01

    Two water-soluble polysaccharides extracted from corn bran were chemically modified to obtain their carboxymethylated derivatives (C-CBP1, C-CBP2). Theresults of degree of substitution and FT-IR analysis showed the carboxymethylation of polysaccharides were successful. The average molecular weight (Mw) of C-CBP1 and C-CBP2 were 368 and 263kDa, respectively. The degree of substitution (DS) of C-CBP1 and C-CBP2 were determined to be 0.44 and 0.46. The results showed that derivatives were effective in antioxidant and bile acidbinding activityin a dose dependent way. And C-CBP2 had the higher activity for hydroxyl radical, superoxide anion scavenging activities and bile acid capacity, as lower molecular weight plays a critical role in antioxidant activities and bile acid capacity. The results suggest that the carboxymethylated derivatives are potential natural antioxidant and blood fat reduce agent that can be used as drugs or functional food ingredients.

  13. Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

    Science.gov (United States)

    Fuerstenau-Sharp, Maya; Zimmermann, Martina E; Stark, Klaus; Jentsch, Nico; Klingenstein, Melanie; Drzymalski, Marzena; Wagner, Stefan; Maier, Lars S; Hehr, Ute; Baessler, Andrea; Fischer, Marcus; Hengstenberg, Christian

    2015-01-01

    Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

  14. Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Maya Fuerstenau-Sharp

    Full Text Available Induced pluripotent stem (iPS cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V. In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

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

    Directory of Open Access Journals (Sweden)

    Zunino Annalisa

    2010-02-01

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

  16. Ultrastructural, protein composition, and antigenic comparison of psittacine beak and feather disease virus purified from four genera of psittacine birds.

    Science.gov (United States)

    Ritchie, B W; Niagro, F D; Latimer, K S; Lukert, P D; Steffens, W L; Rakich, P M; Pritchard, N

    1990-04-01

    Psittacine beak and feather disease (PBFD) virus, was purified from diseased tissues of a lesser sulphur-crested cockatoo (Cacatua sulphurea), a black palm cockatoo (Probosiger aterrimus), a red-lored Amazon parrot (Amazona autumnalis), and a peach-faced lovebird (Agapornis roseicollis). The histopathology of diseased feathers and follicular epithelium from the different species was compared; basophilic intranuclear inclusion bodies were identified in the follicular epithelium and intracytoplasmic globular inclusions were observed within macrophages located in the feather pulp from the four species. Psittacine beak and feather disease virus antigen was specifically detected by colloidal gold immunoelectron microscopy. The different preparations of purified virions displayed an icosahedral symmetry, were non-enveloped, and had a mean diameter that varied from 12 to 15 nm when negatively stained. Two major viral-associated proteins with approximate molecular weights of 26 and 23 kilodaltons (kd) were consistently demonstrated from the four viral preparations. Purified virions from the four genera were antigenically related. These findings suggest that the PBFD virus purified from numerous genera of diseased birds is similar based on ultrastructural characteristics, protein composition and antigenic reactivity.

  17. 16-BAC/SDS-PAGE analysis of membrane proteins of yeast mitochondria purified by free flow electrophoresis.

    Science.gov (United States)

    Braun, Ralf J; Kinkl, Norbert; Zischka, Hans; Ueffing, Marius

    2009-01-01

    Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain. High-resolution separation and analysis of such membrane proteins, for example, by two-dimensional gel electrophoresis (2-DE), is hampered by their hydrophobicity and tendency for aggregation. Here, we describe the separation of mitochondrial membrane proteins of Saccharomyces cerevisiae by 16-benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE). This method enables the separation of membrane proteins owing to the solubilizing power of the ionic detergents 16-BAC and SDS, respectively. Mitochondria were isolated from yeast cultures by differential centrifugation and were further purified by free flow electrophoresis (FFE) in zone-electrophoretic mode (ZE). Subsequently, membrane proteins from ZE-FFE-purified mitochondria were enriched by carbonate extraction and subjected to 16-BAC/SDS-PAGE. The resulting protein spot patterns were visualized by a highly sensitive fluorescence stain with ruthenium-II-bathophenantroline disulfonate chelate (RuBP), and by colloidal Coomassie staining. Proteins were identified by Maldi-Tof mass spectrometry and peptide mass fingerprinting.

  18. A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates.

    Science.gov (United States)

    Blin, Guillaume; Nury, David; Stefanovic, Sonia; Neri, Tui; Guillevic, Oriane; Brinon, Benjamin; Bellamy, Valérie; Rücker-Martin, Catherine; Barbry, Pascal; Bel, Alain; Bruneval, Patrick; Cowan, Chad; Pouly, Julia; Mitalipov, Shoukhrat; Gouadon, Elodie; Binder, Patrice; Hagège, Albert; Desnos, Michel; Renaud, Jean-François; Menasché, Philippe; Pucéat, Michel

    2010-04-01

    Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

  19. Animal-derived pharmaceutical proteins.

    Science.gov (United States)

    Redwan, el-Rashdy M

    2009-01-01

    Livestock animals have made a significant contribution to human health and well-being throughout humankind's history. A significant contribution of farm animals to human health are the longstanding use of bovine and porcine for production of insulin (for treatment of diabetes), gelatin (for pharmaceutical and other purposes), as well as horse and sheep antibody against natural venoms, toxins, drugs and microbial peptides. Gelatin being the biggest animal protein consumed in human health, follows with antibodies fragments. The chronic problem of animal-derived therapeutics, especially those of high molecular weight, is the immunogenicity induction in addition to their biosafety. However, the invertebrates and lower vertebrates donate the human being a several crucial emergency saving life small-peptides or their analogs such as Refludan, Prialt, Exendin. Not only, but the farm animals are enormously using as models for novel surgical strategies, testing of biodegradable implants and sources of tissue replacements, such as skin and heart valves. Recently, they are being harnessing as bioreactor for production of biopharmaceutical related products through gene farming with efficiency far greater than any conventional microbial or cell-culture production systems. Only 16 transgenic cows would be covering the worldwide needs from human growth hormone. The transgenic, especially animal, technology would be solving a several biopharmaceutical products disadvantages, such as cost, biosafety, immunogenicity and the availability dimensions.

  20. Purifying Nanomaterials

    Science.gov (United States)

    Hung, Ching-Cheh (Inventor); Hurst, Janet (Inventor)

    2014-01-01

    A method of purifying a nanomaterial and the resultant purified nanomaterial in which a salt, such as ferric chloride, at or near its liquid phase temperature, is used to penetrate and wet the internal surfaces of a nanomaterial to dissolve impurities that may be present, for example, from processes used in the manufacture of the nanomaterial.

  1. A novel buckwheat protein with a beneficial effect in atherosclerosis was purified from Fagopyrum tataricum (L. Gaertn.

    Directory of Open Access Journals (Sweden)

    Zhou Xiaoli

    2013-01-01

    Full Text Available Buckwheat seeds contain many kinds of functional compounds that are of benefit to patients with cardiovascular disease. In this research, a water-soluble buckwheat protein was isolated and purified through a DEAE-Sepharose anion exchange column and Sephadex G-75 gel chromatography. The isolated buckwheat protein fractions exhibited hypocholesterolemic activity in a HepG2 cell model and demonstrated prominent bile acid salt-binding activity in an in vitro assay. The antioxidative activity of protein fractions with hypolipidemic effects was detected in a free radical scavenging experiment. The buckwheat protein fraction with the most obvious hypolipidemic activity and free radical scavenging activity was named as WSBWP. Its molecular weight was estimated by SDS-PAGE electrophoresis to be 38 kDa. It could become a potential candidate in the treatment of atherosclerosis.

  2. Soy Flour Adhesive Strength Compared with That of Purified Soy Proteins*

    Science.gov (United States)

    Linda Lorenz; Michael Birkeland; Chera Daurio; Charles R. Frihart

    2015-01-01

    Except for the substitution of soy flour in phenolic resins (Frihart et al. 2013) and the use of soy flour at high pHs (Lambuth 2003), the literature on soy protein properties for adhesives has mainly focused on soy protein isolate and specific protein fractions (Sun 2005b). The assumption is that proteins are the main portion of soy flour giving bond strength and the...

  3. Identification and characterization of a chitin-binding protein purified from coelomic fluid of the lugworm Arenicola marina defining a novel protein sequence family

    DEFF Research Database (Denmark)

    Vitashenkova, Nina; Moeller, Jesper Bonnet; Leth-Larsen, Rikke

    2012-01-01

    We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm Arenicola marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass...... by coelomocytes, in the nephridium and in round cells in epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components....

  4. Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane.

    Science.gov (United States)

    Zhao, Yingxin; Zhang, Wei; White, Michael A; Zhao, Yingming

    2003-08-01

    Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.

  5. Electrophoretic separation of purified myelin: a method to improve the protein pattern resolving.

    Science.gov (United States)

    Ravera, Silvia; Bartolucci, Martina; Barbarito, Giulia; Calzia, Daniela; Panfoli, Isabella

    2013-01-01

    Myelin sheath is a lipid-rich membrane, consisting of 70% lipid and 30% proteins, that is involved in physiological and pathological processes. For this reason its protein composition has been often investigated, principally by two-dimensional electrophoresis; however, the consistent lipid content makes it difficult to obtain good proteins separation. To improve the resolution of myelin proteins in a denaturing monodimensional gel electrophoresis, we examined several mixtures for the denaturation of the sample, utilizing different detergents and reducing agents. The definition of the protein pattern was analyzed by both "Blue Silver" Coomassie staining and Western Blot analysis against myelin basic protein, one of the most represented myelin proteins. The best resolution is observed when the sample was incubated with a mixture containing 1.25% dithiothreitol, 4 M urea, and 1% dodecyl maltoside or 1 % 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, prior to addition of denaturing agents. In conclusion, this work describes a novel method to improve the separation of myelin proteins in a monodimensional gel electrophoresis. It may be also useful for investigating other lipid-rich samples.

  6. Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins*

    Science.gov (United States)

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias

    2014-01-01

    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope–labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A–coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope–labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed. PMID:24722731

  7. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins.

    Science.gov (United States)

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias

    2014-06-01

    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  8. [Role of a purified Sertoli cell protein (CMB-21) in the biosynthesis of Leydig cell testosterone in the immature rat].

    Science.gov (United States)

    Boujrad, N; Papadopoulos, V; Drosdowsky, M A; Carreau, S

    1989-09-01

    The existence of Sertoli cell factors which modulate the rat Leydig cell function prompted us to study the biological activity of selected proteins called CMB proteins and produced by immature rat Sertoli cells. Percoll purified Leydig cells (10(5)) from 20 days-old rats have been incubated 5 h at 32 degrees C in 1 ml Ham F12/DME medium with increasing concentrations of partially purified CMB proteins (0-1,000 ng/ml) either in presence or absence of oLH (25 ng/ml). Among the CMB proteins tested, only CMB-21 produces a dose related increase of testosterone production: from 2 to 500 pg/ml of CMB-21, testosterone output is unchanged (51 pg/10(5) cells) but 1 to 1,000 ng/ml of this protein produces a linear increase of testosterone productions (86 to 870 pg). In the presence of oLH which induces a 10-fold increase of testosterone production (499 pg), increasing doses of CMB-21 further stimulate testosterone output (775 to 2.272 pg/10(5) cells). Whatever the concentration of oLH used (0 to 50 ng/ml), CMB-21 (500 ng/ml) leads to a further 2 fold augmentation of testosterone synthesis; similarly, in the presence of dbcAMP (1 mM), CMB 21 increases the testosterone production but no effect is observed when Leydig cells are incubated in the presence of 22R-hydroxycholesterol (30 microM). The cAMP levels which are increased more than 4 fold by oLH, remain unchanged in the presence of CMB-21 either alone or with oLH, as observed when Sertoli cell culture medium is used.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Vacuolar targeting of r-proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice.

    Science.gov (United States)

    Palaniswamy, Harunipriya; Syamaladevi, Divya P; Mohan, Chakravarthi; Philip, Anna; Petchiyappan, Anushya; Narayanan, Subramonian

    2016-02-01

    Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well-developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high-value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar-targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N-terminal 78-bp-long putative vacuolar-targeting sequence from the N-terminal domain of unknown function (DUF) in Triticum aestivum 6-SFT (sucrose: fructan 6-fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar-targeting determinants at the N-terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His-tagged β-glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar-targeting determinant could localize recombinant proteins (r-proteins) to the vacuole in high concentrations and such targeted r-proteins can be purified from the juice with a few simple steps.

  10. Purifying selection in porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation.

    Science.gov (United States)

    Robinson, Sally R; Abrahante, Juan E; Johnson, Craig R; Murtaugh, Michael P

    2013-12-01

    Porcine reproductive and respiratory syndrome virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3466 type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.

    Science.gov (United States)

    Lira, C B B; Gui, K E; Perez, A M; da Silveira, R C V; Gava, L M; Ramos, C H I; Cano, M I N

    2009-02-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

  12. Studies on Locusts' Pathogenesis and Energy Metabolic Inhibition Induced by the Insecticidal Protein Purified from Pseudomonas Pseudoalcaligenes

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The insecticidal protein produced by Pseudomonas pseudoalcaligenes is purified from the suspension of the bacterial culture. As an intact molecule, the protein acts on the foregut, midgut, hindgut, vasa Malpighii and fat body, and kills locusts. The disinsection rates of feeding and injection are 63.3% and 65.7%, respectively. After 24h to 48h, it is observed that all these tissues and cells show pathological changes in varying degrees, and so did the host cellular organs of these cells, such as cytoblast, mitochondria, endoplasmic reticulum and ribosome. Particularly, the changes of the mitochondria are much more serious than those of others. Detection of oxygen electrode shows the efficiency of oxidative phosphorylation and the ATP synthesis decreases, while the activity of mitochondrial ATPase is almost not affected. That means the energy utilization of locusts is in gear, but the shortage of the supplying of energy results in their death. This research forms the substantial basis for controlling locusts.

  13. Exploiting the 21st amino acid-purifying and labeling proteins by selenolate targeting.

    Science.gov (United States)

    Johansson, Linda; Chen, Chunying; Thorell, Jan-Olov; Fredriksson, Anna; Stone-Elander, Sharon; Gafvelin, Guro; Arnér, Elias S J

    2004-10-01

    Selenium is essential to human life and occurs in selenoproteins as selenocysteine (Sec), the 21st amino acid. The selenium atom endows selenocysteine with unique biochemical properties, including a low pK(a) and a high reactivity with many electrophilic agents. Here we describe the introduction of selenocysteine into recombinant non-selenoproteins produced in Escherichia coli, as part of a small tetrapeptide motif at the C terminus. This selenocysteine-containing motif could subsequently be used as a protein tag for purification of the recombinant protein, selenolate-targeted labeling with fluorescent compounds or radiolabeling with either gamma-emitting (75)Se or short-lived positron emitters such as (11)C. The results presented here thus show how a wide range of biotechnological applications can be developed starting from the insertion of selenocysteine into proteins.

  14. In vitro inhibition of vesicular stomatitis virus replication by purified porcine Mx1 protein fused to HIV-1 Tat protein transduction domain (PTD).

    Science.gov (United States)

    Zhang, Xiao-min; He, Dan-Ni; Zhou, Bin; Pang, Ran; Liu, Ke; Zhao, Jin; Chen, Pu-yan

    2013-08-01

    Vesicular stomatitis virus (VSV) is the causative agent of Vesicular stomatitis (VS), a highly contagious fatal disease of human and pigs. Few effective antiviral drugs are currently available against VSV infection. Mx proteins are interferon (IFN)-induced dynamin-like GTPases present in all vertebrates with a range of antiviral activities. Previous studies have shown that the transfected cell lines expressing either porcine Mx1 or human MxA acquired a high degree of resistance to VSV. To explore the feasibility of taking porcine Mx1 protein expressed in Escherichia coli as an antiviral agent, we applied the pCold system to express this fusion protein (PTD-poMx1), which consisted of an N-terminal HIV-1 Tat protein transduction domain (PTD) and the full-length porcine Mx1, and investigated its effects on the replication of VSV in Vero cells. The results demonstrated that the purified PTD-poMx1 fusion proteins could transduct into cells after incubated for 5h and had no cytotoxic. Furthermore, plaque reduction assay, determination of TCID50, real-time PCR and Western blot analyses were carried out to confirm the antiviral activity of purified fusion proteins in VSV-infected Vero cells. Altogether, these data suggested that PTD-poMx1 fusion proteins might be applicable to inhibit VSV replication as a novel antiviral therapeutic agent.

  15. Characterization of purified recombinant Bet v 1 with authentic N-terminus, cloned in fusion with maltose-binding protein.

    Science.gov (United States)

    Spangfort, M D; Ipsen, H; Sparholt, S H; Aasmul-Olsen, S; Larsen, M R; Mørtz, E; Roepstorff, P; Larsen, J N

    1996-11-01

    A gene encoding the pollen major allergen Bet v 1 from Betula verrucosa (White Birch) has been cloned and expressed in Escherichia coli as a fusion with maltose-binding protein and a Factor Xa proteolytic cleavage site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after cleavage. Fusion protein was isolated by amylose affinity chromatography and enzymatically cleaved by incubation with Factor Xa. Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequencing of the first 20 amino acids showed complete agreement with the deduced Bet v 1 DNA sequence. Mass spectrometry showed that recombinant Bet v 1 has a molecular mass of 17,440 +/- 2 Da; 86% of the recombinant Bet v 1 amino acid sequence could be verified by digestion with Lys-C and mass spectrometric peptide mapping. The yield of purified recombinant Bet v 1 was 10 mg per liter E. coli cell culture. Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and one or two minor spots focusing at slightly different pHs. The immunochemical properties of recombinant protein were indistinguishable from those of naturally occurring Bet v 1 when compared using a panel of murine monoclonal antibodies and serum IgE from birch pollen allergic patients. Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1. Thus, the methods used for bacterial expression and protein purification result in relatively high yields of folded recombinant Bet v 1 with correct N-terminal sequence and molecular mass. Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein from pollen.

  16. Cytochrome 572 is a conspicuous membrane protein with iron oxidation activity purified directly from a natural acidophilic microbial community.

    Science.gov (United States)

    Jeans, Chris; Singer, Steven W; Chan, Clara S; Verberkmoes, Nathan C; Shah, Manesh; Hettich, Robert L; Banfield, Jillian F; Thelen, Michael P

    2008-05-01

    Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5-1.2 and laden with toxic metals. To approach the mechanism of Fe(II) oxidation as a means of cellular energy acquisition, we isolated proteins from natural samples and found a conspicuous and novel cytochrome, Cyt(572), which is unlike any known cytochrome. Both the character of its covalently bound prosthetic heme group and protein sequence are unusual. Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. The purified protein has a cytochrome c-type heme binding motif, CxxCH, but a unique spectral signature at 572 nm, and thus is called Cyt(572). It readily oxidizes Fe(2+) in the physiologically relevant acidic regime, from pH 0.95-3.4. Other physical characteristics are indicative of a membrane-bound multimeric protein. Circular dichroism spectroscopy indicates that the protein is largely beta-stranded, and 2D Blue-Native polyacrylamide gel electrophoresis and chemical crosslinking independently point to a multi-subunit structure for Cyt(572). By analyzing environmental genomic information from biofilms in several distinctly different mine locations, we found multiple genetic variants of Cyt(572). MS proteomics of extracts from these biofilms substantiated the prevalence of these variants in the ecosystem. Due to its abundance, cellular location and Fe(2+) oxidation activity at very low pH, we propose that Cyt(572) provides a critical function for fitness within the ecological niche of these acidophilic microbial communities.

  17. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies.

    Science.gov (United States)

    Kuo, Mario Meng-Chiang; Nguyen, Phuong Hong; Jeon, Yun-Hui; Kim, Subin; Yoon, So-Mi; Choe, Senyon

    2014-02-24

    The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 - Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the

  18. AN INTEGRATIVE WAY OF TEACHING MOLECULAR CELL BIOLOGY AND PROTEIN CHEMISTRY USING ACTIN IMMOBILIZATION ON CHITIN FOR PURIFYING MYOSIN II.

    Directory of Open Access Journals (Sweden)

    M.G. Souza

    2007-05-01

    Full Text Available Our intent is to present our experience on teaching Molecular Cell Biology andProtein Chemistry at UNIRIO through an innovative approach that includes myosin IIextraction and purification. We took advantage of the properties of muscle contractionand propose a simple method for purifying myosin II by affinity chromatography. Thisoriginal method is based on the preparation of an affinity column containing actinmolecules covalently bound to chitin particles. We propose a three-week syllabus thatincludes lectures and bench experimental work. The syllabus favors the activelearning of protein extraction and purification, as well as, of scientific concepts suchas muscle contraction, cytoskeleton structure and its importance for the living cell. Italso promotes the learning of the biotechnological applications of chitin and theapplications of protein immobilization in different industrial fields. Furthermore, theactivities also target the development of laboratorial technical abilities, thedevelopment of problem solving skills and the ability to write up a scientific reportfollowing the model of a scientific article. It is very important to mention that thissyllabus can be used even in places where a facility such as ultra-centrifugation islacking.

  19. Paracrine proangiopoietic effects of human umbilical cord blood-derived purified CD133+ cells--implications for stem cell therapies in regenerative medicine.

    Science.gov (United States)

    Ratajczak, Janina; Kucia, Magda; Mierzejewska, Kasia; Marlicz, Wojciech; Pietrzkowski, Zbigniew; Wojakowski, Wojciech; Greco, Nicholas J; Tendera, Michal; Ratajczak, Mariusz Z

    2013-02-01

    CD133+ cells purified from hematopoietic tissues are enriched mostly for hematopoietic stem/progenitor cells, but also contain some endothelial progenitor cells and very small embryonic-like stem cells. CD133+ cells, which are akin to CD34+ cells, are a potential source of stem cells in regenerative medicine. However, the lack of convincing donor-derived chimerism in the damaged organs of patients treated with these cells suggests that the improvement in function involves mechanisms other than a direct contribution to the damaged tissues. We hypothesized that CD133+ cells secrete several paracrine factors that play a major role in the positive effects observed after treatment and tested supernatants derived from these cells for the presence of such factors. We observed that CD133+ cells and CD133+ cell-derived microvesicles (MVs) express mRNAs for several antiapoptotic and proangiopoietic factors, including kit ligand, insulin growth factor-1, vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. These factors were also detected in a CD133+ cell-derived conditioned medium (CM). More important, the CD133+ cell-derived CM and MVs chemoattracted endothelial cells and display proangiopoietic activity both in vitro and in vivo assays. This observation should be taken into consideration when evaluating clinical outcomes from purified CD133+ cell therapies in regenerative medicine.

  20. Purified Humanism

    DEFF Research Database (Denmark)

    Nickelsen, Niels Christian Mossfeldt

    2016-01-01

    Abstract. The aim of the Leicester Conference is to help managers by way of experiential learning to acquire the prerequisites to influence effectively organizational change. For some time there has been an ongoing debate on the innovative potential of social psychological experiments...... and techniques. This article discusses the analytical possibilities of the notion “purified humanism” as part of an alternative analysis of the effective mechanisms of a widely used social psychological experiment. The article unfolds a number of ideas in relation to the socio-material provocations and maneuvers...

  1. Oligomeric state of purified transient receptor potential melastatin-1 (TRPM1), a protein essential for dim light vision.

    Science.gov (United States)

    Agosto, Melina A; Zhang, Zhixian; He, Feng; Anastassov, Ivan A; Wright, Sara J; McGehee, Jennifer; Wensel, Theodore G

    2014-09-26

    Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.

  2. Purified humanism

    DEFF Research Database (Denmark)

    Nickelsen, Niels Christian Mossfeldt

    2016-01-01

    Abstract. The aim of the Leicester Conference is to help managers by way of experiential learning to acquire the prerequisites to influence effectively organizational change. For some time there has been an ongoing debate on the innovative potential of social psychological experiments...... and culturally specific attitudes in relation to leadership and the question of authority among participants. Keywords: The Leicester Conference, experiential learning, authority, socio-materiality, social techniques...... and techniques. This article discusses the analytical possibilities of the notion “purified humanism” as part of an alternative analysis of the effective mechanisms of a widely used social psychological experiment. The article unfolds a number of ideas in relation to the socio-material provocations and maneuvers...

  3. Molecular approaches towards development of purified natural products and their structurally known derivatives as efficient anti-cancer drugs: current trends.

    Science.gov (United States)

    Saha, Santu Kumar; Khuda-Bukhsh, Anisur Rahman

    2013-08-15

    Several natural products and their derivatives, either in purified or structurally identified form, exhibit immense pharmacological and biological properties, some of them showing considerable anticancer potential. Although the molecular mechanisms of action of some of these products are yet to be elucidated, extensive research in this area continues to generate new data that are clinically exploitable. Recent advancement in molecular biology, high throughput screening, biomarker identifications, target selection and genomic approaches have enabled us to understand salient interactions of natural products and their derivatives with cancer cells vis-à-vis normal cells. In this review we highlight the recent approaches and application of innovative technologies made to improve quality as well as efficiency of structurally identified natural products and their derivatives, particularly in small molecular forms capable of being used in "targeted therapies" in oncology. These products preferentially involve multiple mechanistic pathways and overcome chemo-resistance in tumor types with cumulative action. We also mention briefly a few physico-chemical features that compare natural products with drugs in recent natural product discovery approaches. We further report here a few purified natural products as examples that provide molecular interventions in cancer therapeutics to give the reader a glimpse of the current trends of approach for discovering useful anticancer drugs.

  4. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    Science.gov (United States)

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Identification of Onosma visianii Roots Extract and Purified Shikonin Derivatives as Potential Acaricidal Agents against Tetranychus urticae.

    Science.gov (United States)

    Sut, Stefania; Pavela, Roman; Kolarčik, Vladislav; Cappellacci, Loredana; Petrelli, Riccardo; Maggi, Filippo; Dall'Acqua, Stefano; Benelli, Giovanni

    2017-06-16

    There is an increasing need for the discovery of reliable and eco-friendly pesticides and natural plant-derived products may play a crucial role as source of new active compounds. In this research, a lipophilic extract of Onosma visianii roots extract containing 12% of shikonin derivatives demonstrated significant toxicity and inhibition of oviposition against Tetranychus urticae mites. Extensive chromatographic separation allowed the isolation of 11 naphthoquinone derivatives that were identified by spectral techniques and were tested against Tetranychus urticae. All the isolated compounds presented effects against the considered mite and isobutylshikonin (1) and isovalerylshikonin (2) were the most active, being valuable model compounds for the study of new anti-mite agents.

  6. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    Science.gov (United States)

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  7. Neurocognitive derivation of protein surface property from protein aggregate parameters

    Science.gov (United States)

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as protein marker. The protocol used multiple ILMFD inputs obtained for a protein to produce a set of mapped outputs as possible SRI candidates. The outputs were further clustered and largest cluster centre after normalization was found to be a close approximation of expected SRI that was calculated from known PDB structure. The outcome showed that faster derivation of individual protein’s surface property might be possible using its bulk form, heat denatured aggregates. PMID:21572883

  8. Characterization of protein acyltransferase function of recombinant purified GlnA1 from Mycobacterium tuberculosis: a moon lighting property.

    Science.gov (United States)

    Baghel, Anil S; Tandon, Rashmi; Gupta, Garima; Kumar, Ajit; Sharma, Raman K; Aggarwal, Neha; Kathuria, Abha; Saini, Neeraj K; Bose, Mridula; Prasad, Ashok K; Sharma, Sunil K; Nath, Mahendra; Parmar, Virinder S; Raj, Hanumantharao G

    2011-12-20

    The protein acetyltransferase (MTAase) function of glutamine synthetase of Mycobacterium smegmatis was established earlier. In this paper, studies were undertaken to examine MTAase function of recombinant glutamine synthetase (rGlnA1) of Mycobacterium tuberculosis, which showed >80% similarity with M. smegmatis GlnA. The specificity of MTAase to several acyl derivative of coumarins was examined. The results clearly indicated that MTAase exhibited differential specificities to several acyloxycoumarins. Further, MTAase was also found capable of transferring propionyl and butyryl groups from propoxy and butoxy derivatives of 4-methylcoumarin. These observations characterized MTAase in general as a protein acyltransferase. MTAase catalyzed acetylation of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model acetoxy coumarin was confirmed by MALDI-TOF-MS as well as western blot analysis using acetylated lysine polyclonal antibody. In order to validate the active site of rGlnA1 for TAase activity, effect of DAMC and L-methionine-S-sulfoximine (MSO) on GS and TAase activity of rGlnA1 were studied. The results indicated that the active sites of GS and TAase were found different. Acetyl CoA, a universal biological acetyl group donor, was also found to be a substrate for MTAase. These results appropriately characterize glutamine synthetase of Mtb exhibiting transacylase action as a moonlighting protein.

  9. N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

    Science.gov (United States)

    Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

    2010-07-01

    Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

  10. Chromatofocusing profile of purified human alpha-fetoprotein and albumin differs from those of crude samples: effect of protein concentration of the elution of the sample.

    Science.gov (United States)

    Leal, J A; Eddy, K B; Keel, B A

    1991-02-01

    Chromatofocusing was utilized to characterize charge microheterogeneity of purified human alpha-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4.57 (52%), 4.27 (43%), and less than 4.00 (5%), respectively. In contrast, 10 micrograms of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With greater than or equal to 5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with greater than or equal to 5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins with pIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.

  11. Chicken liver TGGCA protein purified by preparative mobility shift electrophoresis (PMSE) shows a 36.8 to 29.8 kd microheterogeneity.

    Science.gov (United States)

    Rupp, R A; Sippel, A E

    1987-12-10

    The TGGCA protein, the chicken homologue of HeLa cell NF-I, was purified to homogeneity from liver tissue by a procedure which includes preparative mobility shift electrophoresis (PMSE) as the final step. PMSE was here adjusted for the isolation of the TGGCA protein, but can be used as a general method to characterize the protein moiety of specific DNA-binding proteins. The TGGCA protein is a family of 6 protein species, which show minor differences in molecular weight from 36.8kd to 29.8kd. This microheterogeneity differs from the size distribution reported for HeLa cell NF-I polypeptides. All species of the TGGCA protein bind identically to a synthetic DNA-binding site and appear to be highly related in primary structure. We discuss the possible functional importance of this microheterogeneity.

  12. Pyrolysis-GC/MS of charred purified condensed tannin: towards identification of tannin-derived black carbon in environmental samples

    Science.gov (United States)

    Kaal, Joeri; Nierop, Klaas G. J.; Kraal, Peter; Preston, Caroline M.

    2010-05-01

    Tannins account for a significant proportion of plant biomass and are therefore a possible source of Black C in the charred remains from wildfires. Nonetheless, in contrast with other major biocomponents such as lignin and cellulose, the thermal degradation of tannins has not been investigated in laboratory charring experiments. We used pyrolysis-GC/MS to investigate the effects of furnace charring (30 min at fixed temperatures up to 600 °C under limited oxygen supply) on the degradation of pure condensed tannin (CT) isolated from Corsican pine (Pinus nigra) needles. The experiments showed a rapid loss (at 300 °C and higher) of the pyrogallol moieties of the B-ring of prodelphinidin-type CT, due to dehydroxylation. The relative abundance of catechols (from procyanidin-type CT) decreased at 350 °C and higher temperatures. This led to the formation of phenols that were strongly enriched between 300 and 400 °C. At higher temperatures, further dehydroxylation caused a decline in contributions of phenols producing a series of monocyclic aromatics ((alkyl)benzenes) and condensation of aromatics produced polycyclic aromatic hydrocarbons (PAHs), i.e. the typical pyrolysis fingerprint of strongly charred biomass. We conclude that (i) the thermal degradation of CT can be successfully monitored by pyrolysis-GC/MS, (ii) thermal degradation of CT is characterized by dehydroxylation of phenolic groups and condensation of aromatics that increase with temperature and (iii) CT-derived Black C may be recognized by catechol enrichments at low temperatures and possibly (relative) abundance of phenol and biphenyl at higher levels of thermal breakdown. Applying the same method to natural charcoal from gorse bushfires indicated that pyrolysis-GC/MS fingerprinting may allow for tannin identification in environmental Black C samples.

  13. Study of CO2 cyclic absorption stability of CaO-based sorbents derived from lime mud purified by sucrose method.

    Science.gov (United States)

    Ma, AiHua; Jia, QingMing; Su, HongYing; Zhi, YunFei; Tian, Na; Wu, Jing; Shan, ShaoYun

    2016-02-01

    Using lime mud (LM) purified by sucrose method, derived from paper-making industry, as calcium precursor, and using mineral rejects-bauxite-tailings (BTs) from aluminum production as dopant, the CaO-based sorbents for high-temperature CO2 capture were prepared. Effects of BTs content, precalcining time, and temperature on CO2 cyclic absorption stability were illustrated. The cyclic carbonation behavior was investigated in a thermogravimetric analyzer (TGA). Phase composition and morphologies were analyzed by XRD and SEM. The results reflected that the as-synthesized CaO-based sorbent doped with 10 wt% BTs showed a superior CO2 cyclic absorption-desorption conversion during multiple cycles, with conversion being >38 % after 50 cycles. Occurrence of Ca12Al14O33 phase during precalcination was probably responsible for the excellent CO2 cyclic stability.

  14. Purified JC virus T antigen derived from insect cells preferentially interacts with binding site II of the viral core origin under replication conditions.

    Science.gov (United States)

    Bollag, B; Mackeen, P C; Frisque, R J

    1996-04-01

    The human polyomavirus JC virus (JCV) establishes persistent, asymptomatic infections in most individuals, but in severely immunocompromised hosts it may cause the fatal demyelinating brain disease progressive multifocal leukoencephalopathy. In cell culture JCV multiplies inefficiently and exhibits a narrow host range. This restricted behavior occurs, in part, at the level of DNA replication, which is regulated by JCV's multifunctional large tumor protein (TAg). To prepare purified JCV TAg (JCT) for biochemical analyses, the recombinant baculovirus B-JCT was generated by cotransfection of insect cells with wild-type baculovirus and the vector pVL-JCT(Int-) containing the JCT-coding sequence downstream of the efficient polyhedrin promoter. JCT expressed in infected cells was immunoaffinity purified using the anti-JCT monoclonal antibody PAb 2000. Characterization of the viral oncoprotein indicated that it exists in solution as a mixture of monomeric and oligomeric species. With the addition of ATP, the population of monomers decreased and that of hexamers and double hexamers increased. A DNA mobility shift assay indicated that origin binding occurred primarily with the double-hexamer form. A comparison of the specific DNA-binding activities of JCT and SV40 TAg (SVT) revealed that JCT generally exhibited greater affinity for binding site II relative to binding site I (B.S. I) of both viral origin regions, whereas SVT preferentially bound B.S. I. Furthermore, JCT bound nonviral DNA more efficiently than did SVT. These functional differences between the two TAgs may contribute to the reduced DNA replication potential of JCV in vitro, and to the virus' ability to establish persistent infections in vivo.

  15. Characterization of a beta-actinin-like protein in purified non-muscle cell membranes. Its activity on (Na+ + K+)-ATPase.

    Science.gov (United States)

    Geny, B; Paraf, A; Fedon, Y; Charlemagne, D

    1982-11-22

    Treatment by EDTA of purified plasma membranes from MF2S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of (Na+ + K+)-ATPase to ouabain. When added back with Ca2+ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of Mr 32000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca2+ it was able to restore the original sensitivity to ouabain of (Na+ + K+)-ATPase from EDTA-treated membrane. This protein was shown to be similar to the beta-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca2+ the same quantitative biological activity on ouabain sensitivity of the (Na+ + K+)-ATPase. A possible interaction between beta-actinin, calmodulin and membrane-bound (Na+ + K+)-ATPase is discussed.

  16. MULTIFRACTAL ANALYSIS OF PROTEIN AGGREGATES TO DERIVE PROTEIN-SPECIFIC SIGNATURE

    OpenAIRE

    Hrishikesh Mishra, Tapobrata Lahiri*

    2010-01-01

    Deriving a property of a protein that is unique to it has well known significance since the study on ab initio model based derivation of protein structure where uniqueness of protein sequence is taken as the source of specificity of protein structure. In this direction, Heat denatured protein aggregates (HDPA) of proteins were studied with an objective to derive some multi-fractal markers specific to constituent protein that may be further utilized to extract information of the seed protein. ...

  17. Production of Self-Purifying Proteins in a Variety of Expression Hosts with Focus on Organophosphorus Hydrolase

    Science.gov (United States)

    2012-08-17

    Princeton University to Ohio State University, and the renewal effort was effectively split into two years and Princeton followed by one year at OSU...identifying protein-protein interactions through protein fishing experiments. This work was published in Protein Science26. In our other demonstration, the...impact the pharmaceutical industry in the next 10 years, and has spawned new conference tracks on Tag Technologies in Cambridge Healthtech Institute

  18. Kinetic and thermodynamic characterization of the interactions between the components of human plasma kinin-forming system and isolated and purified cell wall proteins of Candida albicans.

    Science.gov (United States)

    Seweryn, Karolina; Karkowska-Kuleta, Justyna; Wolak, Natalia; Bochenska, Oliwia; Kedracka-Krok, Sylwia; Kozik, Andrzej; Rapala-Kozik, Maria

    2015-01-01

    Cell wall proteins of Candida albicans, besides their best known role in the adhesion of this fungal pathogen to host's tissues, also bind some soluble proteins, present in body fluids and involved in maintaining the biochemical homeostasis of the human organism. In particular, three plasma factors - high-molecular-mass kininogen (HK), factor XII (FXII) and prekallikrein (PPK) - have been shown to adhere to candidal cells. These proteins are involved in the surface-contact-catalyzed production of bradykinin-related peptides (kinins) that contribute to inflammatory states associated with microbial infections. We recently identified several proteins, associated with the candidal cell walls, and probably involved in the binding of HK. In our present study, a list of potential FXII- and PPK-binding proteins was proposed, using an affinity selection (on agarose-coupled FXII or PPK) from a whole mixture of β-1,3-glucanase-extrated cell wall-associated proteins and the mass-spectrometry protein identification. Five of these fungal proteins, including agglutinin-like sequence protein 3 (Als3), triosephosphate isomerase 1 (Tpi1), enolase 1 (Eno1), phosphoglycerate mutase 1 (Gpm1) and glucose-6-phosphate isomerase 1 (Gpi1), were purified and characterized in terms of affinities to the human contact factors, using the surface plasmon resonance measurements. Except Gpm1 that bound only PPK, and Als3 that exhibited an affinity to HK and FXII, the other isolated proteins interacted with all three contact factors. The determined dissociation constants for the identified protein complexes were of 10(-7) M order, and the association rate constants were in a range of 10(4)-10(5) M(-1)s(-1). The identified fungal pathogen-host protein interactions are potential targets for novel anticandidal therapeutic approaches.

  19. Highly purified human-derived follicle-stimulating hormone (Bravelle® has equivalent efficacy to follitropin-beta (Follistim ® in infertile women undergoing in vitro fertilization

    Directory of Open Access Journals (Sweden)

    Webster Bobby W

    2003-10-01

    Full Text Available Abstract Background These data compare the efficacy and safety of highly purified human-derived follicle-stimulating hormone (Bravelle(R and recombinant follitropin-β (Follistim(R in women undergoing in vitro fertilization. Methods This report describes the pooled data from two, nearly identical, randomized, controlled, parallel-group, multicenter studies conducted in a total of 19 academic and private IVF-ET centers in the United States. Infertile premenopausal women underwent pituitary down-regulation using leuprolide acetate followed by a maximum of 12 days of subcutaneous Bravelle(R (n = 120 or Follistim(R (n = 118, followed by administration of human chorionic gonadotropin, oocyte retrieval and embryo transfer. The primary efficacy measure was the mean number of oocytes retrieved; secondary efficacy measures included the total dose and duration of gonadotropin treatment; peak serum estradion levels; embryo transfer and implantation rates; chemical, clinical and continuing pregnancies; and live birth rates. All adverse events were recorded and injection site pain was recorded daily using a patient, self-assessment diary. Results Similar efficacy responses were observed for all outcome parameters in the two treatment groups. Although patients receiving Bravelle(R consistently reported a greater number of chemical, clinical and continuing pregnancies, as well as an increased rate of live birth, the data did not attain statistical significance (P > 0.05. The overall incidence of adverse events was similar in both groups, but compared to Follistim(R, injections of Bravelle(R were reported by patients to be significantly less painful (P Conclusions Bravelle(R and Follistim(R had comparable efficacy in controlled ovarian hyperstimulation in women undergoing IVF-ET. There were no differences in the nature or number of adverse events between the treatment groups although Bravelle(R injections were reported to be significantly less painful.

  20. Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

    Institute of Scientific and Technical Information of China (English)

    Hui-Na ZHOU; Ying ZHAO; Shao-Min BIAN; Jian-Feng ZHAO; Fei REN; Huang-Ping WANG; Ju-Fu HUANG

    2005-01-01

    To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the partially purified MoFe protein (Av 1) preparation was obtained from Azotobacter vinelandii Lipmann OP by chromatography on DEAE-cellulose (DE52) and Sephacryl S-200 columns and analyzed by PAGE and matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Ay 1 band, the preparation was shown to have three other main bands that migrated slower than Avl. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase (PGI). In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaCl concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.

  1. Ligand binding characteristics and aggregation behavior of purified cow's milk folate binding protein depends on the presence of amphiphatic substances including cholesterol, phospholipids, and synthetic detergents.

    Science.gov (United States)

    Holm, Jan; Hansen, Steen Ingemann

    2002-01-01

    Folate binding protein was purified from cow's milk by a combination of cation exchange chromatography and methotrexate-AH-sepharose affinity chromatography. Dilution of the preparation to concentrations of protein less than 10 nM resulted in drastic changes of radioligand (folate) binding characteristics, i.e., a decrease in binding affinity with a change from upward to downward convex Scatchard plots and increased ligand dissociation combined with appearance of weak-affinity aggregated forms of the binding protein on gel filtration. These findings, consistent with a model predicting dimerization between unliganded and liganded monomers, were reversed in the presence of material eluted from the affinity column after adsorption of the protein(cofactor) or cholesterol, phospholipids, and synthetic detergents. The latter amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers in the surrounding aqueous medium and thereby prevent association between these monomeric forms prevailing at low concentrations of the protein. Our data have some bearings on studies which show that cholesterol and phospholipids are necessary for the clustering of folate receptors in the cell membrane; a process required for optimum receptor function and internalization of folate.

  2. A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production

    Science.gov (United States)

    Neto, João X.S.; Pereira, Mirella L.; Oliveira, Jose T. A.; Rocha-Bezerra, Lady C. B.; Lopes, Tiago D. P.; Costa, Helen P. S.; Sousa, Daniele O. B.; Rocha, Bruno A. M.; Grangeiro, Thalles B.; Freire, José E. C.; Monteiro-Moreira, Ana Cristina O.; Lobo, Marina D. P.; Brilhante, Raimunda S. N.; Vasconcelos, Ilka M.

    2017-01-01

    Candida species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. M. oleifera Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. The purified protein, named Mo-CBP2, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,309 Da. However, by SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of Mo-CBP2 under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9) with 4.1% (m/m) sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3 and flocculating proteins, and 2S albumins from different species. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis, with MIC50 and MIC90 values ranging between 9.45–37.90 and 155.84–260.29 μM, respectively. In addition, Mo-CBP2 (18.90 μM) increased the cell membrane permeabilization and reactive

  3. A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production

    Directory of Open Access Journals (Sweden)

    João X.S. Neto

    2017-06-01

    Full Text Available Candida species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. M. oleifera Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. The purified protein, named Mo-CBP2, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,309 Da. However, by SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of Mo-CBP2 under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9 with 4.1% (m/m sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3 and flocculating proteins, and 2S albumins from different species. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis, with MIC50 and MIC90 values ranging between 9.45–37.90 and 155.84–260.29 μM, respectively. In addition, Mo-CBP2 (18.90 μM increased the cell membrane permeabilization

  4. U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes.

    Science.gov (United States)

    Dybkov, Olexandr; Will, Cindy L; Deckert, Jochen; Behzadnia, Nastaran; Hartmuth, Klaus; Lührmann, Reinhard

    2006-04-01

    The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5' end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5' end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.

  5. In Vitro Assay for the Rap GTPase-Activating Protein Activity of the Purified Cytoplasmic Domain of Plexin.

    Science.gov (United States)

    Pascoe, Heath G; Wang, Yuxiao; Zhang, Xuewu

    2017-01-01

    Plexins are cell surface receptors that bind semaphorins and regulate essential processes such as axon guidance and angiogenesis. The cytoplasmic regions of plexins contain a functionally essential GTPase-activating protein (GAP) domain, which initiates downstream signaling by specifically inactivating the Rap GTPase. Here we describe the methods for expression and purification of the plexin cytoplasmic region in E. coli, and characterization of its GAP activity using a photometric assay. We also provide a protocol for measuring GAP activity of single-chain constructs with Rap covalently linked to the plexin cytoplasmic region.

  6. Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

    Directory of Open Access Journals (Sweden)

    George V. Hillyer

    1995-04-01

    Full Text Available Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44 identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

  7. Neurocognitive derivation of protein surface property from protein aggregate parameters

    OpenAIRE

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as...

  8. Process steps for the preparation of purified fractions of alpha-lactalbumin and beta-lactoglobulin from whey protein concentrates.

    Science.gov (United States)

    Gésan-Guiziou, G; Daufin, G; Timmer, M; Allersma, D; van der Horst, C

    1999-05-01

    Fractions enriched with alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were produced by a process comprising the following successive steps: clarification-defatting of whey protein concentrate, precipitation of alpha-lactalbumin, separation of soluble beta-lactoglobulin, washing the precipitate, solubilization of the precipitate, concentration and purification of alpha-la. The present study evaluated the performance of the process, firstly on a laboratory scale with acid whey and then on a pilot scale with Gouda cheese whey. In both cases soluble beta-lg was separated from the precipitate using diafiltration or microfiltration and the purities of alpha-la and beta-lg were in the range 52-83 and 85-94% respectively. The purity of the beta-lg fraction was higher using acid whey, which does not contain caseinomacropeptide, than using sweet whey. With the pilot scale plant, the recoveries (6% for alpha-la; 51% for beta-lg) were disappointing, but ways of improving each step in the process are discussed.

  9. Antioxidant activities of oyster plasma and the purified protein%牡蛎血浆及其纯化蛋白抗氧化活性研究

    Institute of Scientific and Technical Information of China (English)

    张泽; 高淑悦; 程继龙; 颜士慧; 李翀; 李玉婷; 张莉

    2012-01-01

    Blood was withdrawn from the adductor muscle hemolymph sinus using a syringe equipped with a needle and what was got after the blood was centrifuged with low temperature condition was the plasma. A purified protein with low molecular weight from plasma was obtained by the method of the ammonium sulfate precipitation and gel filtration chromatography. By SDS-PAGE, the protein showed itself a single gel belt with 15 ku in molecular weight. The protein showed the capability of reducing power, superoxide anion radical scavenging and was stable at the temperature of 30-50 ℃.%用配有针头的注射器从牡蛎的闭壳肌静脉窦抽取血液,低温条件下离心所得上清液即为牡蛎血浆。上清液经95%硫酸铵沉淀后通过凝胶过滤法分离提纯获得一种小分子蛋白质,SDS-PAGE电泳为单一带,相对分子质量约为15ku。牡蛎血浆及其纯化后蛋白均具有一定的还原能力和清除超氧阴离子自由基的能力,并在30~50℃温度下具有良好的稳定性。

  10. The effects of general anesthetics on ESR spectra of spin labels in phosphatidylcholine vesicles containing purified Na,K-ATPase or microsomal protein

    Energy Technology Data Exchange (ETDEWEB)

    Shibuya, Makiko, E-mail: shibu@den.hokudai.ac.jp [Department of Dental Anesthesiology, Graduate School of Dental Medicine, Hokkaido University (Japan); Hiraoki, Toshifumi [Division of Applied Physics, Graduate School of Engineering, Hokkaido University (Japan); Kimura, Kunie; Fukushima, Kazuaki [Department of Dental Anesthesiology, Graduate School of Dental Medicine, Hokkaido University (Japan); Suzuki, Kuniaki [Department of Molecular Cell Pharmacology, Graduate School of Dental Medicine, Hokkaido University (Japan)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer We studied the effects of general anesthetics on liposome using ESR spectra. Black-Right-Pointing-Pointer Two spin labels, 5-DSA and 16-DSA, were located in different position in liposome. Black-Right-Pointing-Pointer Anesthetics did not change the environment around the spin labels in the liposome. Black-Right-Pointing-Pointer Anesthetics remained on the surface of the lipid bilayer of liposome. Black-Right-Pointing-Pointer Proteins in the liposome did not change the effects of anesthetics on liposome. - Abstract: We investigated the effects of general anesthetics on liposome containing spin labels, 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA), and purified Na,K-ATPase or membrane protein of microsome using an electron spin resonance (ESR) spectroscopy. The spectra of 16-DSA in liposomes with both proteins showed three sharp signals compared with 5-DSA. The difference in the order parameter S value of 5-DSA and 16-DSA suggested that the nitroxide radical location of 5-DSA and 16-DSA were different in the membrane bilayer. The results were almost the same as those obtained in liposomes without proteins. The addition of sevoflurane, isoflurane, halothane, ether, ethanol and propofol increased the intensity of the signals, but the clinical concentrations of anesthetics did not significantly alter the S and {tau} values, which are indices of the fluidity of the membrane. These results suggest that anesthetics remain on the surface of the lipid bilayer and do not act on both the inside hydrophobic area and the relatively hydrophilic area near the surface. These results and others also suggest that the existence of Na,K-ATPase and microsomal proteins did not affect the environment around the spin labels in the liposome and the effects of anesthetics on liposome as a model membrane.

  11. Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides

    NARCIS (Netherlands)

    Berkhout, B; van Wamel, JLB; Beljaars, L; Meijer, DKF; Visser, Servaas; Floris, R

    2002-01-01

    In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition

  12. Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

    Directory of Open Access Journals (Sweden)

    O Rickard Nilsson

    Full Text Available Recent studies indicate that defective activity of complement factor H (FH is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

  13. Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

    Science.gov (United States)

    Nilsson, O Rickard; Lannergård, Jonas; Morgan, B Paul; Lindahl, Gunnar; Gustafsson, Mattias C U

    2013-01-01

    Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

  14. Preparation of the selenome-thionine derivative of tab- toxin resistance protein

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se- Met) derivative of TTR was overexpressed in E. coli strain M15, with pQE-30 plasmid, through IPTG induction in M9 medium containing Se-Met. The product was purified to an estimated homogeneity of greater than 95% according to SDS-PAGE, by a Ni-NTA metal affinity followed by a Mono Q anion exchange column chromatography. The successful substitution of Se-Met for methionine (Met) was confirmed by MALDI-TOF and ESI-Quadrupole Mass Spectrometry analysis. The derivative crystal was obtained using similar conditions as those for the native.

  15. Expression in Escherichia coli of fusion protein comprising α-conotoxin TxIB and preservation of selectivity to nicotinic acetylcholine receptors in the purified product.

    Science.gov (United States)

    Yu, Jinpeng; Zhu, Xiaopeng; Yang, Yang; Luo, Sulan; Zhangsun, Dongting

    2017-09-11

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, which are widely distributed in the central and peripheral nervous system. The α6β2* nAChR is an important subtype, which is closely associated with nicotine addiction and movement disorders etc. α-conotoxin TxIB with 16-amino acid residues specifically targets α6β2* nAChR with no obvious effect on other nAChR subtypes. However, chemical synthesis of TxIB is expensive, and the quantity of native TxIB extracted from cone snail is limited. In the present study, we attempted to obtain TxIB using biological method based on the recombinant expression in Escherichia coli (E. coli). The synthetic gene encoding mature peptide of TxIB was inserted in pET-31b(+) vector and transformed into E. coli strain BLR(DE3)pLysS for expression. The recombinant fusion protein KSI-TxIB-His6 (KSI, ketosteroid isomerase) was expressed successfully as inclusion body in E. coli, which was purified by Ni-NTA affinity chromatography column and cleaved by cyanogen bromide (CNBr) to release recombinant α-conotoxin TxIB (rTxIB). Then, rTxIB was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and was identified by electrospray ionization mass spectrometry (ESI-MS). Pharmacological activity of rTxIB was assessed by electrophysiological approaches. The results indicated that it preserved about 50% of potency, but, was even more important, had the same selectivity as the natural conotoxin which may provide an alternative method for quantity production of small peptides with low cost on the premise of not changing their potency. © 2017 John Wiley & Sons Ltd.

  16. Detection of prion protein in urine-derived injectable fertility products by a targeted proteomic approach.

    Directory of Open Access Journals (Sweden)

    Alain Van Dorsselaer

    Full Text Available BACKGROUND: Iatrogenic transmission of human prion disease can occur through medical or surgical procedures, including injection of hormones such as gonadotropins extracted from cadaver pituitaries. Annually, more than 300,000 women in the United States and Canada are prescribed urine-derived gonadotropins for infertility. Although menopausal urine donors are screened for symptomatic neurological disease, incubation of Creutzfeldt-Jakob disease (CJD is impossible to exclude by non-invasive testing. Risk of carrier status of variant CJD (vCJD, a disease associated with decades-long peripheral incubation, is estimated to be on the order of 100 per million population in the United Kingdom. Studies showing infectious prions in the urine of experimental animals with and without renal disease suggest that prions could be present in asymptomatic urine donors. Several human fertility products are derived from donated urine; recently prion protein has been detected in preparations of human menopausal gonadotropin (hMG. METHODOLOGY/PRINCIPAL FINDINGS: Using a classical proteomic approach, 33 and 34 non-gonadotropin proteins were identified in urinary human chorionic gonadotropin (u-hCG and highly-purified urinary human menopausal gonadotropin (hMG-HP products, respectively. Prion protein was identified as a major contaminant in u-hCG preparations for the first time. An advanced prion protein targeted proteomic approach was subsequently used to conduct a survey of gonadotropin products; this approach detected human prion protein peptides in urine-derived injectable fertility products containing hCG, hMG and hMG-HP, but not in recombinant products. CONCLUSIONS/SIGNIFICANCE: The presence of protease-sensitive prion protein in urinary-derived injectable fertility products containing hCG, hMG, and hMG-HP suggests that prions may co-purify in these products. Intramuscular injection is a relatively efficient route of transmission of human prion disease, and

  17. Association of coatomer proteins with the beta-receptor for platelet-derived growth factor

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Rorsman, C

    1997-01-01

    of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha......The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... noticed that components of 136 and 97 kDa bound to a peptide from this region of the receptor in a phosphorylation-independent manner. These components have now been purified and identified as alpha- and beta'-coatomer proteins (COPs), respectively. COPs are a family of proteins involved in the regulation...

  18. PURIFICATION OF ANGIOTENSIN CONVERTING ENZYME INHIBITORY PEPTIDE DERIVED FROM KACANG GOAT MEAT PROTEIN HYDROLYSATE

    Directory of Open Access Journals (Sweden)

    J. Jamhari

    2014-10-01

    Full Text Available The objective of this study was to identify the Angiotensin Converting Enzyme (ACE inhibitorypeptide derived from Kacang goat meat protein hydrolysate. Kacang goat meat loin section washydrolyzed with pepsin, trypsin and chymotrypsin. Protein hydrolysate of Kacang goat meat was thentested the protein concentration and ACE inhibitory activity. ACE inhibitory peptide of the proteinhydrolysate was purified through several steps of purification by column SEP-PAK Plus C18 Cartridgeand RP-HPLC using a Cosmosil column 5PE-SM, 4.6 x 250 mm. The sequence of amino acid of ACEinhibitory peptide was identified by amino acid sequencer. The results showed that amino acidssequence of ACE inhibitory peptide derived from protein hydrolysate of Kacang goat meat was leu-thrglu-ala-pro-leu-asn-pro-lys-ala-arg- asn-glu-lys. It had a molecular weight (MW of 1581 and occurredat the position of 20th to 33rd residues of b-actin of goat meat protein (Capra hircus. The ACE inhibitoryactivity (IC50 of the peptide was 190 mg/mL or 120 mM.

  19. Biomimetic repeat protein derived from Xenopus tropicalis for fibrous scaffold fabrication.

    Science.gov (United States)

    Kwon, Yunkyeoung; Yang, Yun Jung; Jung, Dooyup; Hwang, Byeong Hee; Cha, Hyung Joon

    2015-12-01

    Collagen, silk, and elastin are the fibrous proteins consist of representative amino acid repeats. Because these proteins exhibited distinguishing mechanical properties, they have been utilized in diverse applications, such as fiber-based sensors, filtration membranes, supporting materials, and tissue engineering scaffolds. Despite their infinite prevalence and potential, most studies have only focused on a few repeat proteins. In this work, the hypothetical protein with a repeat motif derived from the frog Xenopus tropicalis was obtained and characterized for its potential as a novel protein-based material. The codon-optimized recombinant frog repeat protein, referred to as 'xetro', was produced at a high rate in a bacterial system, and an acid extraction-based purified xetro protein was successfully fabricated into microfibers and nanofibers using wet spinning and electrospinning, respectively. Specifically, the wet-spun xetro microfibers demonstrated about 2- and 1.5-fold higher tensile strength compared with synthetic polymer polylactic acid and cross-linked collagen, respectively. In addition, the wet-spun xetro microfibers showed about sevenfold greater stiffness than collagen. Therefore, the mass production potential and greater mechanical properties of the xetro fiber may result in these fibers becoming a new promising fiber-based material for biomedical engineering.

  20. The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.

    Science.gov (United States)

    Veldhoven, C H; Meilof, J F; Huisman, J G; Smeenk, R J

    1992-07-01

    A characteristic of patients with autoimmune diseases such as Sjögren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.

  1. Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens

    Directory of Open Access Journals (Sweden)

    Ludwig Nicole

    2010-11-01

    Full Text Available Abstract Background The simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate. Methods Candidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients. Result Methods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96. This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly. Conclusion Protein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays

  2. Comparative serum protein binding of anthracycline derivatives.

    Science.gov (United States)

    Chassany, O; Urien, S; Claudepierre, P; Bastian, G; Tillement, J P

    1996-01-01

    The binding of doxorubicin, iododoxorubicin, daunorubicin, epirubicin, pirarubicin, zorubicin, aclarubicin, and mitoxantrone to 600 microM human serum albumin and 50 microM alpha 1-acid glycoprotein was studied by ultrafiltration at 37 degrees C and pH 7.4. Anthracycline concentrations (total and free) were determined by high-performance liquid chromatography (HPLC) with fluorometric detection. Binding to albumin (600 microM) varied from 61% (daunorubicin) to 94% (iododoxorubicin). The binding to alpha 1-acid glycoprotein (50 microM) was more variable, ranging from 31% (epirubicin) to 64% (zorubicin), and was essentially related to the hydrophobicity of the derivatives. Simulations showed that the total serum binding varied over a broad range from 71% (doxorubicin) to 96% (iododoxorubicin). We recently reported that the binding to lipoproteins of a series of eight anthracycline analogues could be ascribed to chemicophysical determinants of lipophilicity [2]. The present study was conducted to evaluate in vitro the contribution of albumin and alpha 1-acid glycoprotein to the total serum binding of these drugs.

  3. Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein

    DEFF Research Database (Denmark)

    Justesen, Sune; Lamberth, Kasper; Nielsen, Lise-Lotte B

    2009-01-01

    Fusion tags add desirable properties to recombinant proteins, but they are not necessarily acceptable in the final products. Ideally, fusion tags should be removed releasing the intact native protein with no trace of the tag. Unique endoproteinases with the ability to cleave outside their own...... characteristics for the exact removal of fusion tags. It is readily available in highly purified recombinant versions approved by the FDA for preparation of food for human consumption. We suggest that one should consider extending the use of chymosin to the preparation of pharmaceutical proteins....... recognition sequence can potentially cleave at the boundary of any native protein. Chymosin was recently shown to cleave a pro-chymosin derived fusion tag releasing native target proteins. In our hands, however, not all proteins are chymosin-resistant under the acidic cleavage conditions (pH 4.5) used...

  4. Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification.

    Science.gov (United States)

    Chen, Shengxi; Fahmi, Nour Eddine; Bhattacharya, Chandrabali; Wang, Lin; Jin, Yuguang; Benkovic, Stephen J; Hecht, Sidney M

    2013-11-26

    In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational

  5. Comparison of the Effect of Curing Ingredients Derived from Purified and Natural Sources on Inhibition of Clostridium perfringens Outgrowth during Cooling of Deli-Style Turkey Breast.

    Science.gov (United States)

    King, Amanda M; Glass, Kathleen A; Milkowski, Andrew L; Sindelar, Jeffrey J

    2015-08-01

    The antimicrobial impact of purified and natural sources of both nitrite and ascorbate were evaluated against Clostridium perfringens during the postthermal processing cooling period of deli-style turkey breast. The objective of phase I was to assess comparable concentrations of nitrite (0 or 100 ppm) and ascorbate (0 or 547 ppm) from both purified and natural sources. Phase II was conducted to investigate concentrations of nitrite (50, 75, or 100 ppm) from cultured celery juice powder and ascorbate (0, 250, or 500 ppm) from cherry powder to simulate alternative curing formulations. Ground turkey breast (75% moisture, 1.2% salt, pH 6.2) treatments were inoculated with C. perfringens spores (three-strain mixture) to yield 2.5 log CFU/g. Individual 50-g portions were vacuum packaged, cooked to 71.1°C, and chilled from 54.4 to 26.7°C in 5 h and from 26.7 to 7.2°C in 10 additional hours. Triplicate samples were assayed for growth of C. perfringens at predetermined intervals by plating on tryptose-sulfite-cycloserine agar; experiments were replicated three times. In phase I, uncured, purified nitrite, and natural nitrite treatments without ascorbate had 5.3-, 4.2-, and 4.4-log increases in C. perfringens, respectively, at 15 h, but <1-log increase was observed at the end of chilling in treatments containing 100 ppm of nitrite and 547 ppm of ascorbate from either source. In phase II, 0, 50, 75, and 100 ppm of nitrite and 50 ppm of nitrite plus 250 ppm of ascorbate supported 4.5-, 3.9-, 3.5-, 2.2-, and 1.5-log increases in C. perfringens, respectively. In contrast, <1-log increase was observed after 15 h in the remaining phase II treatments supplemented with 50 ppm of nitrite and 500 ppm of ascorbate or ≥75 ppm of nitrite and ≥250 ppm of ascorbate. These results confirm that equivalent concentrations of nitrite, regardless of the source, provide similar inhibition of C. perfringens during chilling and that ascorbate enhances the antimicrobial effect of nitrite on C

  6. Handbook of purified gases

    CERN Document Server

    Schoen, Helmut

    2015-01-01

    Technical gases are used in almost every field of industry, science and medicine and also as a means of control by government authorities and institutions and are regarded as indispensable means of assistance. In this complete handbook of purified gases the physical foundations of purified gases and mixtures as well as their manufacturing, purification, analysis, storage, handling and transport are presented in a comprehensive way. This important reference work is accompanied with a large number of Data Sheets dedicated to the most important purified gases.  

  7. Susceptibility of field populations of the fall armyworm (Lepidoptera: Noctuidae) from Florida and Puerto Rico to purified Cry1F protein and corn leaf tissue containing single and pyramided Bt genes

    Science.gov (United States)

    Larval survival of Cry1F-susceptible (FL), -resistant (PR and Cry1F-RR), and -heterozygous (FL x PR and Cry1F-RS) populations of the fall armyworm, Spodoptera frugiperda (J.E. Smith) to purified Cry1F protein and corn leaf tissue of seven Bacillus thuringiensis (Bt) corn hybrids and five non-Bt corn...

  8. Protein binding prodrugs : Synthesis and protein binding studies of didanonsine derivates

    OpenAIRE

    Olberg, Dag Erlend

    2004-01-01

    A novel series of 5 -O-ester prodrugs of the anti-HIV drug 2 ,3 -dideoxyinosine (ddI,didanosine) were synthesized for the purpose of increasing protein binding. Hope was that these derivates would exhibit superior pharmacodynamic and pharmacokinetic properties against HIV-infection than the parent drug, didanosine. Ten compounds were synthesized, five fatty acid derivates and five dicarboxylic acid monoester derivates. The fatty acid- and dicarboxylic acid derivates had the sam...

  9. The omega-hydroxlyation of lauric acid: oxidation of 12-hydroxlauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase.

    Science.gov (United States)

    Shet, M s; Fisher, C W; Holmans, P L; Estabrook, R W

    1996-06-01

    The recombinant fusion protein rF450[mRat4Al/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b5, and a 20-fold excess of purified NADPH-P450 reductase, catalyzes the omega- oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with a Ki of about 1.9 microM. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described. Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps, b5, and purified NADPH-P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated.

  10. Pharmacological properties and pathophysiological significance of a Kunitz-type protease inhibitor (Rusvikunin-II) and its protein complex (Rusvikunin complex) purified from Daboia russelii russelii venom.

    Science.gov (United States)

    Mukherjee, Ashis K; Mackessy, Stephen P

    2014-10-01

    A 7.1 kDa basic peptide (Rusvikunin-II) was purified from a previously described protein complex (Rusvikunin complex, consists of Rusvikunin and Rusvikunin-II) of Daboia russelii russelii venom. The N-terminal sequence of Rusvikunin-II was found to be blocked, but peptide mass fingerprinting analysis indicated its identity as Kunitz-type basic protease inhibitor 2, previously reported from Russell's Viper venom. A tryptic peptide sequence of Rusvikunin-II containing the N-terminal sequence HDRPTFCNLFPESGR demonstrated significant sequence homology to venom basic protease inhibitors, Kunitz-type protease inhibitors and trypsin inhibitors. The secondary structure of Rusvikunin-II was dominated by β-sheets (60.4%), followed by random coil (38.2%), whereas α-helix (1.4%) contributes the least to its secondary structure. Both Rusvikunin-II and the Rusvikunin complex demonstrated dose-dependent anticoagulant activity; however, the anticoagulant potency of latter was found to be higher. Both inhibited the amidolytic activity of trypsin > plasmin > FXa, fibrinogen clotting activity of thrombin, and, to a lesser extent, the prothrombin activation property of FXa; however, the inhibitory effect of the Rusvikunin complex was more pronounced. Neither Rusvikunin-II nor Rusvikunin complex inhibited the amidolytic activity of chymotrypsin and thrombin. Rusvikunin-II at 10 μg/ml was not cytotoxic to Colo-205, MCF-7 or 3T3 cancer cells; conversely, Rusvikunin complex showed ∼30% reduction of MCF-7 cells under identical experimental conditions. Rusvikunin-II (5.0 mg/kg body weight, i.p. injection) was not lethal to mice or House Geckos; nevertheless, it showed in vivo anticoagulant action in mice. However, the Rusvikunin complex (at 5.0 mg/kg) was toxic to NSA mice, but not to House Geckos, suggesting it has prey-specific toxicity. Rusvikunin complex-treated mice exhibited dyspnea and hind-limb paresis prior to death. The present study indicates that the Kunitz

  11. Partial characterization of hog renin purified by affinity chromatography.

    Science.gov (United States)

    Devaux, C; Ménard, J; Sicard, P; Corvol, P

    1976-05-01

    A method has been set up to purify renin on a large scale by affinity chromatography using Pepstatin, a potent inhibitor of renin, as a ligand. Pepstatin was covalently coupled to Sepharose via six different spacer 'arms'. The Sepharose-hexamethylenediamino-Pepstatin appeared to be the better derivative for renin purification even at a concentration as low as 160 nmol of Pepstatin/ml of moist gel. Renin was extracted from 100 kg of hog kidneys and semi-purified by ammonium sulfate precipitations and chromatography on DEAE-cellulose. The active fraction (48.5 g of proteins) was applied on a 500-ml affinity column. Renin was eluted in the starting buffer containing 6 M urea. Renin was purified 120-fold by the affinity chromatography step with a 79% recovery. Physico-chemical characterization of highly purified renin was performed. Isoelectrofocusing on a pH gradient from 3 to 6 showed a major peak with an isoelectric point (pI) of 4.95 and a minor peak (pI = 4.70). Polyacrylamide gel electrophoresis, pH 7.8, at different gel concentrations, showed a single peak of renin activity which was found in the major protein band. Molecular size estimated on agarose-acrylamide gel filtration was 40 000. All these physical parameters were similar before and after purification.

  12. A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects.

    Science.gov (United States)

    Omura, Kazunobu; Hitosugi, Masahito; Zhu, Xia; Ikeda, Masashi; Maeda, Hiroaki; Tokudome, Shogo

    2005-11-01

    Natto, steamed soybeans fermented by Bacillus subtilis natto, is a traditional Japanese food. We derived a purified protein layer, called NKCP as a trade mark, from B. subtilis natto fermentation. In the present study, we examined the fibrinolytic and antithrombotic effects of NKCP and identified its active component to clarify the fibrinolytic effect of NKCP observed in preliminary clinical trials previously. The active component of NKCP was identified as a 34-kilodalton protein designated bacillopeptidase F. NKCP showed direct degradation of artificial blood clot in saline. The protease activity, accounting for the fibrinolytic effect of NKCP, was examined with a chromogenic substrate for plasmin. Dose-dependent prolongations of both prothrombin time and active partial thromboplastin time were observed in rats with intra-duodenum administration of NKCP. Our in vitro and in vivo studies suggest that NKCP has both a fibrinolytic effect and an antithrombotic effect similar to heparin. Because NKCP is derived from food and has safety data demonstrated by previous animal experiments and preliminary clinical trials, NKCP is considered as safe for clinical use.

  13. MULTIFRACTAL ANALYSIS OF PROTEIN AGGREGATES TO DERIVE PROTEIN-SPECIFIC SIGNATURE

    Directory of Open Access Journals (Sweden)

    Hrishikesh Mishra, Tapobrata Lahiri*

    2010-11-01

    Full Text Available Deriving a property of a protein that is unique to it has well known significance since the study on ab initio model based derivation of protein structure where uniqueness of protein sequence is taken as the source of specificity of protein structure. In this direction, Heat denatured protein aggregates (HDPA of proteins were studied with an objective to derive some multi-fractal markers specific to constituent protein that may be further utilized to extract information of the seed protein. Since Ordinary microscopic images of aggregates were analyzed to extract Intensity Level-based Multifractal Dimension (ILMFD features. ILMFD features include four different features, perimeter fractal dimension (ILMFDP, perimeter-area relationship (ILMFDPAR, Area fractal dimension (ILMFDA and Perimeter-area fractal dimension (ILMFDPA that were calculated using fractal computations considering perimeter, and area of aggregate images. Feed forward backpropagation network was used to classify the proteins using different ILMFD parameters. It was found that ILMFD features could discriminate the proteins used in our study, that points to their potential to serve as unique property or marker of a protein. Further to validate the results, the outputs from ANN were clustered, and the outputs clustered in the largest cluster were found to significantly improve the result in class decision given by ANN.

  14. Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools.

    Science.gov (United States)

    Rajendra, Yashas; Balasubramanian, Sowmya; Peery, Robert B; Swartling, James R; McCracken, Neil A; Norris, Dawn L; Frye, Christopher C; Barnard, Gavin C

    2017-03-01

    Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534-540, 2017. © 2017 American Institute of Chemical Engineers.

  15. Identification of a novel hypocholesterolemic protein, major royal jelly protein 1, derived from royal jelly.

    Directory of Open Access Journals (Sweden)

    Yuri Kashima

    Full Text Available Royal jelly (RJ intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1, MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1 mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine β-sitosterol in rats.

  16. Critical epitopes in the nucleocapsid protein of SFTS virus recognized by a panel of SFTS patients derived human monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Li Yu

    Full Text Available BACKGROUND: SFTS virus (SFTSV is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs recognized the nucleocapsid (N protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. CONCLUSIONS/SIGNIFICANCE: The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.

  17. Generation of protein-derived redox cofactors by posttranslational modification.

    Science.gov (United States)

    Davidson, Victor L

    2011-01-01

    Redox enzymes which catalyze the oxidation and reduction of substrates are ubiquitous in nature. These enzymes typically possess exogenous cofactors to allow them to perform catalytic functions which cannot be accomplished using only amino acid residues. It is now evident that nature also employs an alternative strategy of generating catalytic and redox-active sites in proteins by posttranslational modification of amino acid residues. This review describes the structures and functions of several of these protein-derived cofactors and the diverse mechanisms of posttranslational modification through which they are generated.

  18. Comparative immunoblot analysis with 10 different, partially overlapping recombinant fusion proteins derived from 5 different cytomegalovirus proteins

    NARCIS (Netherlands)

    van Zanten, J.; LAZZAROTTO, T; CAMPISI, B; VORNHAGEN, R; JAHN, G; LANDINI, MP; The, T. Hauw

    Ten fusion proteins derived from five various CMV encoded proteins were used for the detection of specific antibody response by immunoblot technique in sera from renal transplant recipients. The fusion proteins were derived from the following CMV specific proteins: the assembly protein ppUL80a with

  19. Enamel matrix protein derivatives: role in periodontal regeneration

    Directory of Open Access Journals (Sweden)

    Rathva VJ

    2011-12-01

    Full Text Available Vandana J RathvaDepartment of Periodontics, KM Shah Dental College and Hospital, Sumandeep University, Gujarat, IndiaAbstract: The role of regenerative periodontal therapy is the reconstitution of lost periodontal structures, ie, new formation of root cementum, periodontal ligament, and alveolar bone. The outcome of basic research has pointed to the important role of enamel matrix protein derivative (EMD in periodontal wound healing. Histologic results from animal and human studies have shown that treatment with EMD promotes periodontal regeneration. Moreover, clinical studies have indicated that treatment with EMD positively influences periodontal wound healing in humans. The goal of this paper is to review the existing literature on EMD.Keywords: enamel matrix protein derivative, Emdogain®, periodontal regeneration

  20. Purified water quality study

    Energy Technology Data Exchange (ETDEWEB)

    Spinka, H.; Jackowski, P.

    2000-04-03

    Argonne National Laboratory (HEP) is examining the use of purified water for the detection medium in cosmic ray sensors. These sensors are to be deployed in a remote location in Argentina. The purpose of this study is to provide information and preliminary analysis of available water treatment options and associated costs. This information, along with the technical requirements of the sensors, will allow the project team to determine the required water quality to meet the overall project goals.

  1. Poultry feed based on protein hydrolysate derived from chrome-tanned leather solid waste: creating value from waste.

    Science.gov (United States)

    Chaudhary, Rubina; Pati, Anupama

    2016-04-01

    Leather industry generates huge amount of chrome-containing leather solid waste which creates major environment problems to tanners worldwide. Chrome-tanned leather solid waste is primarily chromium complex of collagen protein. The presence of chromium limits its protein application in animal feed industry. The purified protein hydrolysate with zero chromium could be used in poultry feed. In this study, an attempt has been made to assess performance of poultry with purified protein hydrolysate as a feed derived from chrome-tanned leather waste as partial replacement of soyabean meal as a sole source of protein for growing broiler chickens. Growth study was conducted to evaluate the effect of feeding protein hydrolysate on performance and physiochemical characteristics of meat of broiler chickens. Two experimental diets containing various levels of protein hydrolysate (EI-20 % and EII-30 %) were evaluated. The comparative study was performed as control with soyabean meal. Daily feed intake, body weight gain and feed conversion ratio were measured from day 8 to day 35. At the end of the study, birds were randomly selected and slaughtered to evaluate for physiochemical characteristics of meat. Diet had significant effects on feed intake and body weight gain. Birds fed with 20 and 30 % protein hydrolysate consumed 9.5 and 17.5 % higher amount of feed and gained 6.5 and 16.6 % higher than soyabean meal-fed birds. The current study produced evidence that protein hydrolysate can replace up to 75 % of soyabean meal in broiler diets without affecting either growth performance or meat characteristics.

  2. Human cord blood derived immature basophils show dual characteristics, expressing both basophil and eosinophil associated proteins.

    Directory of Open Access Journals (Sweden)

    Jeanette Grundström

    Full Text Available Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95% and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. β-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP, charcot leyden crystal (CLC, eosinophil cationic protein (ECP, eosinophil derived neurotoxin (EDN and eosinophil peroxidase (EPO. Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that

  3. Human cord blood derived immature basophils show dual characteristics, expressing both basophil and eosinophil associated proteins.

    Science.gov (United States)

    Grundström, Jeanette; Reimer, Jenny M; Magnusson, Sofia E; Nilsson, Gunnar; Wernersson, Sara; Hellman, Lars

    2012-01-01

    Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. β-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and

  4. Raising purity of adipose tissue-derived stem cells by purifying adipose tissue in the laboratory%纯化取材脂肪提高实验中干细胞培养的纯度

    Institute of Scientific and Technical Information of China (English)

    宋小飞; 傅强

    2011-01-01

    BACKGROUND: In the past, only vascular tissues on fat surface were removed during the primary culture of adiposetissue-derived stem cells (ADSCs) reported in most literatures. However, blood vessels within the fat tissues, especially thelymph nodes around the blood vessels removed little.OBJECTIVE: To investigate the feasibility of raising purity of cultured ADSCs in the laboratory through purifying adipose tissues.METHODS: Adipose tissues derived from rat inguinal groove were divided into two parts: one for purifying, rejecting superficialblood vessel, skins and muscular tissues, rejecting blood vessel inside and surrounding elliptic nodal tissues; the other one onlyfor rejecting superficial blood vessels and possible adherent skins and muscular tissues outside. Primary culture of ADSCs wasperformed.RESULTS AND CONCLUSION: Elliptic nodal tissues stained by hematoxylin-eosin were proved to be lymphatic tissues undermicroscope. The cells cultured from purified adipose tissues were uniformity. Cell photos, immunofluorescence and flowcytometry results can prove that the cell's purity from the purified adipose tissues was higher than that from the unpurifiedadipose tissues. The purity of primary cultured ADSCs can be improved by purifying adipose tissues in the laboratory.%背景:在以往脂肪干细胞原代培养的文献中大多只是剔除脂肪表面的血管组织,而对于脂肪内部的血管,尤其是血管周围淋巴结的剔除很少有文献报道.目的:探讨通过脂肪纯化来提高培养干细胞纯度的可行性.方法:将取材自同一大鼠腹股沟处的脂肪组织分成两份,一份给予纯化处理,剔除表面的血管、皮肤和肌肉组织的同时一并剔除组织内血管及其周围结节样组织.而另一份仅剔除表面血管及可能黏附的皮肤和肌肉组织,分别对两份脂肪行干细胞原代培养.结果与结论:经苏木精-伊红染色显微镜下观察,证实血管周围结节样组织为淋巴结.纯化脂

  5. A mass spectrometric-derived cell surface protein atlas.

    Science.gov (United States)

    Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anetta; Van Eyk, Jennifer E; Bourquin, Jean-Pierre; Aebersold, Ruedi; Boheler, Kenneth R; Zandstra, Peter; Wollscheid, Bernd

    2015-01-01

    Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

  6. A mass spectrometric-derived cell surface protein atlas.

    Directory of Open Access Journals (Sweden)

    Damaris Bausch-Fluck

    Full Text Available Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa. The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

  7. Bacteriophages and Phage-Derived Proteins – Application Approaches

    Science.gov (United States)

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

    2015-01-01

    Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes – peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases – that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general. PMID:25666799

  8. Bacteriophages and phage-derived proteins--application approaches.

    Science.gov (United States)

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

    2015-01-01

    Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes - peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases - that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general.

  9. Extraction and downstream processing of plant-derived recombinant proteins.

    Science.gov (United States)

    Buyel, J F; Twyman, R M; Fischer, R

    2015-11-01

    Plants offer the tantalizing prospect of low-cost automated manufacturing processes for biopharmaceutical proteins, but several challenges must be addressed before such goals are realized and the most significant hurdles are found during downstream processing (DSP). In contrast to the standardized microbial and mammalian cell platforms embraced by the biopharmaceutical industry, there are many different plant-based expression systems vying for attention, and those with the greatest potential to provide inexpensive biopharmaceuticals are also the ones with the most significant drawbacks in terms of DSP. This is because the most scalable plant systems are based on the expression of intracellular proteins in whole plants. The plant tissue must therefore be disrupted to extract the product, challenging the initial DSP steps with an unusually high load of both particulate and soluble contaminants. DSP platform technologies can accelerate and simplify process development, including centrifugation, filtration, flocculation, and integrated methods that combine solid-liquid separation, purification and concentration, such as aqueous two-phase separation systems. Protein tags can also facilitate these DSP steps, but they are difficult to transfer to a commercial environment and more generic, flexible and scalable strategies to separate target and host cell proteins are preferable, such as membrane technologies and heat/pH precipitation. In this context, clarified plant extracts behave similarly to the feed stream from microbes or mammalian cells and the corresponding purification methods can be applied, as long as they are adapted for plant-specific soluble contaminants such as the superabundant protein RuBisCO. Plant-derived pharmaceutical proteins cannot yet compete directly with established platforms but they are beginning to penetrate niche markets that allow the beneficial properties of plants to be exploited, such as the ability to produce 'biobetters' with tailored

  10. Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin

    Directory of Open Access Journals (Sweden)

    Renata Angeli

    2009-01-01

    Full Text Available A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A and Cratylia mollis (Cramoll 1 and Cramoll 1,4 did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.

  11. A Purified Recombinant Lipopeptide as Adjuvant for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Ying-Chyi Song

    2014-01-01

    Full Text Available Synthetic lipopeptides have been widely used as vaccine adjuvants to enhance immune responses. The present study demonstrated that the tryptic N-terminal fragment of the lipoprotein rlipo-D1E3 (lipo-Nter induces superior antitumor effects compared to a synthetic lipopeptide. The lipo-Nter was purified and formulated with protein or peptide vaccines to determine if lipo-Nter could be used as a novel adjuvant and could induce antitumor immunity in a cervical cancer model. Purified lipo-Nter activated the maturation of bone marrow-derived dendritic cells (BM-DCs, leading to the secretion of TNF-α through TLR2/6 but not TLR1/2. A recombinant mutant HPV16 E7 (rE7m protein was mixed with lipo-Nter to immunize the mice; the anti-E7 antibody titers were increased, and the T helper cells were skewed toward the Th1 fate (increased IL-2 and decreased IL-5 secretion. Single-dose injection of rE7m and lipo-Nter inhibited tumor growth, but the injection of rE7m alone did not. Accordingly, lipo-Nter also enhanced the antitumor immunity of the E7-derived peptide but not the synthetic lipopeptide (Pam3CSK4. We demonstrated that the lipo-Nter of a bacterial-derived recombinant lipoprotein is a novel adjuvant that could be used for the development of a new generation of vaccines.

  12. Effect of purified β-glucans derived from Laminaria digitata, Laminaria hyperborea and Saccharomyces cerevisiae on piglet performance, selected bacterial populations, volatile fatty acids and pro-inflammatory cytokines in the gastrointestinal tract of pigs.

    Science.gov (United States)

    Sweeney, T; Collins, C B; Reilly, P; Pierce, K M; Ryan, M; O'Doherty, J V

    2012-10-01

    β-Glucans have been identified as natural biomolecules with immunomodulatory activity. The first objective of the present study was to compare the effects of purified β-glucans derived from Laminaria digitata, L. hyperborea and Saccharomyces cerevisiae on piglet performance, selected bacterial populations and intestinal volatile fatty acid (VFA) production. The second aim was to compare the gene expression profiles of the markers of pro- and anti-inflammation in both unchallenged and lipopolysaccharide (LPS)-challenged ileal and colonic tissues. β-Glucans were included at 250 mg/kg in the diets. The β-glucans derived from L. hyperborea, L. digitata and S. cerevisiae all reduced the Enterobacteriaceae population (P0·05) in the ileum and colon. There was a significant interaction between gastrointestinal region and β-glucan source in the expression of cytokine markers, IL-1α (yeast sources reduce Enterobacteriaceae counts and pro-inflammatory markers in the colon, though the mechanisms of action may be different between the soluble and insoluble fibre sources.

  13. Protein interactions with nanoporous sol-gel derived bioactive glasses.

    Science.gov (United States)

    Lin, Sen; Van den Bergh, Wouter; Baker, Simon; Jones, Julian R

    2011-10-01

    Sol-gel derived bioactive glasses are excellent candidates for bone regenerative implant materials as they bond with bone, stimulate bone growth and degrade in the body. Their interactions with proteins are critical to understanding their performance after implantation. This study focuses on the interactions between fibrinogen and sol-gel glass particles of the 70S30C (70 mol.% SiO(2), 30 mol.% CaO composition). Sol-gel silica and melt-derived Bioglass® were also used for comparison. Fibrinogen penetration into the nanoporous glasses was observed by live tracking the fluorescent-labelled fibrinogen with confocal microscopy. The effect of pore size on protein penetration was investigated. Nanoporous networks with modal pore diameters larger than 6 nm were accessible to fibrinogen. When the modal nanopore diameter was decreased to 2 nm or less, the penetration of fibrinogen was inhibited. The surface properties of the glasses, which can be modulated by media pH, glass composition and final stabilisation temperature in the sol-gel process, have effects on fibrinogen adsorption via long-range Coulombic forces before the adsorption and via short-range interactions such as hydrogen bonding after the adsorption.

  14. Natural Air Purifier

    Science.gov (United States)

    1993-01-01

    NASA environmental research has led to a plant-based air filtering system. Dr. B.C. Wolverton, a former NASA engineer who developed a biological filtering system for space life support, served as a consultant to Terra Firma Environmental. The company is marketing the BioFilter, a natural air purifier that combines activated carbon and other filter media with living plants and microorganisms. The filter material traps and holds indoor pollutants; plant roots and microorganisms then convert the pollutants into food for the plant. Most non-flowering house plants will work. After pollutants have been removed, the cleansed air is returned to the room through slits in the planter. Terra Firma is currently developing a filter that will also disinfect the air.

  15. Improved "optical highlighter" probes derived from discosoma red fluorescent protein.

    Science.gov (United States)

    Robinson, Lisbeth C; Marchant, Jonathan S

    2005-02-01

    The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda highlighter" probe for tracking live cells, organelles, and fusion proteins. This color change results from selective bleaching of the "mature" red-emitting species of DsRed and an enhancement of emission from the "immature" green species, likely caused by dequenching of fluorescence resonance energy transfer occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more vivid, and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for fluorescence resonance energy transfer-based probes.

  16. Milk-derived proteins and peptides in clinical trials

    Directory of Open Access Journals (Sweden)

    Jolanta Artym

    2013-08-01

    Full Text Available Clinical trials are reviewed, involving proteins and peptides derived from milk (predominantly bovine, with the exception of lactoferrin, which will be the subject of another article. The most explored milk fraction is α-lactalbumin (LA, which is often applied with glycomacropeptide (GMP – a casein degradation product. These milk constituents are used in health-promoting infant and adult formulae as well as in a modified form (HAMLET to treat cancer. Lactoperoxidase (LCP is used as an additive to mouth hygiene products and as a salivary substitute. Casein derivatives are applied, in addition, in the dry mouth syndrome. On the other hand, casein hydrolysates, containing active tripeptides, found application in hypertension and in type 2 diabetes. Lysozyme is routinely used for food conservation and in pharmaceutical products. It was successfully used in premature infants with concomitant diseases to improve health parameters. When used as prophylaxis in patients with scheduled surgery, it significantly reduced the incidence of hepatitis resulting from blood transfusion. Lysozyme was also used in infected children as an antimicrobial agent showing synergistic effects in combination with different antibiotics. Proline-rich polypeptide (PRP was introduced to therapy of Alzheimer’s disease patients. The therapeutic value of PRP was proved in several clinical trials and supported by studies on its mechanism of action. Concentrated immunoglobulin preparations from colostrum and milk of hyperimmunized cows showed efficacy in prevention of infections by bacteria, viruses and protozoa. A nutrition formula with milk-derived TGF-β2 (Modulen IBD® found application in treatment of pediatric Crohn’s disease. In conclusion, the preparations containing milk-derived products are safe and effective measures in prevention and treatment of infections as well as autoimmune and neoplastic diseases.

  17. An Improved Method for Purifying Cry1Ac Protein from Bacillus thuringiensis%一种纯化苏云金杆菌Cry1Ac蛋白的方法

    Institute of Scientific and Technical Information of China (English)

    王发祥; 刘永乐; 丁学知; 夏立秋

    2011-01-01

    The research of structure and function of Cry proteins was greatly restricted by their purification method. Therefore, a method that purifying Bacillus thuringiensis CrylAc protoxin by using isoelectric precipitation and purifying Bacillus thuringiensis CrylAc activated toxin by using molecular sieve chromatography was proposed. The results showed that the CrylAc protoxin had good effects of precipitation and purification when Ph value was 5.05; activating the purified protoxin to conduct molecular sieve chromatography, high concentration of CrylAc activated toxin could be obtained from the maximum peak of chromatography. This improved method not only has good purification effect, but also contribute a renovation to the purification method of Cry protein, which is significant for promoting researches on structure and function of Cry protein.%Cry蛋白的纯化技术在很大程度上制约了其结构与功能的研究.为此,提出了一种以等电点沉淀方法纯化苏云金杆菌Cry1Ac的原毒素,用分子筛层析纯化其活性毒素的方法.结果表明:Cry1Ac的原毒素在pH值5.05条件下沉淀纯化效果好;以纯化的原毒素激活进行分子筛层析,收集其最大峰便可获得较高浓度的活性毒素;该方法不仅纯化效果好,而且省去层析后的浓缩步骤,是Cry蛋白的纯化方法研究中的一次革新,对推动Cry蛋白的结构和功能研究具有重要的意义.

  18. INTERACTION OF ALDEHYDES DERIVED FROM LIPID PEROXIDATION AND MEMBRANE PROTEINS.

    Directory of Open Access Journals (Sweden)

    Stefania ePizzimenti

    2013-09-01

    Full Text Available A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

  19. Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics.

    Directory of Open Access Journals (Sweden)

    Serena Nicolai

    Full Text Available The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER known as transcription coupled repair (TCR. CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

  20. Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB) Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics.

    Science.gov (United States)

    Nicolai, Serena; Filippi, Silvia; Caputo, Manuela; Cipak, Lubos; Gregan, Juraj; Ammerer, Gustav; Frontini, Mattia; Willems, Daniela; Prantera, Giorgio; Balajee, Adayabalam S; Proietti-De-Santis, Luca

    2015-01-01

    The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER) known as transcription coupled repair (TCR). CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP) technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

  1. Monoaminylation of Fibrinogen and Glia-Derived Proteins: Indication for Similar Mechanisms in Posttranslational Protein Modification in Blood and Brain.

    Science.gov (United States)

    Hummerich, René; Costina, Victor; Findeisen, Peter; Schloss, Patrick

    2015-07-15

    Distinct proteins have been demonstrated to be posttranslationally modified by covalent transamidation of serotonin (5-hydropxytryptamin) to glutamine residues of the target proteins. This process is mediated by transglutaminase (TGase) and has been termed "serotonylation." It has also been shown that other biogenic amines, including the neurotransmitters dopamine and norepinephrine, can substitute for serotonin, implying a more general mechanism of "monoaminylation" for this kind of protein modification. Here we transamidated the autofluorescent monoamine monodansylcadaverine (MDC) to purified plasma fibrinogen and to proteins from a primary glia cell culture. Electrophoretic separation of MDC-conjugated proteins followed by mass spectrometry identified three fibrinogen subunits (Aα, Bβ, γ), a homomeric Aα2 dimer, and adducts of >250 kDa molecular weight, as well as several glial proteins. TGase-mediated MDC incorporation was strongly reduced by serotonin, underlining the general mechanism of monoaminylation.

  2. Solubility of a cruciferin-rich protein product purified from rapeseed pressed cake (Brassica napus L.) by an aqueous processing method

    DEFF Research Database (Denmark)

    Rehder, Alina; Sulewska, Anna Maria; Markedal, Keld Ejdrup

    2017-01-01

    in 50 mm phosphate buffer, pH 8.0 with varying NaCl concentration (0-500 mm). Unexpectedly, a salting-out effect was observed of the globulin proteins, as 15.9 ± 0.6% protein was in solution at 500 mm added NaCl, whereas 21.5 ± 1.1% was solubilised without added NaCl; whether the observed effects...

  3. Device for purifying drilling mud

    Energy Technology Data Exchange (ETDEWEB)

    Surkov, V.T.; Dorosh, M.M.; Khariv, I.Yu.; Makedonov, N.I.

    1982-01-01

    A device is proposed for purifying drilling mud which includes a dynamic filter made in the form of a spiral-shaped tube with input and output sleeves, and a container for purified solution with outlet sleeve. It is distinguished by the fact that in order to simplify the design, the spiral-shaped tube is perforated from the inside and is installed in the container for the purified solution.

  4. Application of Near-Infrared and Fourier Transform Infrared Spectroscopy in the Characterization of Ligand-Induced Conformation Changes in Folate Binding Protein Purified from Bovine Milk

    DEFF Research Database (Denmark)

    Bruun, Susanne Wrang; Holm, Jan; Hansen, Steen Ingemann

    2006-01-01

    Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a beta-sheet to alpha-helix transition upon ligation in acetate...... indicated similar changes at the two pH values. Therefore, we suggest that FT-IR and NIR spectroscopy may complement each other, such that the two techniques in combination may give information on all three types of protein conformational changes. While the secondary structure changes are revealed by FT...

  5. Actin Immobilization on Chitin for Purifying Myosin II: A Laboratory Exercise That Integrates Concepts of Molecular Cell Biology and Protein Chemistry

    Science.gov (United States)

    de Souza, Marcelle Gomes; Grossi, Andre Luiz; Pereira, Elisangela Lima Bastos; da Cruz, Carolina Oliveira; Mendes, Fernanda Machado; Cameron, Luiz Claudio; Paiva, Carmen Lucia Antao

    2008-01-01

    This article presents our experience on teaching biochemical sciences through an innovative approach that integrates concepts of molecular cell biology and protein chemistry. This original laboratory exercise is based on the preparation of an affinity chromatography column containing F-actin molecules immobilized on chitin particles for purifying…

  6. Application of Near-Infrared and Fourier Transform Infrared Spectroscopy in the Characterization of Ligand-Induced Conformation Changes in Folate Binding Protein Purified from Bovine Milk

    DEFF Research Database (Denmark)

    Bruun, Susanne Wrang; Holm, Jan; Hansen, Steen Ingemann;

    2006-01-01

    Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a beta-sheet to alpha-helix transition upon ligation in acetate...

  7. Actin Immobilization on Chitin for Purifying Myosin II: A Laboratory Exercise That Integrates Concepts of Molecular Cell Biology and Protein Chemistry

    Science.gov (United States)

    de Souza, Marcelle Gomes; Grossi, Andre Luiz; Pereira, Elisangela Lima Bastos; da Cruz, Carolina Oliveira; Mendes, Fernanda Machado; Cameron, Luiz Claudio; Paiva, Carmen Lucia Antao

    2008-01-01

    This article presents our experience on teaching biochemical sciences through an innovative approach that integrates concepts of molecular cell biology and protein chemistry. This original laboratory exercise is based on the preparation of an affinity chromatography column containing F-actin molecules immobilized on chitin particles for purifying…

  8. Truncation Derivatives of the S-Layer Protein of Sporosarcina ureae ATCC 13881 (SslA): Towards Elucidation of the Protein Domain Responsible for Self-Assembly.

    Science.gov (United States)

    Varga, Melinda

    2016-08-24

    The cell surface of Sporosarcina ureae ATCC 13881 is covered by an S-layer (SslA) consisting of identical protein subunits that assemble into lattices exhibiting square symmetry. In this work the self-assembly properties of the recombinant SslA were characterised with an emphasis on the identification of protein regions responsible for self-assembly. To this end, recombinant mature SslA (aa 31-1097) and three SslA truncation derivatives (one N-terminal, one C-terminal and one CN-terminal) were produced in a heterologous expression system, isolated, purified and their properties analysed by in vitro recrystallisation experiments on a functionalised silicon wafer. As a result, recombinant mature SslA self-assembled into crystalline monolayers with lattices resembling the one of the wild-type SslA. The study identifies the central protein domain consisting of amino acids 341-925 self-sufficient for self-assembly. Neither the first 341 amino acids nor the last 172 amino acids of the protein sequence are required to self-assemble into lattices.

  9. Truncation Derivatives of the S-Layer Protein of Sporosarcina ureae ATCC 13881 (SslA: Towards Elucidation of the Protein Domain Responsible for Self-Assembly

    Directory of Open Access Journals (Sweden)

    Melinda Varga

    2016-08-01

    Full Text Available The cell surface of Sporosarcina ureae ATCC 13881 is covered by an S-layer (SslA consisting of identical protein subunits that assemble into lattices exhibiting square symmetry. In this work the self-assembly properties of the recombinant SslA were characterised with an emphasis on the identification of protein regions responsible for self-assembly. To this end, recombinant mature SslA (aa 31-1097 and three SslA truncation derivatives (one N-terminal, one C-terminal and one CN-terminal were produced in a heterologous expression system, isolated, purified and their properties analysed by in vitro recrystallisation experiments on a functionalised silicon wafer. As a result, recombinant mature SslA self-assembled into crystalline monolayers with lattices resembling the one of the wild-type SslA. The study identifies the central protein domain consisting of amino acids 341-925 self-sufficient for self-assembly. Neither the first 341 amino acids nor the last 172 amino acids of the protein sequence are required to self-assemble into lattices.

  10. AN OVERVIEW ON BLOOD PURIFIER

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    Sabia Chauhan

    2013-09-01

    Full Text Available Blood is a connective tissue which protects us from different problems. Without blood body cannot functions at all and blood doesn’t purify itself. When blood does not purifies itself that times kidney, liver and lymphatic system work together that they help’s to purifiers the blood. Causes which are included in blood impurities are modern life style, junk food, alcohol etc. If the blood becomes impure it causes different problems e.g. acne, rashes, allergic etc. There is not any proper synthetic medication for blood impurities. Only herbal formulations are used for the blood purifier. In this review article we discussed about the market formulations and the different plants which are used in them with their different activities which are helpful in purifies the blood and also protects from other problems.

  11. Studies of the Mu-Opioid Receptor/G-protein Complex Affinity Co-Purified and Membrane Preparations from 7315c Cells

    Science.gov (United States)

    1989-06-09

    stimulation of insulin secretion from islet cells of the pancreas (Katada and Ui, 1979b). The toxin became known as Islet Activating Protein, IAP...reduced 20-fold by using a Millipore CX-30 ultrafiltration device. Phospholipid vesicle formation and receptor/G-p~otein complex reconstitution was...100 ~ GTP. The eluted solution was subjected to ultrafiltration using Millipore CX30 ultrafiltration units to reduce the volume to 1 ml. This

  12. Rational Design, Synthesis and Evaluation of Coumarin Derivatives as Protein-protein Interaction Inhibitors.

    Science.gov (United States)

    De Luca, Laura; Agharbaoui, Fatima E; Gitto, Rosaria; Buemi, Maria Rosa; Christ, Frauke; Debyser, Zeger; Ferro, Stefania

    2016-09-01

    Herein we describe the design and synthesis of a new series of coumarin derivatives searching for novel HIV-1 integrase (IN) allosteric inhibitors. All new obtained compounds were tested in order to evaluate their ability to inhibit the interaction between the HIV-1 IN enzyme and the nuclear protein lens epithelium growth factor LEDGF/p75. A combined approach of docking and molecular dynamic simulations has been applied to clarify the activity of the new compounds. Specifically, the binding free energies by using the method of molecular mechanics-generalized Born surface area (MM-GBSA) was calculated, whereas hydrogen bond occupancies were monitored throughout simulations methods.

  13. Monoclonal antibody-glial-derived neurotrophic factor fusion protein penetrates the blood-brain barrier in the mouse.

    Science.gov (United States)

    Zhou, Qing-Hui; Boado, Ruben J; Lu, Jeff Zhiqiang; Hui, Eric Ka-Wai; Pardridge, William M

    2010-04-01

    Glial-derived neurotrophic factor (GDNF) is a potent neuroprotective agent for multiple brain disorders, including Parkinson's disease. However, GDNF drug development is difficult because GDNF does not cross the blood-brain barrier (BBB). To enable future drug development of GDNF in mouse models, the neurotrophin was re-engineered as an IgG fusion protein to enable penetration through the BBB after intravenous administration. The 134-amino acid GDNF was fused to the heavy chain of a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR) designated the cTfRMAb. This antibody undergoes receptor-mediated transport across the BBB and acts as a molecular Trojan horse to ferry the GDNF into mouse brain. The cTfRMAb-GDNF fusion protein was expressed by stably transfected Chinese hamster ovary cells, affinity-purified, and the biochemical identity was confirmed by mouse IgG and GDNF Western blotting. The cTfRMAb-GDNF fusion protein was bifunctional and bound with high affinity to both the GDNF receptor alpha1, ED(50) = 1.7 +/- 0.2 nM, and the mouse TfR, ED(50) = 3.2 +/- 0.3 nM. The cTfRMAb-GDNF fusion protein was rapidly taken up by brain, and the brain uptake was 3.1 +/- 0.2% injected dose/g brain at 60 min after intravenous injection of a 1-mg/kg dose of the fusion protein. Brain capillary depletion analysis showed the majority of the fusion protein was transcytosed across the BBB with penetration into brain parenchyma. The brain uptake results indicate it is possible to achieve therapeutic elevations of GDNF in mouse brain with intravenous administration of the cTfRMAb-GDNF fusion protein.

  14. Technological options for the production of health-promoting proteins and peptides derived from milk and colostrum.

    Science.gov (United States)

    Korhonen, H; Pihlanto, A

    2007-01-01

    Milk proteins are known to exert a wide range of nutritional, functional and biological activities. Apart from being a balanced source of valuable amino acids, milk proteins contribute to the consistency and sensory properties of various dairy products. Furthermore, many milk proteins possess specific biological properties which make them potential ingredients of health-promoting foods. These properties are attributed to both native protein molecules and to physiologically active peptides encrypted in the protein molecules. Considerable progress has been made over the last twenty years in technologies aimed at separation, fractionation and isolation in a purified form of many interesting proteins occurring in bovine colostrum and milk. Industrial-scale methods have been developed for native whey proteins such as immunoglobulins, lactoferrin, lactoperoxidase, alpha-lactalbumin and beta-lactoglobulin. Their large-scale manufacture and commercial exploitation is still limited although validated research data about their physiological health benefits is rapidly accumulating. Promising product concepts and novel fields of use have emerged recently, and some of these molecules have already found commercial applications. The same applies to bioactive peptides derived from different milk proteins. Active peptides can be liberated during gastrointestinal digestion or milk fermentation with proteolytic enzymes. Such peptides may exert a number of physiological effects in vivo on the gastrointestinal, cardiovascular, endocrine, immune, nervous and other body systems. However, at present the industrial-scale production of such peptides is limited by a lack of suitable technologies. On the other hand, a number of bioactive peptides have been identified in fermented dairy products, and there are already a few commercial dairy products enriched with blood pressure-reducing milk protein peptides. There is a need to develop methods to optimise the activity of bioactive peptides in

  15. Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1 and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis.

    Directory of Open Access Journals (Sweden)

    Roger Konrad

    Full Text Available Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1 were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa. Furthermore, recombinant OC-1 (rOC-1, the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1% in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products.

  16. Sabin株脊髓灰质炎病毒纯化液蛋白成分分析%Analysis of protein composition of purified poliovirus Sabin strain

    Institute of Scientific and Technical Information of China (English)

    李爱灵; 赵玉秀; 刘小娟; 董圆; 李婉莉; 梁宏阳; 于守智; 岳卓; 赵硕

    2016-01-01

    目的 对本公司研发的Ⅰ、Ⅱ、Ⅲ型Sabin株脊髓灰质炎病毒纯化液进行蛋白成分分析及鉴定.方法 按照疫苗生产工艺,应用NBS篮式生物反应器生产单价脊髓灰质炎病毒原液,经超滤浓缩及分子筛和阴离子交换两步柱层析纯化方法,收集并灭活蛋白峰.透射电镜下观察病毒形态及大小;ELISA方法检测宿主细胞蛋白(host cell protein,HCP)及残留牛血清白蛋白含量;HPLC法分析单价灭活病毒纯化液纯度;液相色谱(liquid chromatography,LC)与电喷雾电离质谱(electrospray ionization-mass spectrometry,ESI-MS)相结合的技术鉴定样品中所含蛋白成分.结果 Ⅰ、Ⅱ、Ⅲ型Sabin株脊髓灰质炎病毒纯化液中可见球形病毒颗粒,直径25~ 30 nm;HCP、残余牛血清白蛋白含量分别低于50 ng/ml、2.5μg/ml;纯度大于95%;单价脊髓灰质炎病毒样品中含有相应血清型的脊髓灰质炎病毒基因组编码蛋白,且相应病毒蛋白成分数据库匹配相对分值在所含蛋白组分中均最高.结论 Ⅰ、Ⅱ、Ⅲ型Sabin株脊髓灰质炎病毒液经纯化后,有效去除了杂质蛋白保留了病毒蛋白组分,单价病毒纯化液病毒蛋白成分明确,纯度较高,为后续该疫苗的研发与生产提供了参考.

  17. Engineering a high-performance, metagenomic-derived novel xylanase with improved soluble protein yield and thermostability.

    Science.gov (United States)

    Qian, Changli; Liu, Ning; Yan, Xing; Wang, Qian; Zhou, Zhihua; Wang, Qianfu

    2015-03-01

    The novel termite gut metagenomic-derived GH11 xylanase gene xyl7 was expressed in Escherichia coli BL21, and the purified XYL7 enzyme exhibited high specific activity (6340U/mg) and broad pH active range of 5.5-10.0. Directed evolution was employed to enhance the thermostability of XYL7; two mutants (XYL7-TC and XYL7-TS) showed a 250-fold increase in half-life at 55°C, with a 10°C increase in optimal temperature compared to that of wild-type XYL7. A truncated enzyme (XYL7-Tr3) acquired by protein engineering showed similar catalytic properties as the wild-type, with a tenfold increase in soluble protein yield by the mutant. The reducing sugar produced by XYL7-TC was about fourfold greater than that produced by their parents when incubated with xylan at 60°C for 4h. The engineered novel xylanase exhibited superior enzymatic performance and showed promise as an excellent candidate for industrial application due to its high specific activity, stability and soluble protein yield.

  18. Steroidogenesis in amlodipine treated purified Leydig cells

    Energy Technology Data Exchange (ETDEWEB)

    Latif, Rabia, E-mail: rabialatif08@hotmail.com [Department of Physiology, Army Medical College, National University of Sciences and Technology, Islamabad (Pakistan); Lodhi, Ghulam Mustafa, E-mail: drmustafa786@gmail.com [Department of Physiology, Wah Medical College, Wah (Pakistan); Hameed, Waqas, E-mail: waqham@hotmail.com [Department of Physiology, Rehman Medical College, Peshawar (Pakistan); Aslam, Muhammad, E-mail: professormaslam@yahoo.com [Department of Physiology, Shifa College of Medicine, Islamabad (Pakistan)

    2012-01-01

    Drugs have been shown to adversely affect male fertility and recently anti-hypertensive drugs were added to the list. The anti-fertility effects of amlodipine, a calcium channel blocker, are well-illustrated in in vivo experiments but lack an in vitro proof. The present study was designed to experimentally elucidate the effects of amlodipine on Leydig cell steroidogenesis and intracellular calcium in vitro. Leydig cells of Sprague–Dawley rats were isolated and purified by Percoll. Cells were incubated for 3 h with/without amlodipine in the presence/absence of LH, dbcAMP, Pregnenolone and 25-Hydroxycholesterol. Cytosolic calcium was measured in purified Leydig cells by fluorometric technique. The results showed significantly reduced (P < 0.05) steroidogenesis and intracellular calcium in amlodipine exposed rats. The site of amlodipine induced steroidogenic inhibition seems to be prior to the formation of Pregnenolone at the level of StAR protein. -- Highlights: ► Inhibition of steroidogenesis in isolated and purified Leydig cells by amlodipine. ► Site of inhibition was before Pregnenolone formation, at the level of StAR protein. ► Inhibition of LH stimulated rise in cytosolic calcium by amlodipine.

  19. Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN). Detection by enzyme-linked immunosorbent assay and purification from normal human urine

    DEFF Research Database (Denmark)

    Reimert, C M; Minuva, U; Kharazmi, A

    1991-01-01

    subsequently raised in rabbits. The anti-EPX-antibodies raised in rabbits showed no reactivity with other proteins in the granule extract. The sandwich ELISA utilized the biotin/avidin amplification system and measured EPX over the range of 60-2000 pg/ml. The intra- and interassay coefficients of variation......Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN) is one of the cationic proteins found in the granules of the human eosinophilic granulocytes. EPX was purified from extracts of granules isolated from blood buffy coat cells of healthy donors. Polyclonal anti-EPX antibodies were...... procedure involving affinity chromatography on heparin Sepharose and size exclusion chromatography on Sephadex G-50 superfine. Extracted EPX and U-EPX had ribonuclease activity and comigrated on agarose electrophoresis. They also showed immunological identity when evaluated with rabbit anti-EPX antibodies...

  20. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

    Science.gov (United States)

    Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

    2011-11-22

    Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41.

  1. Protein dynamics derived from clusters of crystal structures.

    OpenAIRE

    van Aalten, D M; Conn, D A; de Groot, B L; Berendsen, H J; Findlay, J B; Amadei, A

    1997-01-01

    A method is presented to mathematically extract concerted structural transitions in proteins from collections of crystal structures. The "essential dynamics" procedure is used to filter out small-amplitude fluctuations from such a set of structures; the remaining large conformational changes describe motions such as those important for the uptake/release of substrate/ligand and in catalytic reactions. The method is applied to sets of x-ray structures for a number of proteins, and the results ...

  2. Proteomic analysis of purified coronavirus infectious bronchitis virus particles

    Directory of Open Access Journals (Sweden)

    Shu Dingming

    2010-06-01

    Full Text Available Abstract Background Infectious bronchitis virus (IBV is the coronavirus of domestic chickens causing major economic losses to the poultry industry. Because of the complexity of the IBV life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. Toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified IBV particles. Results Apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%, molecular chaperone (18%, macromolcular biosynthesis proteins (17%, cytoskeletal proteins (15%, signal transport proteins (15%, protein degradation (8%, chromosome associated proteins (2%, ribosomal proteins (2%, and other function proteins (3%. Interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, TENP protein, ovalbumin, and scavenger receptor protein. Following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified IBV preparation was verified by western blotting and immunogold labeling detection. Conclusions The results present the first standard proteomic profile of IBV and may facilitate the understanding of the pathogenic mechanisms.

  3. Light-induced protein degradation in human-derived cells.

    Science.gov (United States)

    Sun, Wansheng; Zhang, Wenyao; Zhang, Chao; Mao, Miaowei; Zhao, Yuzheng; Chen, Xianjun; Yang, Yi

    2017-05-27

    Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Enhanced antitumor immunity of nanoliposome-encapsulated heat shock protein 70 peptide complex derived from dendritic tumor fusion cells.

    Science.gov (United States)

    Zhang, Yunfei; Luo, Wen; Wang, Yucai; Chen, Jun; Liu, Yunyan; Zhang, Yong

    2015-06-01

    Tumor-derived heat shock proteins peptide complex (HSP.PC-Tu) has been regarded as a promising antitumor agent. However, inadequate immunogenicity and low bioavailability limit the clinical uses of this agent. In a previous study, we first produced an improved HSP70.PC-based vaccine purified from dendritic cell (DC)-tumor fusion cells (HSP70.PC-Fc) which had increased immunogenicity due to enhanced antigenic tumor peptides compared to HSP70.PC-Tu. In order to increase the bioavailability of HSP70.PC-Fc, the peptide complex was encapsulated with nanoliposomes (NL-HSP70.PC-Fc) in this study. After encapsulation, the tumor immunogenicity was observed using various assays. It was demonstrated that the NL-HSP70.PC-Fc has acceptable stability. The in vivo antitumor immune response was increased with regard to T-cell activation, CTL response and tumor therapy efficiency compared to that of HSP70.PC-Fc. In addition, it was shown that DC maturation was improved by NL-HSP70.PC-Fc, which added to the antitumor immunity. The results obtained for NL-HSP70.PC-Fc, which improved immunogenicity and increases the bioavailability of HSP70.PC, may represent superior heat shock proteins (HSPs)-based tumor vaccines. Such vaccines deserve further investigation and may provide a preclinical rationale to translate findings into early phase trials for patients with breast tumors.

  5. Proteomic analysis of purified Newcastle disease virus particles

    Directory of Open Access Journals (Sweden)

    Ren Xiangpeng

    2012-05-01

    Full Text Available Abstract Background Newcastle disease virus (NDV is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles. Results In addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase and one tumor-associated protein (tumor protein D52. The presence of five selected cellular proteins (i.e., β-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin associated with the purified NDV particles was validated by Western blot or immunogold labeling assays. Conclusions The current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.

  6. Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1996-01-01

    to respond to mycobacterial antigens. The immunological dysfunction leading to reduced mycobacterial-specific cytotoxic activity in healthy HIV-seropositive subjects is caused either by a reduction in the number of antigen-responsive CD4+ T cells (memory) or by an impairment of their ability to respond...

  7. Interpreting meta-analysis according to the adequacy of sample size. An example using isoniazid chemoprophylaxis for tuberculosis in purified protein derivative negative HIV-infected individuals

    DEFF Research Database (Denmark)

    Thorlund, Kristian; Anema, Aranka; Mills, Edward

    2010-01-01

    To illustrate the utility of statistical monitoring boundaries in meta-analysis, and provide a framework in which meta-analysis can be interpreted according to the adequacy of sample size. To propose a simple method for determining how many patients need to be randomized in a future trial before ...

  8. Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

    Directory of Open Access Journals (Sweden)

    Rafał Biedroń

    Full Text Available The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl, causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin and glycoproteins (human apo-transferrin, ovalbumin gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206, scavenger receptors A (CD204 and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system

  9. Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus.

    Science.gov (United States)

    Gao, Feng-Shan; Fang, Qin-Mei; Li, Yun-Gang; Li, Xin-Sheng; Hao, Hui-Fang; Xia, Chun

    2006-10-15

    No experimental system to date is available to identify viral T-cell epitopes in swine. In order to reconstruct the system for identification of short antigenic peptides, the swine SLA-2 gene was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence (G4S)3, using splicing overlap extension-PCR (SOE-PCR). The maltose binding protein (MBP)-SLA-2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/Escherichia coli TB1 system. The purified MBP-SLA-2-(G4S)3-beta(2)m protein was cleaved by factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the SLA-2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD) spectrum. In addition, the refolded SLA-2-(G4S)3-beta(2)m protein was used to bind three nonameric peptides derived from the foot-and-mouth disease virus (FMDV) O subtype VP1. The SLA-2-(G4S)3-beta(2)m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and secondary spectra, respectively. The results indicate that the SLA-2-(G4S)3-beta(2)m was 41.6kDa, and its alpha-helix, beta-sheet, turn, and random coil by CD estimation were 78 aa, 149 aa, 67 aa, and 93 aa, respectively. SLA-2-(G4S)3-beta(2)m protein was able to bind the nonameric peptides derived from the FMDV VP1 region: 26-34 (RRQHTDVSF) and 157-165 (RTLPTSFNY). The experimental system demonstrated that the reconstructed SLA-2-(G4S)3-beta(2)m protein complex can be used to identify nonameric peptides, including T-cell epitopes in swine.

  10. Neuromuscular electrical stimulation prior to presleep protein feeding stimulates the use of protein-derived amino acids for overnight muscle protein synthesis.

    Science.gov (United States)

    Dirks, Marlou L; Groen, Bart B L; Franssen, Rinske; van Kranenburg, Janneau; van Loon, Luc J C

    2017-01-01

    Short periods of muscle disuse result in substantial skeletal muscle atrophy. Recently, we showed that both neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. In this study, we test our hypothesis that NMES can augment the use of presleep protein-derived amino acids for overnight muscle protein synthesis in older men. Twenty healthy, older [69 ± 1 (SE) yr] men were subjected to 24 h of bed rest, starting at 8:00 AM. In the evening, volunteers were subjected to 70-min 1-legged NMES, while the other leg served as nonstimulated control (CON). Immediately following NMES, 40 g of intrinsically l-[1-(13)C]-phenylalanine labeled protein was ingested prior to sleep. Blood samples were taken throughout the night, and muscle biopsies were obtained from both legs in the evening and the following morning (8 h after protein ingestion) to assess dietary protein-derived l-[1-(13)C]-phenylalanine enrichments in myofibrillar protein. Plasma phenylalanine concentrations and plasma l-[1-(13)C]-phenylalanine enrichments increased significantly following protein ingestion and remained elevated for up to 6 h after protein ingestion (P stimulated compared with the control leg (0.0344 ± 0.0019 vs. 0.0297 ± 0.0016 MPE, respectively; P stimulates the use of dietary protein-derived amino acids for overnight muscle protein synthesis in older men. Neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. Here we demonstrate that in older men after a day of bed rest, the application of NMES prior to presleep protein feeding stimulates the use of dietary protein-derived amino acids for overnight muscle protein synthesis by 18% compared with presleep protein feeding only. Copyright © 2017 the American Physiological Society.

  11. Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography.

    Science.gov (United States)

    Robichon, Carine; Luo, Jianying; Causey, Thomas B; Benner, Jack S; Samuelson, James C

    2011-07-01

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

  12. Erythrocyte-derived microparticles supporting activated protein C-mediated regulation of blood coagulation.

    Science.gov (United States)

    Koshiar, Ruzica Livaja; Somajo, Sofia; Norström, Eva; Dahlbäck, Björn

    2014-01-01

    Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte-derived microparticle

  13. Interaction of Berberine derivative with protein POT1 affect telomere function in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Nannan; Chen, Siqi; Ma, Yan; Qiu, Jun; Tan, Jia-Heng; Ou, Tian-Miao; Gu, Lian-Quan; Huang, Zhi-Shu [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou University City, Waihuan East Road 132, Guangzhou 510006 (China); Li, Ding, E-mail: liding@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou University City, Waihuan East Road 132, Guangzhou 510006 (China)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer The protein POT1 plays an important role in telomere protection. Black-Right-Pointing-Pointer Functional POT1 was overexpressed in Escherichia coli for the first time, and purified. Black-Right-Pointing-Pointer Compound Sysu-00692 was found to be the first POT1-binding ligand. Black-Right-Pointing-Pointer Sysu-00692 could interfere with the binding activity of POT1 in vivo. Black-Right-Pointing-Pointer Sysu-00692 had inhibition on telomerase and cell proliferation. -- Abstract: The protein POT1 plays an important role in telomere protection, which is related with telomere elongation and cell immortality. The protein has been recognized as a promising drug target for cancer treatment. In the present study, we cloned, overexpressed in Escherichia coli for the first time, and purified recombinant human POT1. The protein was proved to be active through filter binding assay, FRET and CD experiments. In the initial screening for protein binding ligands using SPR, compound Sysu-00692 was found to bind well with the POT1, which was confirmed with EMSA. Its in vivo activity study showed that compound Sysu-00692 could interfere with the binding between human POT1 and the telomeric DNA through chromatin immunoprecipitation. Besides, the compound showed mild inhibition on telomerase and cell proliferation. As we know, compound Sysu-00692 is the first reported POT1-binding ligand, which could serve as a lead compound for further improvement. This work offered a potentially new approach for drug design for the treatment of cancers.

  14. Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

    Directory of Open Access Journals (Sweden)

    Joseph Minu

    2012-03-01

    Full Text Available Abstract Background The N-terminal proline-rich domain (Zera of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER-derived protein bodies (PBs when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles. Results We analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed. Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer. Conclusions This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of

  15. Antistaphylococcal activity of bacteriophage derived chimeric protein P128

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    Vipra Aradhana A

    2012-03-01

    Full Text Available Abstract Background Bacterial drug resistance is one of the most significant challenges to human health today. In particular, effective antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA are urgently needed. A causal relationship between nasal commensal S. aureus and infection has been reported. Accordingly, elimination of nasal S. aureus reduces the risk of infection. Enzymes that degrade bacterial cell walls show promise as antibacterial agents. Bacteriophage-encoded bacterial cell wall-degrading enzymes exhibit intrinsic bactericidal activity. P128 is a chimeric protein that combines the lethal activity of the phage tail-associated muralytic enzyme of Phage K and the staphylococcal cell wall targeting-domain (SH3b of lysostaphin. Here we report results of in vitro studies evaluating the susceptibility of staphylococcal strains to this novel protein. Results Using the broth microdilution method adapted for lysostaphin, we found that P128 is effective against S. aureus clinical strains including MRSA, methicillin-sensitive S. aureus (MSSA, and a mupirocin-resistant S. aureus. Minimum bactericidal concentrations and minimum inhibitory concentrations of P128 (1-64 μg/mL were similar across the 32 S. aureus strains tested, demonstrating its bactericidal nature. In time-kill assays, P128 reduced colony-forming units by 99.99% within 1 h and inhibited growth up to 24 h. In an assay simulating topical application of P128 to skin or other biological surfaces, P128 hydrogel was efficacious when layered on cells seeded on solid media. P128 hydrogel was lethal to Staphylococci recovered from nares of healthy people and treated without any processing or culturing steps, indicating its in situ efficacy. This methodology used for in vitro assessment of P128 as an agent for eradicating nasal carriage is unique. Conclusions The novel chimeric protein P128 is a staphylococcal cell wall-degrading enzyme under development for

  16. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

    NARCIS (Netherlands)

    Shaner, Nathan C; Campbell, Robert E; Steinbach, Paul A; Giepmans, Ben N G; Palmer, Amy E; Tsien, Roger Y

    2004-01-01

    Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red

  17. Process for purifying geothermal steam

    Science.gov (United States)

    Li, C.T.

    Steam containing hydrogen sulfide is purified and sulfur recovered by passing the steam through a reactor packed with activated carbon in the presence of a stoichiometric amount of oxygen which oxidizes the hydrogen sulfide to elemental sulfur which is adsorbed on the bed. The carbon can be recycled after the sulfur has been recovered by vacuum distillation, inert gas entrainment or solvent extraction. The process is suitable for the purification of steam from geothermal sources which may also contain other noncondensable gases.

  18. Conotoxins Are Purified and Cloned

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ A group of CAS scientists have succeeded in purifying many conotoxins and cloning more than 100 new genes from six species of cone snails living in waters off the coast of the South China Sea, paving the way for the development of new drugs to relieve neuropathic pains. The work has been honored with a first prize from the 2005 Awards for S&T Progress in Shanghai.

  19. Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF.

    Science.gov (United States)

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

    2008-07-01

    Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years.

  20. The future supply of animal-derived protein for human consumption

    NARCIS (Netherlands)

    Boland, M.; Rae, A.; Vereijken, J.; Meuwissen, M.P.M.; Fischer, A.R.H.; Boekel, van M.A.J.S.; Rutherfurd, S.M.; Gruppen, H.; Moughan, P.J.; Hendriks, W.H.

    2013-01-01

    The combined effects of population increase and increasing standards of living in developing countries are expected to create a high demand for animal-derived protein by 2050. New initiatives will be required to produce the necessary quantities of high quality protein. We explore a range of initiati

  1. Protein composition of the occlusion derived virus of Chrysodeixis chalcites nucleopolyhedrovirus

    NARCIS (Netherlands)

    Xu, F.; Ince, I.A.; Boeren, S.; Vlak, J.M.; Oers, van M.M.

    2011-01-01

    Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) is a group II NPV and its genome has 151 predicted open reading frames. In this study, the protein composition of ChchNPV occlusion derived virus (ODV) was determined by LC–MS/MS. Fifty-three proteins were identified in ChchNPV ODV particles. One

  2. 131-Iodine-Labeled Derivatives of the Sonic Hedgehog Protein

    Directory of Open Access Journals (Sweden)

    Jennifer Sims-Mourtada

    2012-01-01

    Full Text Available Activation of hedgehog (HH pathway signaling is observed in many tumors. Due to a feedback loop, the HH receptor Patched (PTCH-1 is overexpressed in tumors with activated HH signaling. Therefore, we sought to radiolabel the PTCH-1 ligand sonic (SHH for detection of cancer cells with canonical HH activity. Receptor binding of 131I-SHH was increased in cell lines with high HH pathway activation. Our findings also show that PTCH-1 receptor expression is decreased upon treatment with HH signaling inhibitors, and receptor binding of 131I-SHH is significantly decreased following treatment with cyclopamine. In vivo imaging and biodistribution studies revealed significant accumulation of 131I-SHH within tumor tissue as compared to normal organs. Tumor-to-muscle ratios were approximately 8 : 1 at 5 hours, while tumor to blood and tumor to bone were 2 : 1 and 5 : 1, respectively. Significant uptake was also observed in liver and gastrointestinal tissue. These studies show that 131I-SHH is capable of in vivo detection of breast tumors with high HH signaling. We further demonstrate that the hedgehog receptor PTCH-1 is downregulated upon treatment with hedgehog inhibitors. Our data suggests that radiolabeled SHH derivatives may provide a method to determine response to SHH-targeted therapies.

  3. Antibodies Directed against a Peptide Epitope of a Klebsiella pneumoniae-Derived Protein Are Present in Ankylosing Spondylitis

    Science.gov (United States)

    Tinazzi, Elisa; Moretta, Francesca; D’Angelo, Salvatore; Olivieri, Ignazio; Lunardi, Claudio

    2017-01-01

    Ankylosing spondylitis (AS) is a chronic inflammatory arthritis of unknown origin. Its autoimmune origin has been suggested but never proven. Several reports have implicated Klebsiella pneumoniae as a triggering or perpetuating factor in AS; however, its role in the disease pathogenesis remains debated. Moreover, despite extensive investigations, a biomarker for AS has not yet been identified. To clarify these issues, we screened a random peptide library with pooled IgGs obtained from 40 patients with AS. A peptide (AS peptide) selected from the library was recognized by serum IgGs from 170 of 200 (85%) patients with AS but not by serum specimens from 100 healthy controls. Interestingly, the AS peptide shows a sequence similarity with several molecules expressed at the fibrocartilaginous sites that are primarily involved in the AS inflammatory process. Moreover, the peptide is highly homologous to a Klebsiella pneumoniae dipeptidase (DPP) protein. The antibody affinity purified against the AS peptide recognizes the autoantigens and the DPP protein. Furthermore, serum IgG antibodies against the Klebsiella DPP121-145 peptide epitope were detected in 190 of 200 patients with AS (95%), 3 of 200 patients with rheumatoid arthritis (1.5%) and only 1 of 100 (1%) patients with psoriatic arthritis. Such reactivity was not detected in healthy control donors. Our results show that antibodies directed against an epitope of a Klebsiella pneumoniae-derived protein are present in nearly all patients with AS. In the absence of serological biomarkers for AS, such antibodies may represent a useful tool in the diagnosis of the disease. PMID:28135336

  4. Synthesis and characterization of novel 2, 2'-bipyrimidine fluorescent derivative for protein binding

    Directory of Open Access Journals (Sweden)

    Padalkar Vikas S

    2011-11-01

    Full Text Available Abstract Background Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility. Results Novel 4, 4'-(1, 1'-(5-(2-methoxyphenoxy-[2, 2'-bipyrimidine]-4, 6-diylbis(1H-pyrazol-3, 1-diyl dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2-methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375 and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry. Conclusion In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.

  5. Bioactive peptides derived from milk proteins and their health beneficial potentials: an update.

    Science.gov (United States)

    Nagpal, Ravinder; Behare, Pradip; Rana, Rajiv; Kumar, Ashwani; Kumar, Manoj; Arora, Sanu; Morotta, Fransesco; Jain, Shalini; Yadav, Hariom

    2011-01-01

    It has been well recognized that dietary proteins provide a rich source of biologically active peptides. Today, milk proteins are considered the most important source of bioactive peptides and an increasing number of bioactive peptides have been identified in milk protein hydrolysates and fermented dairy products. Bioactive peptides derived from milk proteins offer a promising approach for the promotion of health by means of a tailored diet and provide interesting opportunities to the dairy industry for expansion of its field of operation. The potential health benefits of milk protein-derived peptides have been a subject of growing commercial interest in the context of health-promoting functional foods. Hence, these peptides are being incorporated in the form of ingredients in functional and novel foods, dietary supplements and even pharmaceuticals with the purpose of delivering specific health benefits.

  6. Cinnamic Acid and Its Derivatives Inhibit Fructose-Mediated Protein Glycation

    Directory of Open Access Journals (Sweden)

    Sirintorn Yibchok-anun

    2012-02-01

    Full Text Available Cinnamic acid and its derivatives have shown a variety of pharmacologic properties. However, little is known about the antiglycation properties of cinnamic acid and its derivatives. The present study sought to characterize the protein glycation inhibitory activity of cinnamic acid and its derivatives in a bovine serum albumin (BSA/fructose system. The results demonstrated that cinnamic acid and its derivatives significantly inhibited the formation of advanced glycation end products (AGEs by approximately 11.96–63.36% at a concentration of 1 mM. The strongest inhibitory activity against the formation of AGEs was shown by cinnamic acid. Furthermore, cinnamic acid and its derivatives reduced the level of fructosamine, the formation of Nε-(carboxymethyl lysine (CML, and the level of amyloid cross β-structure. Cinnamic acid and its derivatives also prevented oxidative protein damages, including effects on protein carbonyl formation and thiol oxidation of BSA. Our findings may lead to the possibility of using cinnamic acid and its derivatives for preventing AGE-mediated diabetic complications.

  7. Screening of Protein A Resins for Purifying Combinant Antibody%几种Protein A亲和层析填料纯化重组单克隆抗体效果的比较

    Institute of Scientific and Technical Information of China (English)

    何凌冰; 李乐; 邓义熹; 蒙国基; 于玉根

    2015-01-01

    随着表达技术的进步以及发酵技术的优化,细胞发酵上清液中抗体的滴度大幅度提高,必然对下游纯化工艺的处理能力有更高的要求.Protein A亲和层析是分离单克隆抗体的一种标准纯化方法.目前已经有很多种protein A亲和填料,其配体基本上都是大肠杆菌发酵的重组protein A.不同厂家生产的protein A填料有各自不同的基质或键合技术,从而造成填料对抗体亲和力以及对碱耐受性的差异.针对正在研发的WLB303单克隆抗体,比较了6种不同的protein A填料的动态载量,并从洗脱溶液种类和pH两个方面进行单克隆抗体纯化条件的筛选,分析纯化后样品SEC纯度和回收率.综合考虑载量、除杂效果、回收率三个因素,MabSelect填料是最适合本抗体纯化的,SEC纯度可以达到98%,回收率可以达到100%左右.基于这些数据,对MabSelect填料进行了使用寿命试验,200次循环之后载量仅衰减了5%.

  8. Amplified optical transduction of proteins derived from Mo6S9-xIx nanowires

    Institute of Scientific and Technical Information of China (English)

    Nijuan Sun; Martin McMullan; Pagona Papakonstantinou; Dragan Mihailovic; Meixian Li

    2013-01-01

    We demonstrate that Mo6S9-xIx nanowires (MoSI NWs) enable the detection of proteins with cytochrome c as a model protein using UV-vis spectrometry. The association of cytochrome c with the nanowires was verified by scanning electroctron microscopy, X-ray photoelectron, light scattering and micro-FTIR spectroscopies. Our results show that MoSI NWs is a promising nanostructure material for the development of ultrasensitive sensors for detecting proteins. The new MoSI NW derived amplification bioassay is expected to provide a straightforward and effective strategy for protein analysis and biosensor construction.

  9. Characterization of an Extremely Basic Protein Derived from Granulosis Virus Nucleocapsids †

    Science.gov (United States)

    Tweeten, Kathleen A.; Bulla, Lee A.; Consigli, Richard A.

    1980-01-01

    Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex. Images PMID:16789190

  10. S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk.

    Directory of Open Access Journals (Sweden)

    Ruisong Li

    Full Text Available BACKGROUND: Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF, and glial cell line-derived neurotrophic factor (GDNF in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05. In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05. Delivery modes were negatively associated with the concentration of GDNF in human milk. CONCLUSIONS: S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.

  11. Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells.

    Science.gov (United States)

    Noessner, Elfriede; Gastpar, Robert; Milani, Valeria; Brandl, Anna; Hutzler, Peter J S; Kuppner, Maria C; Roos, Miriam; Kremmer, Elisabeth; Asea, Alexzander; Calderwood, Stuart K; Issels, Rolf D

    2002-11-15

    Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr(+)) but not from tyrosinase-negative (HSP70-PC/tyr(-)) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-alpha), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.

  12. Structure-property studies on carbohydrate-derived polymers for use as protein-resistant biomaterials.

    Science.gov (United States)

    Metzke, Mark; Guan, Zhibin

    2008-01-01

    Here we describe structure-property studies on our carbohydrate-derived side-chain ether polymers as protein-resistant biomaterials. A series of side-chain ether polymers, including two polyesters and two polyamides, were prepared by condensation polymerization of monomers derived from simple carbohydrates. The two side-chain permethoxylated polyesters having different stereochemical repeating units demonstrate excellent resistance toward nonspecific protein adsorption as shown by surface plasmon resonance, indicating that the polymer stereochemistry does not have much effect on its protein-resistant properties. The introduction of amide bonds to polymer backbones leads to more pronounced effects. While the polymer degradation stability is significantly enhanced by replacing ester with amide linkages, the protein resistance for the polymer is greatly reduced by introduction of amide bonds. Finally, our results suggest that free hydroxyl and amide groups, while both are hydrogen-bond donors, seem to have different effects on protein resistant properties for polymers. It appears that free amide groups have more detrimental effect on protein resistance than free hydroxyl groups. These results show that the protein-resistant properties of this family of polymers can be tailored by modifying the backbone and side chain functionalities. In combination with the biodegradability and functionalizability, this family of carbohydrate-derived polymers shows promise as versatile biomaterials for biomedical applications.

  13. A machine learning approach for the identification of odorant binding proteins from sequence-derived properties

    Directory of Open Access Journals (Sweden)

    Suganthan PN

    2007-09-01

    Full Text Available Abstract Background Odorant binding proteins (OBPs are believed to shuttle odorants from the environment to the underlying odorant receptors, for which they could potentially serve as odorant presenters. Although several sequence based search methods have been exploited for protein family prediction, less effort has been devoted to the prediction of OBPs from sequence data and this area is more challenging due to poor sequence identity between these proteins. Results In this paper, we propose a new algorithm that uses Regularized Least Squares Classifier (RLSC in conjunction with multiple physicochemical properties of amino acids to predict odorant-binding proteins. The algorithm was applied to the dataset derived from Pfam and GenDiS database and we obtained overall prediction accuracy of 97.7% (94.5% and 98.4% for positive and negative classes respectively. Conclusion Our study suggests that RLSC is potentially useful for predicting the odorant binding proteins from sequence-derived properties irrespective of sequence similarity. Our method predicts 92.8% of 56 odorant binding proteins non-homologous to any protein in the swissprot database and 97.1% of the 414 independent dataset proteins, suggesting the usefulness of RLSC method for facilitating the prediction of odorant binding proteins from sequence information.

  14. Methods for Purifying Enzymes for Mycoremediation

    Science.gov (United States)

    Cullings, Kenneth W. (Inventor); DeSimone, Julia C. (Inventor); Paavola, Chad D. (Inventor)

    2014-01-01

    A process for purifying laccase from an ectomycorrhizal fruiting body is disclosed. The process includes steps of homogenization, sonication, centrifugation, filtration, affinity chromatography, ion exchange chromatography, and gel filtration. Purified laccase can also be separated into isomers.

  15. Evaluation of a portable air purifier.

    OpenAIRE

    Lawrence, J.C.; Lilly, H. A.; Wilkins, M. D.

    1981-01-01

    A portable air purifier significantly reduced mal odour in a small room. If the atmosphere was deliberately contaminated with Serratia marcescens the unit rapidly removed this organism. However, if incorrectly sited, the purifier could disperse organisms into the atmosphere.

  16. Republished: Importance of carcinoma-associated fibroblast-derived proteins in clinical oncology.

    Science.gov (United States)

    Valcz, Gabor; Sipos, Ferenc; Tulassay, Zsolt; Molnar, Bela; Yagi, Yukako

    2015-05-01

    Carcinoma-associated fibroblast (CAF) as prominent cell type of the tumour microenvironment has complex interaction with both the cancer cells and other non-neoplastic surrounding cells. The CAF-derived regulators and extracellular matrix proteins can support cancer progression by providing a protective microenvironment for the cancer cells via reduction of chemotherapy sensitivity. On the other hand, these proteins may act as powerful prognostic markers as well as potential targets of anticancer therapy. In this review, we summarise the clinical importance of the major CAF-derived signals influencing tumour behaviour and determining the outcome of chemotherapy.

  17. Importance of carcinoma-associated fibroblast-derived proteins in clinical oncology.

    Science.gov (United States)

    Valcz, Gabor; Sipos, Ferenc; Tulassay, Zsolt; Molnar, Bela; Yagi, Yukako

    2014-12-01

    Carcinoma-associated fibroblast (CAF) as prominent cell type of the tumour microenvironment has complex interaction with both the cancer cells and other non-neoplastic surrounding cells. The CAF-derived regulators and extracellular matrix proteins can support cancer progression by providing a protective microenvironment for the cancer cells via reduction of chemotherapy sensitivity. On the other hand, these proteins may act as powerful prognostic markers as well as potential targets of anticancer therapy. In this review, we summarise the clinical importance of the major CAF-derived signals influencing tumour behaviour and determining the outcome of chemotherapy.

  18. Milk-derived proteins and minerals alter serum osteocalcin in prepubertal boys after 7 days

    DEFF Research Database (Denmark)

    Mark, A.B.; Hoppe, Camilla; Michaelsen, K.F.

    2010-01-01

    We have previously shown that at equal protein content, milk, but not meat, decreased bone turnover in boys. This suggested that milk-derived components are important for bone metabolism. In the present study, we hypothesized that milk-derived proteins (whey and casein) affect bone turnover during...... growth depending on the content of milk minerals (calcium and phosphorus). This was a randomized, parallel, double-blind study. Eight-year-old boys (n = 57) received 1 of 4 milk drinks: whey protein with low or high content of minerals, or casein protein with low or high content of minerals. The amount...... of whey and casein was identical to their content in 1.5 L of milk. We measured serum osteocalcin (sOC), bone-specific alkaline phosphatase, and C-terminal telopeptides of type 1 collagen (immunoassay) and estimated dietary intake (3-day weighed food record) at baseline and after 7 days. Only s...

  19. Dendritic-tumor fusion cells derived heat shock protein70-peptide complex has enhanced immunogenicity.

    Science.gov (United States)

    Zhang, Yunfei; Zhang, Yong; Chen, Jun; Liu, Yunyan; Luo, Wen

    2015-01-01

    Tumor-derived heat shock protein70-peptide complexes (HSP70.PC-Tu) have shown great promise in tumor immunotherapy due to numerous advantages. However, large-scale phase III clinical trials showed that the limited immunogenicity remained to be enhanced. In previous research, we demonstrated that heat shock protein 70-peptide complexes (HSP70.PC-Fc) derived from dendritic cell (DC)-tumor fusions exhibit enhanced immunogenicity compared with HSP70.PCs from tumor cells. However, the DCs used in our previous research were obtained from healthy donors and not from the patient population. In order to promote the clinical application of these complexes, HSP70.PC-Fc was prepared from patient-derived DC fused directly with patient-derived tumor cells in the current study. Our results showed that compared with HSP70.PC-Tu, HSP70.PC-Fc elicited much more powerful immune responses against the tumor from which the HSP70 was derived, including enhanced T cell activation, and CTL responses that were shown to be antigen specific and HLA restricted. Our results further indicated that the enhanced immunogenicity is related to the activation of CD4+ T cells and increased association with other heat shock proteins, such as HSP90. Therefore, the current study confirms the enhanced immunogenicity of HSP70.PC derived from DC-tumor fusions and may provide direct evidence promoting their future clinical use.

  20. Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Linlin [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001 (China); Liu, Ziwen [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Department of Surgery, Peking Union Medical College Hospital, Beijing 100730 (China); Yan, Ruilan [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Johnson, Stephen [Carbon Dynamics Institute, LLC, 2835 via Verde Drive, Springfield, IL 62703-4325 (United States); Zhao, Yupei [Department of Surgery, Peking Union Medical College Hospital, Beijing 100730 (China); Fang, Xiubin [Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001 (China); Cao, Deliang, E-mail: dcao@siumed.edu [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States)

    2009-09-18

    Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

  1. Bioavailability of milk protein-derived bioactive peptides: a glycaemic management perspective.

    Science.gov (United States)

    Horner, Katy; Drummond, Elaine; Brennan, Lorraine

    2016-06-01

    Milk protein-derived peptides have been reported to have potential benefits for reducing the risk of type 2 diabetes. However, what the active components are and whether intact peptides exert this bioactivity has received little investigation in human subjects. Furthermore, potentially useful bioactive peptides can be limited by low bioavailability. Various peptides have been identified in the gastrointestinal tract and bloodstream after milk-protein ingestion, providing valuable insights into their potential bioavailability. However, these studies are currently limited and the structure and sequence of milk peptides exerting bioactivity for glycaemic management has received little investigation in human subjects. The present article reviews the bioavailability of milk protein-derived peptides in human studies to date, and examines the evidence on milk proteins and glycaemic management, including potential mechanisms of action. Areas in need of advancement are identified. Only by establishing the bioavailability of milk protein-derived peptides, the active components and the mechanistic pathways involved can the benefits of milk proteins for the prevention or management of type 2 diabetes be fully realised in future.

  2. Cargo proteins of plasma astrocyte-derived exosomes in Alzheimer's disease.

    Science.gov (United States)

    Goetzl, Edward J; Mustapic, Maja; Kapogiannis, Dimitrios; Eitan, Erez; Lobach, Irina V; Goetzl, Laura; Schwartz, Janice B; Miller, Bruce L

    2016-11-01

    Efficient intercellular transfer of RNAs, proteins, and lipids as protected exosomal cargo has been demonstrated in the CNS, but distinct physiologic and pathologic roles have not been well defined for this pathway. The capacity to isolate immunochemically human plasma neuron-derived exosomes (NDEs), containing neuron-specific cargo, has permitted characterization of CNS-derived exosomes in living humans. Constituents of the amyloid β-peptide (Aβ)42-generating system now are examined in 2 distinct sets of human neural cells by quantification in astrocyte-derived exosomes (ADEs) and NDEs, enriched separately from plasmas of patients with Alzheimer's disease (AD) or frontotemporal dementia (FTD) and matched cognitively normal controls. ADE levels of β-site amyloid precursor protein-cleaving enzyme 1 (BACE-1), γ-secretase, soluble Aβ42, soluble amyloid precursor protein (sAPP)β, sAPPα, glial-derived neurotrophic factor (GDNF), P-T181-tau, and P-S396-tau were significantly (3- to 20-fold) higher than levels in NDEs for patients and controls. BACE-1 levels also were a mean of 7-fold higher in ADEs than in NDEs from cultured rat type-specific neural cells. Levels of BACE-1 and sAPPβ were significantly higher and of GDNF significantly lower in ADEs of patients with AD than in those of controls, but not significantly different in patients with FTD than in controls. Abundant proteins of the Aβ42 peptide-generating system in ADEs may sustain levels in neurons. ADE cargo proteins may be useful for studies of mechanisms of cellular interactions and effects of BACE-1 inhibitors in AD.-Goetzl, E. J., Mustapic, M., Kapogiannis, D., Eitan, E., Lobach, I. V., Goetzl, L., Schwartz, J. B., Miller, B. L. Cargo proteins of plasma astrocyte-derived exosomes in Alzheimer's disease.

  3. Purification, crystallization and preliminary X-ray diffraction analysis of an oomycete-derived Nep1-­like protein

    Science.gov (United States)

    Luberacki, Borries; Weyand, Michael; Seitz, Ulrich; Koch, Wolfgang; Oecking, Claudia; Ottmann, Christian

    2008-01-01

    The elicitor protein Nep1-like protein from the plant pathogen Pythium aphanidermatum was purified and crystallized using the hanging-drop vapour-diffusion method. A native data set was collected to 1.35 Å resolution at 100 K using synchrotron radiation. Since selenomethionine-labelled protein did not crystallize under the original conditions, a second crystal form was identified that yielded crystals that diffracted to 2.1 Å resolution. A multiple-wavelength anomalous dispersion (MAD) experiment was performed at 100 K and all four selenium sites were identified, which allowed solution of the structure. PMID:19052381

  4. Purification, crystallization and preliminary X-ray diffraction analysis of an oomycete-derived Nep1-like protein.

    Science.gov (United States)

    Luberacki, Borries; Weyand, Michael; Seitz, Ulrich; Koch, Wolfgang; Oecking, Claudia; Ottmann, Christian

    2008-12-01

    The elicitor protein Nep1-like protein from the plant pathogen Pythium aphanidermatum was purified and crystallized using the hanging-drop vapour-diffusion method. A native data set was collected to 1.35 A resolution at 100 K using synchrotron radiation. Since selenomethionine-labelled protein did not crystallize under the original conditions, a second crystal form was identified that yielded crystals that diffracted to 2.1 A resolution. A multiple-wavelength anomalous dispersion (MAD) experiment was performed at 100 K and all four selenium sites were identified, which allowed solution of the structure.

  5. Ability of silybin and its derivatives to prevent protein oxidation in different model systems

    DEFF Research Database (Denmark)

    Purchartová, K.; Baron, C.P.; Křen, V.

    2013-01-01

    to prevent activation of hemoglobin (Hb) to highly reactive hypervalent heme protein species (ferrylHb and perferrylHb) was examined. Indeed, Hb cytotoxicity has been associated with the generation of protein radicals, which are formed when the ferric iron of Hb (Fe3+) is oxidised by H2O2 to (Fe4+) to form...... perferrylHb and ferrylHb, with the later also bearing a radical on its protein. The relationship between the structural properties of silybin and its derivatives and their ability to prevent oxidation of Hb was investigated in model system in the presence or the absence of lipids. The antioxidant activities...

  6. Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

    Science.gov (United States)

    Bonino, F; Heermann, K H; Rizzetto, M; Gerlich, W H

    1986-01-01

    Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d. Images PMID:3701932

  7. The application of an enamel matrix protein derivative (Emdogain) in regenerative periodontal therapy: a review.

    NARCIS (Netherlands)

    Sculean, A.; Schwarz, F.; Becker, J.; Brecx, M.

    2007-01-01

    Regenerative periodontal therapy aims at reconstitution of the lost periodontal structures such as new formation of root cementum, periodontal ligament and alveolar bone. Findings from basic research indicate that enamel matrix protein derivative (EMD) has a key role in periodontal wound healing. Hi

  8. Targeting diverse protein-protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold.

    Science.gov (United States)

    Checco, James W; Kreitler, Dale F; Thomas, Nicole C; Belair, David G; Rettko, Nicholas J; Murphy, William L; Forest, Katrina T; Gellman, Samuel H

    2015-04-14

    Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF165-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

  9. Targeted mass spectrometry analysis of neutrophil-derived proteins released during sepsis progression.

    Science.gov (United States)

    Malmström, E; Davidova, A; Mörgelin, M; Linder, A; Larsen, M; Qvortrup, K; Nordenfelt, P; Shannon, O; Dzupova, O; Holub, M; Malmström, J; Herwald, H

    2014-12-01

    Early diagnosis of severe infectious diseases is essential for timely implementation of lifesaving therapies. In a search for novel biomarkers in sepsis diagnosis we focused on polymorphonuclear neutrophils (PMNs). Notably, PMNs have their protein cargo readily stored in granules and following systemic stimulation, an immediate increase of neutrophil-borne proteins can be observed into the circulation of sepsis patients. We applied a combination of mass spectrometry (MS) based approaches, LC-MS/MS and selected reaction monitoring (SRM), to characterise and quantify the neutrophil proteome in healthy or disease conditions. With this approach we identified a neutrophil-derived protein abundance pattern in blood plasma consisting of 20 proteins that can be used as a protein signature for severe infectious diseases. Our results also show that SRM is highly sensitive, specific, and reproducible and, thus, a promising technology to study a complex, dynamic and multifactorial disease such as sepsis.

  10. Sulforaphane, a cruciferous vegetable-derived isothiocyanate, inhibits protein synthesis in human prostate cancer cells.

    Science.gov (United States)

    Wiczk, Aleksandra; Hofman, Dagmara; Konopa, Grażyna; Herman-Antosiewicz, Anna

    2012-08-01

    Sulforaphane (SFN) is a compound derived from cruciferous plants. Its anticancer properties have been demonstrated both, in cancer cell lines as well as tumors in animal models. It has been shown that SFN inhibits cell proliferation, induces apoptosis, autophagy, and sensitizes cancer cells to therapies. As induction of catabolic processes is often related to perturbation in protein synthesis we aimed to investigate the impact of SFN on this process in PC-3 human prostate cancer cells. In the present study we show that SFN inhibits protein synthesis in PC-3 cells in a dose- and time-dependent manner which is accompanied by a decreased phosphorylation of mTOR substrates. Translation inhibition is independent of mitochondria-derived ROS as it is observed in PC-3 derivatives devoid of functional mitochondrial respiratory chain (Rho0 cells). Although SFN affects mitochondria and slightly decreases glycolysis, the ATP level is maintained on the level characteristic for control cells. Inhibition of protein synthesis might be a protective response of prostate cancer cells to save energy. However, translation inhibition contributes to the death of PC-3 cells due to decreased level of a short-lived protein, survivin. Overexpression of this anti-apoptotic factor protects PC-3 cells against SFN cytotoxicity. Protein synthesis inhibition by SFN is not restricted to prostate cancer cells as we observed similar effect in SKBR-3 breast cancer cell line. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Cellular Functions and Gene and Protein Expression Profiles in Endothelial Cells Derived from Moyamoya Disease-Specific iPS Cells

    Science.gov (United States)

    Hamauchi, Shuji; Shichinohe, Hideo; Uchino, Haruto; Yamaguchi, Shigeru; Nakayama, Naoki; Kazumata, Ken; Osanai, Toshiya; Abumiya, Takeo; Houkin, Kiyohiro; Era, Takumi

    2016-01-01

    Background and purpose Moyamoya disease (MMD) is a slow, progressive steno-occlusive disease, arising in the terminal portions of the cerebral internal carotid artery. However, the functions and characteristics of the endothelial cells (ECs) in MMD are unknown. We analyzed these features using induced pluripotent stem cell (iPSC)-derived ECs. Methods iPSC lines were established from the peripheral blood of three patients with MMD carrying the variant RNF213 R4810K, and three healthy persons used as controls. After the endothelial differentiation of iPSCs, CD31+CD144+ cells were purified as ECs using a cell sorter. We analyzed their proliferation, angiogenesis, and responses to some angiogenic factors, namely VEGF, bFGF, TGF-β, and BMP4. The ECs were also analyzed using DNA microarray and proteomics to perform comprehensive gene and protein expression analysis. Results Angiogenesis was significantly impaired in MMD regardless of the presence of any angiogenic factor. On the contrary, endothelial proliferation was not significant between control- and MMD-derived cells. Regarding DNA microarray, pathway analysis illustrated that extracellular matrix (ECM) receptor-related genes, including integrin β3, were significantly downregulated in MMD. Proteomic analysis revealed that cytoskeleton-related proteins were downregulated and splicing regulation-related proteins were upregulated in MMD. Conclusions Downregulation of ECM receptor-related genes may be associated with impaired angiogenic activity in ECs derived from iPSCs from patients with MMD. Upregulation of splicing regulation-related proteins implied differences in splicing patterns between control and MMD ECs. PMID:27662211

  12. Complex Assembly Behavior During the Encapsulation of Green Fluorescent Protein Analogs in Virus Derived Protein Capsules

    NARCIS (Netherlands)

    Minten, Inge J.; Nolte, Roeland J.M.; Cornelissen, Jeroen J.L.M.

    2010-01-01

    Enzymes encapsulated in nanocontainers are a better model of the conditions inside a living cell than free enzymes in solution. In a first step toward the encapsulation of multiple enzymes inside the cowpea chlorotic mottle virus (CCMV) capsid, enhanced green fluorescent protein (EGFP) was attached

  13. Excretion of purine base derivatives after intake of bacterial protein meal in pigs

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, A.

    2007-01-01

    Bacterial protein meal has a high content ofprotein but also of RNA and DNA. Sixteen barrows were allocated to four diets containing increasing levels of bacterial protein meal (BPM), from weaning to 80 kg live weight, to evaluate whether the RNA and DNA contents of BPM influenced the retention...... of nitrogen. It was hypothesised that an increased intake of RNA and DNA would lead to an increased urinary excretion of purine base derivatives and increased plasma concentrations. Retention of nitrogen was unaffected by dietary content of BPM (P=0.08) and the urinary excretion of purine base derivatives...... increased with increasing dietary content of BPM. No differences in fasting plasma concentration of uric acid, xanthine and hypoxanthine were observed. It can therefore be concluded that increasing levels of dietary BPM maintained protein accretion and led to changes in excretion of purine detrivatices...

  14. QSAR Study on Caffeine Derivatives Docked on Poly(ARNA Polymerase Protein Cid1

    Directory of Open Access Journals (Sweden)

    Teodora E. Harsa

    2016-06-01

    Full Text Available Caffeine is the most commonly ingested alkylxantine and is recognized as a psycho-stimulant. It improves some aspects of cognitive performance, however it reduces the cerebral blood flow both in animals and humans. In this paper a QSAR study on caffeine derivatives, docked on the Poly(ARNA polymerase protein cid1, is reported. A set of forty caffeine derivatives, downloaded from PubChem, was modeled, within the hypermolecule strategy; the predicted activity was LD50 and prediction was done on similarity clusters with the leaders chosen as the best docked ligands on the Poly(ARNA polymerase protein cid1. It was concluded that LD50 of the studied caffeines is not influenced by their binding to the target protein. This work is licensed under a Creative Commons Attribution 4.0 International License.

  15. Label-free aptasensor for platelet-derived growth factor (PDGF) protein

    Energy Technology Data Exchange (ETDEWEB)

    Degefa, Tesfaye Hailu [Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701 (Korea, Republic of); Kwak, Juhyoun [Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701 (Korea, Republic of)], E-mail: Juhyoun_Kwak@kaist.ac.kr

    2008-04-21

    A label-free aptasensor for platelet-derived growth factor (PDGF) protein is reported. The aptasensor uses mixed self-assembled monolayers (SAMs) composed of a thiol-modified PDGF binding aptamer and 6-mercaptohexanol (MCH) on a gold electrode. The SAMs were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) before and after binding of the protein using [Fe(CN){sub 6}]{sup 3-/4-}, a redox marker ion as an indicator for the formation of a protein-aptamer complex. The CVs at the PDGF modified electrode showed significant differences, such as changes in the peak currents and peak-to-peak separation, before and after binding of the target protein. The EIS spectra, in the form of Nyquist plots, were analyzed with a Randles circuit while the electron transfer resistance R{sub ct} was used to monitor the binding of the target protein. The results showed that, without any modification to the aptamer, the target protein can be recognized effectively at the PDGF binding aptamer SAMs at the electrode surface. Control experiments using non-binding oligonucleotides assembled at the electrode surfaces also confirmed the results and showed that there was no formation of an aptamer-protein complex. The DPV signal at the aptamer functionalized electrode showed a linearly decreased marker ion peak current in a protein concentrations range of 1-40 nM. Thus, label-free detection of PDGF protein at an aptamer modified electrode has been demonstrated.

  16. Angiotensin I-converting enzyme inhibitor derived from cross-linked oyster protein.

    Science.gov (United States)

    Xie, Cheng-Liang; Kim, Jin-Soo; Ha, Jong-Myung; Choung, Se-Young; Choi, Yeung-Joon

    2014-01-01

    Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE) inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50) of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR). The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension.

  17. Angiotensin I-Converting Enzyme Inhibitor Derived from Cross-Linked Oyster Protein

    Directory of Open Access Journals (Sweden)

    Cheng-Liang Xie

    2014-01-01

    Full Text Available Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50 of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR. The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension.

  18. Exosomal Secretion of Cytoplasmic Prostate Cancer Xenograft-derived Proteins *S⃞

    Science.gov (United States)

    Jansen, Flip H.; Krijgsveld, Jeroen; van Rijswijk, Angelique; van den Bemd, Gert-Jan; van den Berg, Mirella S.; van Weerden, Wytske M.; Willemsen, Rob; Dekker, Lennard J.; Luider, Theo M.; Jenster, Guido

    2009-01-01

    Novel markers for prostate cancer (PCa) are needed because current established markers such as prostate-specific antigen lack diagnostic specificity and prognostic value. Proteomics analysis of serum from mice grafted with human PCa xenografts resulted in the identification of 44 tumor-derived proteins. Besides secreted proteins we identified several cytoplasmic proteins, among which were most subunits of the proteasome. Native gel electrophoresis and sandwich ELISA showed that these subunits are present as proteasome complexes in the serum from xenograft-bearing mice. We hypothesized that the presence of proteasome subunits and other cytoplasmic proteins in serum of xenografted mice could be explained by the secretion of small vesicles by cancer cells, so-called exosomes. Therefore, mass spectrometry and Western blotting analyses of the protein content of exosomes isolated from PCa cell lines was performed. This resulted in the identification of mainly cytoplasmic proteins of which several had previously been identified in the serum of xenografted mice, including proteasome subunits. The isolated exosomes also contained RNA, including the gene fusion TMPRSS2-ERG product. These observations suggest that although their function is not clearly defined cancer-derived exosomes offer possibilities for the identification of novel biomarkers for PCa. PMID:19204029

  19. Protein structure and evolution: are they constrained globally by a principle derived from information theory?

    Science.gov (United States)

    Hatton, Leslie; Warr, Gregory

    2015-01-01

    That the physicochemical properties of amino acids constrain the structure, function and evolution of proteins is not in doubt. However, principles derived from information theory may also set bounds on the structure (and thus also the evolution) of proteins. Here we analyze the global properties of the full set of proteins in release 13-11 of the SwissProt database, showing by experimental test of predictions from information theory that their collective structure exhibits properties that are consistent with their being guided by a conservation principle. This principle (Conservation of Information) defines the global properties of systems composed of discrete components each of which is in turn assembled from discrete smaller pieces. In the system of proteins, each protein is a component, and each protein is assembled from amino acids. Central to this principle is the inter-relationship of the unique amino acid count and total length of a protein and its implications for both average protein length and occurrence of proteins with specific unique amino acid counts. The unique amino acid count is simply the number of distinct amino acids (including those that are post-translationally modified) that occur in a protein, and is independent of the number of times that the particular amino acid occurs in the sequence. Conservation of Information does not operate at the local level (it is independent of the physicochemical properties of the amino acids) where the influences of natural selection are manifest in the variety of protein structure and function that is well understood. Rather, this analysis implies that Conservation of Information would define the global bounds within which the whole system of proteins is constrained; thus it appears to be acting to constrain evolution at a level different from natural selection, a conclusion that appears counter-intuitive but is supported by the studies described herein.

  20. Protein structure and evolution: are they constrained globally by a principle derived from information theory?

    Directory of Open Access Journals (Sweden)

    Leslie Hatton

    Full Text Available That the physicochemical properties of amino acids constrain the structure, function and evolution of proteins is not in doubt. However, principles derived from information theory may also set bounds on the structure (and thus also the evolution of proteins. Here we analyze the global properties of the full set of proteins in release 13-11 of the SwissProt database, showing by experimental test of predictions from information theory that their collective structure exhibits properties that are consistent with their being guided by a conservation principle. This principle (Conservation of Information defines the global properties of systems composed of discrete components each of which is in turn assembled from discrete smaller pieces. In the system of proteins, each protein is a component, and each protein is assembled from amino acids. Central to this principle is the inter-relationship of the unique amino acid count and total length of a protein and its implications for both average protein length and occurrence of proteins with specific unique amino acid counts. The unique amino acid count is simply the number of distinct amino acids (including those that are post-translationally modified that occur in a protein, and is independent of the number of times that the particular amino acid occurs in the sequence. Conservation of Information does not operate at the local level (it is independent of the physicochemical properties of the amino acids where the influences of natural selection are manifest in the variety of protein structure and function that is well understood. Rather, this analysis implies that Conservation of Information would define the global bounds within which the whole system of proteins is constrained; thus it appears to be acting to constrain evolution at a level different from natural selection, a conclusion that appears counter-intuitive but is supported by the studies described herein.

  1. Augmentation of protein-derived acetic acid production by heat-alkaline-induced changes in protein structure and conformation.

    Science.gov (United States)

    Wang, Xu; Li, Yanbo; Liu, Junxin; Ren, Nan-Qi; Qu, Jiuhui

    2016-01-01

    Waste-derived acetic acid (HAc) is an attractive feedstock for microbe-mediated biofuel production. However, fermentative conversion of HAc from waste-activated sludge (WAS) has low yield because of the high concentration of proteins not readily utilizable by microorganisms without prior hydrolysis. We investigated a combined technology for HAc augmentation during sludge protein fermentation. The maximal HAc yield increased over two-fold, reaching 0.502 ± 0.021 g/g protein (0.36 ± 0.01 g COD/g COD, ∼52% of the total volatile fatty acids) when synthetic sludge protein was heated at 120 °C for 30 min, treated at pH 12 for 24 h, and fermented at pH 9 for 72 h. Comprehensive analysis illustrated that the heat-alkaline pretreatment significantly induced protein fragmentation, simultaneously increasing the efficiency of protein biohydrolysis (from 35.5% to 85.9%) by inducing conformational changes indicative of protein unfolding. Consequently, the native α-helix content was decreased from 67.3% to 32.5% by conversion to an unordered shape, whose content increased from 27.5% to 45.5%; disulfide bonds were cleaved, whereas the main S-S stretching pattern was altered from gauche-gauche-gauche to gauche-gauche-trans, consequently causing increased protein susceptibility to proteolytic hydrolysis (76.3% vs. 47.0%). Economic analysis indicated that anaerobic fermentation with appropriate heat-alkaline pretreatment is a cost-effective approach for waste conversion to energy sources such as HAc.

  2. Posthoc phosphorylation of proteins derived from ischemic rat hippocampus, striatum and neocortex.

    Science.gov (United States)

    Kirschenbaum, B; Pulsinelli, W A

    1990-03-12

    Disruption of the brain's protein phosphorylation system by ischemia may cause irreversible metabolic and structural alterations leading eventually to cell death. To examine the effect of ischemia on the phosphorylation state of brain proteins, tissue homogenates derived from the hippocampus, striatum and neocortex of normal rats and rats subjected to severe forebrain ischemia were phosphorylated with [gamma-32P]ATP. The phosphorylated proteins were separated by two-dimensional polyacrylamide gel electrophoresis and changes were assessed by autoradiography. Cerebral ischemia caused marked alterations of the phosphorylation state of many brain proteins; phosphorylation of some proteins was increased while phosphorylation of others was decreased. Despite differences in the sensitivity of the hippocampus, striatum and neocortex to ischemic injury the direction and approximate magnitude of protein phosphorylation changes caused by ischemia were similar in all three regions. Since the pattern of protein phosphorylation in the ischemia-vulnerable hippocampus was identical to that in the ischemia-resistant paramedian neocortex we conclude that abnormalities of protein phosphorylation may be necessary for ischemic injury to neurons but none are sufficient to explain the selective vulnerability of certain brain regions to ischemic damage.

  3. The Effect of Plant Proteins Derived from Cereals and Legumes on Heme Iron Absorption

    Directory of Open Access Journals (Sweden)

    Valerie Weinborn

    2015-10-01

    Full Text Available The aim of this study is to determine the effect of proteins from cereals and legumes on heme iron (Fe absorption. The absorption of heme Fe without its native globin was measured. Thirty adult females participated in two experimental studies (15 per study. Study I focused on the effects of cereal proteins (zein, gliadin and glutelin and study II on the effects of legume proteins (soy, pea and lentil on heme Fe absorption. When heme was given alone (as a control, study I and II yielded 6.2% and 11.0% heme absorption (p > 0.05. In study I, heme Fe absorption was 7.2%, 7.5% and 5.9% when zein, gliadin and glutelin were added, respectively. From this, it was concluded that cereal proteins did not affect heme Fe absorption. In study II, heme Fe absorption was 7.3%, 8.1% and 9.1% with the addition of soy, pea and lentil proteins, respectively. Only soy proteins decreased heme Fe absorption (p < 0.05. These results suggest that with the exception of soy proteins, which decreased absorption, proteins derived from cereals and legumes do not affect heme Fe absorption.

  4. Obstructive sleep apnea decreases central nervous system-derived proteins in the cerebrospinal fluid.

    Science.gov (United States)

    Ju, Yo-El S; Finn, Mary Beth; Sutphen, Courtney L; Herries, Elizabeth M; Jerome, Gina M; Ladenson, Jack H; Crimmins, Daniel L; Fagan, Anne M; Holtzman, David M

    2016-07-01

    We hypothesized that one mechanism underlying the association between obstructive sleep apnea (OSA) and Alzheimer's disease is OSA leading to decreased slow wave activity (SWA), increased synaptic activity, decreased glymphatic clearance, and increased amyloid-β. Polysomnography and lumbar puncture were performed in OSA and control groups. SWA negatively correlated with cerebrospinal fluid (CSF) amyloid-β-40 among controls and was decreased in the OSA group. Unexpectedly, amyloid-β-40 was decreased in the OSA group. Other neuronally derived proteins, but not total protein, were also decreased in the OSA group, suggesting that OSA may affect the interaction between interstitial and cerebrospinal fluid. Ann Neurol 2016;80:154-159.

  5. Antimicrobial Activity of Peptides Derived from Olive Flounder Lipopolysaccharide Binding Protein/Bactericidal Permeability-Increasing Protein (LBP/BPI

    Directory of Open Access Journals (Sweden)

    Bo-Hye Nam

    2014-10-01

    Full Text Available We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.

  6. Antimicrobial activity of peptides derived from olive flounder lipopolysaccharide binding protein/bactericidal permeability-increasing protein (LBP/BPI).

    Science.gov (United States)

    Nam, Bo-Hye; Moon, Ji-Young; Park, Eun-Hee; Kim, Young-Ok; Kim, Dong-Gyun; Kong, Hee Jeong; Kim, Woo-Jin; Jee, Young Ju; An, Cheul Min; Park, Nam Gyu; Seo, Jung-Kil

    2014-10-17

    We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.

  7. A comparison of surface proteins in embryonal carcinoma cells and their differentiated derivatives.

    Science.gov (United States)

    Keil-Dlouha, V; Paulin, D; Bagilet, L K; Keil, B

    1980-03-27

    Surface proteins from five cell lines (three embryonal carcinoma cell lines (F9, PCC4 and PCC3), teratocarcinoma-derived endodermal cells (PYS) and fibroblasts (line 3/A/1-D-3 differentiated from PCC3) were compared by two-dimensional polyacrylamide gel electrophoresis after selective iodination with 125I in the presence of lactoperoxidase. The labeled proteins were solubilized either in Nonidet P40/urea/ampholyte/mercaptoethanol solution or in Nonidet P40 only. In total, about thirty major 125I-labeled surface proteins were identified by their isoelectric point and molecular weight. 14 proteins are present in all five cell types, although their quantity or accessibility for labeling differs between differentiated and undifferentiated cells. Three proteins (200, 160 and 150 kilodaltons) are present in undifferentiated cells only. Two of them (160 and 150 kilodaltons) were solubilized by Nonidet P40/urea/ampholyte/mercaptoethanol, but not by Nonidet P40. One protein (50 kilodaltons) was found in nullipotent F9 cells only. About 14--15 proteins (including fibronectin) were released by Nonidet P40/urea/ampholyte/mercaptoethanol but not by Nonidet P40. They are presumably bound to submembrane or cytoskeleton structures by non-covalent bonds.

  8. Adsorption of Heavy Metal Ions, Dyes and Proteins by Chitosan Composites and Derivatives-A Review

    Institute of Scientific and Technical Information of China (English)

    LIU Bingjie; WANG Dongfeng; YU Guangli; MENG Xianghong

    2013-01-01

    Chitosan composites and derivatives have gained wide attentions as effective biosorbents due to their low costs and high contents of amino and hydroxyl functional groups.They have showed significant potentials of removing metal ions,dyes and proteins from various media.Chemical modifications that lead to the formation of the chitosan derivatives and chitosan composites have been extensively studied and widely reported in literatures.The aims of this review were to summarize the important information of the bioactivities of chitosan,highlight the various preparation methods of chitosan-based active biosorbents,and outline its potential applications in the adsorption of heavy metal ions,dyes and proteins from wastewater and aqueous solutions.

  9. Novel cycloheximide derivatives targeting the moonlighting protein Mip exhibit specific antimicrobial activity against Legionella pneumophila

    Directory of Open Access Journals (Sweden)

    Janine eRasch

    2015-03-01

    Full Text Available Mip (macrophage infectivity potentiator and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP family of peptidyl-prolyl-cis/trans-isomerases (PPIases. In L. pneumophila the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development, and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 µM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30 to 40 µM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5‐dimethyladamantan‐1‐[yl]acetamide substitution (MT_30.32, and a 3‐ethyladamantan‐1‐[yl]acetamide substitution (MT_30.51 had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple

  10. Milk protein-derived peptide inhibitors of angiotensin-I-converting enzyme

    OpenAIRE

    Fitzgerald, Richard J; Meisel, Hans

    2000-01-01

    peer-reviewed Numerous casein and whey protein-derived angiotensin-I-converting enzyme (ACE) inhibitory peptides/hydrolysates have been identified. Clinical trials in hypertensive animals and humans show that these peptides/hydrolysates can bring about a significant reduction in hypertension. These peptides/hydrolysates may be classified as functional food ingredients and nutraceuticals due to their ability to provide health benefits i.e. as functional food ingredients in reduc...

  11. Labelling of penicillin-binding proteins from Escherichia coli with photoreactive derivatives of. beta. -lactam antibiotics

    Energy Technology Data Exchange (ETDEWEB)

    Aran, V.; Rodriguez-Tebar, A.; Vazquez, D. (Universidad Autonoma de Madrid (Spain))

    1983-03-21

    The authors have synthesized a number of photoreactive radiolabelled ..beta..-lactams that react and form permanent covalent bonds with the penicillin-binding proteins (PBPs), since photoreactive ligand derivatives have been used to some extent for structural studies on membranes and other biological structures. Chemical and photochemical labelling of a receptor by its ligand are important techniques to elucidate the nature of the ligand-receptor interaction, and for identification and characterization of receptors. They have synthesized two ..beta..-lactam derivatives each containing two different photoreactive moieties. One of them is an aryl azido compound, widely known as a photoreactive reagent for labelling studies, whereas the other one contains a nitroguaiacol derived group used in photochemical studies with other biological materials.

  12. Biogenic mechanisms and utilization of small RNAs derived from human protein-coding genes

    DEFF Research Database (Denmark)

    Valen, Eivind; Preker, Pascal; Andersen, Peter Refsing

    2011-01-01

    Efforts to catalog eukaryotic transcripts have uncovered many small RNAs (sRNAs) derived from gene termini and splice sites. Their biogenesis pathways are largely unknown, but a mechanism based on backtracking of RNA polymerase II (RNAPII) has been suggested. By sequencing transcripts 12......-100 nucleotides in length from cells depleted of major RNA degradation enzymes and RNAs associated with Argonaute (AGO1/2) effector proteins, we provide mechanistic models for sRNA production. We suggest that neither splice site-associated (SSa) nor transcription start site-associated (TSSa) RNAs arise from...... RNAPII backtracking. Instead, SSa RNAs are largely degradation products of splicing intermediates, whereas TSSa RNAs probably derive from nascent RNAs protected by stalled RNAPII against nucleolysis. We also reveal new AGO1/2-associated RNAs derived from 3' ends of introns and from mRNA 3' UTRs...

  13. Feasible protein aggregation of phosphorylated poly-γ-glutamic acid derivative from Bacillus subtilis (natto).

    Science.gov (United States)

    Kurita, Osamu; Sago, Toru; Umetani, Kaori; Kokean, Yasushi; Yamaoka, Chizuru; Takahashi, Nobuyuki; Iwamoto, Hiroyuki

    2017-10-01

    Poly-γ-glutamic acid (PGA) was modified with phosphorylating agents such as sodium metaphosphate and potassium metaphosphate in the culture medium of Bacillus subtilis (natto). The highly phosphorylated PGA derivatives were prepared and investigated for their chemical and physicochemical properties. The PGA derivatives had approximately 7% (W/W) inorganic phosphorus and characteristic absorbance PO2(-) bands at 1082cm(-1) and 1260cm(-1) by Fourier Transform Infrared Spectroscopy. The derivative modified by sodium metaphosphate (J-5) was easily hydrated in water and had extremely low viscosity. The shear rate-induced transition leading to the decrease of viscosity was not observed in J-5 whereas the derivative modified by potassium metaphosphate (J-6) as well as unmodified PGA (J-1) showed the typical decrease of viscosity. In circular dichroism (CD) measurement of J-5, there was a significant loss of the negative chirality CD signal, implying that protein aggregation occured at decreasing pH from 6.2 to 4.4. The thioflavin T fluorescence intensity of the aqueous solution in the J-5 was extremely high despite the absence of heat-treatment. The results indicate that the J-5 is the likeliest type of aggregation by β-sheet cross-linking which is relevant to protein diseases like Alzheimer's disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    Science.gov (United States)

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  15. Strong Purifying Selection in Transmission of Mammalian Mitochondrial DNA

    Science.gov (United States)

    Stewart, James Bruce; Freyer, Christoph; Elson, Joanna L; Wredenberg, Anna; Cansu, Zekiye; Trifunovic, Aleksandra; Larsson, Nils-Göran

    2008-01-01

    There is an intense debate concerning whether selection or demographics has been most important in shaping the sequence variation observed in modern human mitochondrial DNA (mtDNA). Purifying selection is thought to be important in shaping mtDNA sequence evolution, but the strength of this selection has been debated, mainly due to the threshold effect of pathogenic mtDNA mutations and an observed excess of new mtDNA mutations in human population data. We experimentally addressed this issue by studying the maternal transmission of random mtDNA mutations in mtDNA mutator mice expressing a proofreading-deficient mitochondrial DNA polymerase. We report a rapid and strong elimination of nonsynonymous changes in protein-coding genes; the hallmark of purifying selection. There are striking similarities between the mutational patterns in our experimental mouse system and human mtDNA polymorphisms. These data show strong purifying selection against mutations within mtDNA protein-coding genes. To our knowledge, our study presents the first direct experimental observations of the fate of random mtDNA mutations in the mammalian germ line and demonstrates the importance of purifying selection in shaping mitochondrial sequence diversity. PMID:18232733

  16. Clinical Applications of Phage-Derived sFvs and sFv Fusion Proteins

    Directory of Open Access Journals (Sweden)

    K. A. Chester

    2000-01-01

    Full Text Available Single chain Fv antibodies (sFvs have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA, a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2 for use in the ADEPT (antibody directed enzyme prodrug therapy system and MFE::TNFα aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

  17. Hydrogen purifier module with membrane support

    Science.gov (United States)

    A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

    2012-07-24

    A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

  18. High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy.

    Science.gov (United States)

    Parker, Katherine H; Horn, Lucas A; Ostrand-Rosenberg, Suzanne

    2016-09-01

    Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by

  19. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    Science.gov (United States)

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  20. Human malignant melanoma-derived progestagen-associated endometrial protein immunosuppresses T lymphocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Suping Ren

    Full Text Available Progestagen-associated endometrial protein (PAEP is a glycoprotein of the lipocalin family that acts as a negative regulator of T cell receptor-mediated activation. However, the function of tumor-derived PAEP on the human immune system in the tumor microenvironment is unknown. PAEP is highly expressed in intermediate and thick primary melanomas (Breslow's 2.5mm or greater and metastatic melanomas, correlating with its expression in daughter cell lines established in vitro. The current study investigates the role of melanoma cell-secreted PAEP protein in regulating T cell function. Upon the enrichment of CD3+, CD4+ and CD8+ T cells from human peripheral blood mononuclear cells, each subset was then mixed with either melanoma-derived PAEP protein or PAEP-poor supernatant of gene-silenced tumor cells. IL-2 and IFN-γ secretion of CD4+ T cells significantly decreased with the addition of PAEP-rich supernatant. And the addition of PAEP-positive cell supernatant to activated lymphocytes significantly inhibited lymphocyte proliferation and cytotoxic T cell activity, while increasing lymphocyte apoptosis. Our result suggests that melanoma cell-secreted PAEP protein immunosuppresses the activation, proliferation and cytotoxicity of T lymphocytes, which might partially explain the mechanism of immune tolerance induced by melanoma cells within the tumor microenvironment.

  1. Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks.

    Science.gov (United States)

    Sabalza, Maite; Christou, Paul; Capell, Teresa

    2014-12-01

    Molecular pharming is a cost-effective platform for the production of recombinant proteins in plants. Although the biopharmaceutical industry still relies on a small number of standardized fermentation-based technologies for the production of recombinant proteins there is now a greater awareness of the advantages of molecular pharming particularly in niche markets. Here we discuss some of the technical, economic and regulatory barriers that constrain the clinical development and commercialization of plant-derived pharmaceutical proteins. We also discuss strategies to increase productivity and product quality/homogeneity. The advantages of whole plants should be welcomed by the industry because this will help to reduce the cost of goods and therefore expand the biopharmaceutical market into untapped sectors.

  2. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    Science.gov (United States)

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo.

  3. Making novel bio-interfaces through bacterial protein recrystallization on biocompatible polylactide derivative films

    Science.gov (United States)

    Lejardi, Ainhoa; López, Aitziber Eleta; Sarasua, José R.; Sleytr, U. B.; Toca-Herrera, José L.

    2013-09-01

    Fabrication of novel bio-supramolecular structures was achieved by recrystallizing the bacterial surface protein SbpA on amorphous and semicrystalline polylactide derivatives. Differential scanning calorimetry showed that the glass transition temperature (Tg) for (poly-L-lactide)-PLLA, poly(L,D-lactide)-PDLLA, poly(lactide-co-glycolide)-PLGA and poly(lactide-co-caprolactone)-PLCL was 63 °C, 53 °C, 49 °C and 15 °C, respectively. Tensile stress-strain tests indicated that PLLA, PLGA, and PDLLA had a glassy behaviour when tested below Tg. The obtained Young modulus were 1477 MPa, 1330 MPa, 1306 MPa, and 9.55 MPa for PLLA, PLGA, PDLLA, and PLCL, respectively. Atomic force microscopy results confirmed that SbpA recrystallized on every polymer substrate exhibiting the native S-layer P4 lattice (a = b = 13 nm, γ = 90°). However, the polymer substrate influenced the domain size of the S-protein crystal, with the smallest size for PLLA (0.011 μm2), followed by PDLLA (0.034 μm2), and PLGA (0.039 μm2), and the largest size for PLCL (0.09 μm2). quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated that the adsorbed protein mass per unit area (˜1800 ng cm-2) was independent of the mechanical, thermal, and crystalline properties of the polymer support. The slowest protein adsorption rate was observed for amorphous PLCL (the polymer with the weakest mechanical properties and lowest Tg). QCM-D also monitored protein self-assembly in solution and confirmed that S-layer formation takes place in three main steps: adsorption, self-assembly, and crystal reorganization. Finally, this work shows that biodegradable polylactide derivatives films are a suitable support to form robust biomimetic S-protein layers.

  4. Making novel bio-interfaces through bacterial protein recrystallization on biocompatible polylactide derivative films.

    Science.gov (United States)

    Lejardi, Ainhoa; López, Aitziber Eleta; Sarasua, José R; Sleytr, U B; Toca-Herrera, José L

    2013-09-28

    Fabrication of novel bio-supramolecular structures was achieved by recrystallizing the bacterial surface protein SbpA on amorphous and semicrystalline polylactide derivatives. Differential scanning calorimetry showed that the glass transition temperature (T(g)) for (poly-L-lactide)-PLLA, poly(L,D-lactide)-PDLLA, poly(lactide-co-glycolide)-PLGA and poly(lactide-co-caprolactone)-PLCL was 63 °C, 53 °C, 49 °C and 15 °C, respectively. Tensile stress-strain tests indicated that PLLA, PLGA, and PDLLA had a glassy behaviour when tested below T(g). The obtained Young modulus were 1477 MPa, 1330 MPa, 1306 MPa, and 9.55 MPa for PLLA, PLGA, PDLLA, and PLCL, respectively. Atomic force microscopy results confirmed that SbpA recrystallized on every polymer substrate exhibiting the native S-layer P4 lattice (a = b = 13 nm, γ = 90°). However, the polymer substrate influenced the domain size of the S-protein crystal, with the smallest size for PLLA (0.011 μm(2)), followed by PDLLA (0.034 μm(2)), and PLGA (0.039 μm(2)), and the largest size for PLCL (0.09 μm(2)). quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated that the adsorbed protein mass per unit area (~1800 ng cm(-2)) was independent of the mechanical, thermal, and crystalline properties of the polymer support. The slowest protein adsorption rate was observed for amorphous PLCL (the polymer with the weakest mechanical properties and lowest T(g)). QCM-D also monitored protein self-assembly in solution and confirmed that S-layer formation takes place in three main steps: adsorption, self-assembly, and crystal reorganization. Finally, this work shows that biodegradable polylactide derivatives films are a suitable support to form robust biomimetic S-protein layers.

  5. Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation.

    Science.gov (United States)

    White, Michael J V; Roife, David; Gomer, Richard H

    2015-08-15

    To metastasize, tumor cells often need to migrate through a layer of collagen-containing scar tissue which encapsulates the tumor. A key component of scar tissue and fibrosing diseases is the monocyte-derived fibrocyte, a collagen-secreting profibrotic cell. To test the hypothesis that invasive tumor cells may block the formation of the fibrous sheath, we determined whether tumor cells secrete factors that inhibit monocyte-derived fibrocyte differentiation. We found that the human metastatic breast cancer cell line MDA-MB-231 secretes activity that inhibits human monocyte-derived fibrocyte differentiation, whereas less aggressive breast cancer cell lines secrete less of this activity. Purification indicated that Galectin-3 binding protein (LGALS3BP) is the active factor. Recombinant LGALS3BP inhibits monocyte-derived fibrocyte differentiation, and immunodepletion of LGALS3BP from MDA-MB 231 conditioned media removes the monocyte-derived fibrocyte differentiation-inhibiting activity. LGALS3BP inhibits the differentiation of monocyte-derived fibrocytes from wild-type mouse spleen cells, but not from SIGN-R1(-/-) mouse spleen cells, suggesting that CD209/SIGN-R1 is required for the LGALS3BP effect. Galectin-3 and galectin-1, binding partners of LGALS3BP, potentiate monocyte-derived fibrocyte differentiation. In breast cancer biopsies, increased levels of tumor cell-associated LGALS3BP were observed in regions of the tumor that were invading the surrounding stroma. These findings suggest LGALS3BP and galectin-3 as new targets to treat metastatic cancer and fibrosing diseases.

  6. column chromatography separation of crude and purified protein in cobratoxin and analysis of anti-tumor activity of its fractions%眼镜蛇毒粗纯蛋白的柱层析分离及其各馏分的抗肿瘤活性研究

    Institute of Scientific and Technical Information of China (English)

    陈思; 臧宁; 张虹

    2012-01-01

    Objective: To separate crude protein and purified protein in cobratoxin and analyze anti - tumor activity of its fractions. Methods:The crude protein and purified protein in cobratoxin was separated by ion exchange chroma-tography. Qualitative study was made on each fraction by means of analysis by reverse -phase chromatography, content determination and anti -tumor activity test. Results: Four elution peaks were obtained when a linear gradient e-lution was applied to crude protein and purified protein in cobratoxin by adding sodium chloride (0 -0.5 mol · L‐1 ) to Tris - HC1 buffer (20 mmol · L ‐1, pH 8. 0) on DEAE - sephacel ion - exchange column. Among the four elution peaks, the first one showed the highest anti -tumor activity. It contained two types of proteins, which differed in hy-drophobicity and ultraviolet absorption, while the other three elution peaks contained single protein respectively. Conclusion ; The fractions from cobratoxin have some inhibitory effects on tumor cell lines cultured in vitro.%目的:眼镜蛇毒粗纯蛋白的分离及其馏分抗痛活性研究.方法:应用离子交换层析法分离眼镜蛇毒粗纯蛋白,通过高效液相色谱行为分析、含量测定和抗肿瘤活性试验对各馏分进行了性质研究.结果:以Tris-HCI缓冲液(20 mmol·L-1,pH8.0)为洗脱体系,在DEAE-sephacel填料离子交换柱上对眼镜蛇毒粗纯蛋白以O~0.5 mol·L-1氯化钠的Tris-HCL溶液进行线性梯度洗脱,可得到4个洗脱峰.第1个洗脱峰馏分抗癌活性最强,含有两类疏水性、紫外吸收特性各异的蛋白混合物;另3个洗脱峰对应馏分蛋白成分较单一.结论:眼镜蛇毒粗纯蛋白分离馏分对体外培养的癌细胞株有一定的抑制作用.

  7. Freeze-drying of proteins with glass-forming oligosaccharide-derived sugar alcohols.

    Science.gov (United States)

    Kadoya, Saori; Fujii, Kahori; Izutsu, Ken-ichi; Yonemochi, Etsuo; Terada, Katsuhide; Yomota, Chikako; Kawanishi, Toru

    2010-04-15

    Physical properties and protein-stabilizing effects of sugar alcohols in frozen aqueous solutions and freeze-dried solids were studied. Various frozen sugar alcohol solutions showed a glass transition of the maximally freeze-concentrated phase at temperatures (T(g)'s) that depended largely on the solute molecular weights. Some oligosaccharide-derived sugar alcohols (e.g., maltitol, lactitol, maltotriitol) formed glass-state amorphous cake-structure freeze-dried solids. Microscopic observation of frozen maltitol and lactitol solutions under vacuum (FDM) indicated onset of physical collapse at temperatures (T(c)) several degrees higher than their T(g)'s. Freeze-drying of pentitols (e.g., xylitol) and hexitols (e.g., sorbitol, mannitol) resulted in collapsed or crystallized solids. The glass-forming sugar alcohols prevented activity loss of a model protein (LDH: lactate dehydrogenase) during freeze-drying and subsequent storage at 50 degrees C. They also protected bovine serum albumin (BSA) from lyophilization-induced secondary structure perturbation. The glass-forming sugar alcohols showed lower susceptibility to Maillard reaction with co-lyophilized L-lysine compared to reducing and non-reducing disaccharides during storage at elevated temperature. Application of the oligosaccharide-derived sugar alcohols as alternative stabilizers in lyophilized protein formulations was discussed. Copyright 2010 Elsevier B.V. All rights reserved.

  8. Characterization of a probiotic-derived soluble protein which reveals a mechanism of preventive and treatment effects of probiotics on intestinal inflammatory diseases.

    Science.gov (United States)

    Yan, Fang; Polk, D Brent

    2012-01-01

    The beneficial effects of probiotics have been demonstrated in many diseases, such as inflammatory bowel disease. The known mechanisms for probiotic action include blocking pathogenic bacterial effects, enhancing the innate immunity and decreasing pathogen-induced inflammation, and promoting intestinal epithelial cell survival, barrier function, and protective responses. We purified and cloned a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, p40. This protein ameliorated cytokine-induced apoptosis in intestinal epithelial cells through activation of the EGF receptor and its down-stream target, Akt. By using special hydrogel beads to protect p40 from degradation, we showed that p40 reduced intestinal epithelial apoptosis and preserved barrier function in the colon epithelium in an EGF receptor-dependent manner, thereby preventing and treating intestinal inflammation in mouse models of colitis. Further works regarding structural analysis of p40, regulation of EGF receptor activation and immunoregulatory effects by p40 are discussed. These results may provide insights into the clinical application of probiotics for intestinal inflammatory disorders.

  9. Prediction of protein secondary structure from circular dichroism using theoretically derived spectra.

    Science.gov (United States)

    Louis-Jeune, Caroline; Andrade-Navarro, Miguel A; Perez-Iratxeta, Carol

    2012-02-01

    Circular dichroism (CD) is a spectroscopic technique commonly used to investigate the structure of proteins. Major secondary structure types, alpha-helices and beta-strands, produce distinctive CD spectra. Thus, by comparing the CD spectrum of a protein of interest to a reference set consisting of CD spectra of proteins of known structure, predictive methods can estimate the secondary structure of the protein. Currently available methods, including K2D2, use such experimental CD reference sets, which are very small in size when compared to the number of tertiary structures available in the Protein Data Bank (PDB). Conversely, given a PDB structure, it is possible to predict a theoretical CD spectrum from it. The methodological framework for this calculation was established long ago but only recently a convenient implementation called DichroCalc has been developed. In this study, we set to determine whether theoretically derived spectra could be used as reference set for accurate CD based predictions of secondary structure. We used DichroCalc to calculate the theoretical CD spectra of a nonredundant set of structures representing most proteins in the PDB, and applied a straightforward approach for predicting protein secondary structure content using these theoretical CD spectra as reference set. We show that this method improves the predictions, particularly for the wavelength interval between 200 and 240 nm and for beta-strand content. We have implemented this method, called K2D3, in a publicly accessible web server at http://www. ogic.ca/projects/k2d3.

  10. Activation of Adhesion G Protein-coupled Receptors: AGONIST SPECIFICITY OF STACHEL SEQUENCE-DERIVED PEPTIDES.

    Science.gov (United States)

    Demberg, Lilian M; Winkler, Jana; Wilde, Caroline; Simon, Kay-Uwe; Schön, Julia; Rothemund, Sven; Schöneberg, Torsten; Prömel, Simone; Liebscher, Ines

    2017-03-17

    Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the Stachel sequence, can activate the respective receptor. As the conserved core region of the Stachel sequence is highly similar between aGPCRs, the agonist specificity of Stachel sequence-derived peptides was tested between family members using cell culture-based second messenger assays. Stachel peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the Stachel sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the Stachel-derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the Stachel sequence are very similar between aGPCRs allowing for agonist promiscuity of several Stachel-derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for in vitro and in vivo studies. However, it also offers the possibility of a broader use of more potent peptides when the original Stachel sequence is less effective. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs.

    Science.gov (United States)

    Froyen, G; Proost, P; Ronsse, I; Mitera, T; Haelens, A; Wuyts, A; Opdenakker, G; Van Damme, J; Billiau, A

    1997-02-01

    Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The

  12. Ion-Exchange Chromatography for Purifying TRAIL Protein Expressed by Prokaryote%离子交换层析法纯化原核表达的肿瘤坏死因子相关凋亡诱导配体蛋白

    Institute of Scientific and Technical Information of China (English)

    王琼秀; 周菁

    2013-01-01

    Objective To establish a method for purifying the tumor necrosis factor related apoptosis inducing ligand proteins(TRAIL)protein expressed by prokaryote.Methods PET28a-TRAIL was transferred into B121 (DE3),the primary separation was firstly conducted by ammonium sulfate,and then the CM Sepharose cation exchange column was adopted to perform the ion-exchange chromatography for purifying TRAIL protein.Results The expression of TRAIL protein reached the maximal quantity at 20 ℃ with 8 h induction by IPTG(0.5 mM).After the primary separation by ammonium sulfate and the purification by the ion-exchange column chromatography,5.8 mg pure Trail protein was obtained from 1L bacteria liquid.Conclusion It is successful to establish the large-scale purification method of the Trail protein expressed by prokaryote.%目的 纯化原核表达的肿瘤坏死因子相关凋亡诱导配体(TRAIL)蛋白.方法 将pET28a-TRIAL转入BL21(DE3),先进行硫酸铵初级分离,然后利用CM Sepharose阳离子交换柱进行离子交换层析进行纯化.结果 在20℃条件下,使用0.5 mmol/LIPTG诱导8h,TRAIL蛋白表达量达到最大;经硫酸铵初步分离和离子交换层析纯化后,从1L菌液纯化得到5.8mg纯净的TRAIL蛋白.结论 成功建立了一种原核表达TRIAL蛋白的大规模纯化方法.

  13. Investigating isoquinoline derivatives for inhibition of inhibitor of apoptosis proteins for ovarian cancer treatment

    Directory of Open Access Journals (Sweden)

    Chen C

    2017-09-01

    Full Text Available Chen Chen,1,* Jie Wu,2,* Pengfei Zhu,3 Congjian Xu,1 Liangqing Yao1 1Obstetrics and Gynecology Hospital and Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, 2Department of Chemistry, Fudan University, Shanghai, 3Department of Obstetrics and Gynecology, Shangyu City Hospital, Shangyu, Zhejiang Province, People’s Republic of China *These authors contributed equally to this work Objective: To discover novel isoquinoline derivatives for inhibition of inhibitor of apoptosis proteins (IAP for the treatment of ovarian cancer. Methods: We first synthesized 533 isoquinoline derivatives, and screened them using CCK-8 to measure their antiproliferative activity. These compounds were further tested by Hoechst staining and flow cytometric analysis to assess proapoptotic activity. The in vivo antitumor efficacy and safety of the screened compounds were evaluated on the xenograft mouse model. Ki-67 staining and TUNEL assay were used to evaluate proliferation and apoptosis in the resected tumors, respectively. Western blot and polymerase chain reaction (PCR were conducted to evaluate the levels of proliferating cell nuclear antigen (PCNA, caspase-3, PARP, and IAP in resected tumors. Results: Compound B01002 and C26001 displayed antiproliferative and proapoptotic activity on SKOV3 ovarian cancer with an IC50 of 7.65 and 11.68 µg/mL, respectively. Both compounds inhibited tumor growth in a xenografted mouse model with good safety profiles, and tumor growth inhibition (TGI of B01002 and C26001 was 99.53% and 84.23%, respectively. Resected tumors showed that both compounds inhibited tumor cell proliferation and induced apoptosis in vivo. Caspase-3 and PARP were activated, whereas IAP proteins were downregulated at the protein level. Conclusion: Compound B01002 and C26001 could inhibit ovarian tumor growth and promote tumor apoptosis, partly by downregulating the IAPs, and, thus, might be promising candidates for treatment of ovarian

  14. Ligand exchange between proteins. Exchange of biotin and biotin derivatives between avidin and streptavidin.

    Science.gov (United States)

    Pazy, Yael; Kulik, Tikva; Bayer, Edward A; Wilchek, Meir; Livnah, Oded

    2002-08-23

    We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs

  15. Ceramic materials purified by experimental method

    Science.gov (United States)

    1965-01-01

    Crystalline ceramic materials are purified for use as high-temperature electrical insulators. Any impurities migrate to the cathode when a dc voltage is applied across the material while it is heated in an inert gas atmosphere.

  16. Bioactive 4-Oxoheptanedioic Monoamide Derivatives of Proteins and Ethanolaminephospholipids: Products of Docosahexaenoate Oxidation.

    Science.gov (United States)

    Guo, Junhong; Hong, Li; West, Xiaoxia Z; Wang, Hua; Salomon, Robert G

    2016-10-17

    Oxidative stress causes lipid-derived oxidative modification of biomolecules that has been implicated in many pathological states. Phospholipids containing polyunsaturated fatty acids are major targets of free radical-initiated oxidation. Phospholipids that incorporate docosahexaenoate (DHA) are highly enriched in important neural structures including the brain and retina, where DHA comprises 40% and 60% of total fatty acids, respectively. Oxidative fragmentation of 2-docosahexaenoyl-1-palmityl-sn-glycerophosphocholine generates esters of 4-hydroxy-7-oxohept-5-enoic acid (HOHA) and 4-keto-7-oxohept-5-enoic acid (KOHA) with 2-lysophosphatidylcholine, HOHA-PC, and KOHA-PC. Covalent HOHA adducts that incorporate the primary amino groups of proteins and ethanolamine phospholipids in carboxyethylpyrrole (CEP) derivatives were detected immunologically with anti-CEP antibodies in human tumors, retina, and blood. Now, we generated an anti-OHdiA antibody to test the hypothesis that KOHA adducts, which incorporate the primary amino groups of proteins or ethanolamine phospholipids in 4-oxo-heptanedioic (OHdiA) monoamide derivatives, are present in vivo. However, whereas the anti-CEP antibody is highly specific and does not cross-react with the OHdiA monoamide epitope, the anti-OHdiA monoamide antibody cross-reacted with CEP epitopes making it of little value as an analytical tool for OHdiA monoamides but suggesting the possibility that OHdiA monoamides would exhibit receptor-mediated biological activity similar to that of CEP. An LC-MS/MS method was developed that allows quantification of OHdiA derivatives in biological samples. We now find that KOHA-PC forms OHdiA monoamide adducts of proteins and ethanolamine phospholipids and that OHdiA-protein levels are significantly higher than OHdiA-ethanloamine phospholipid levels in blood from healthy human subjects, 0.45 μM and 0.18 μM, respectively (n = 3, and p = 0.027). OHdiA monoamide epitopes are angiogenic, causing TLR2

  17. The application of an enamel matrix protein derivative (Emdogain) in regenerative periodontal therapy: a review.

    Science.gov (United States)

    Sculean, Anton; Schwarz, Frank; Becker, Jurgen; Brecx, Michel

    2007-01-01

    Regenerative periodontal therapy aims at reconstitution of the lost periodontal structures such as new formation of root cementum, periodontal ligament and alveolar bone. Findings from basic research indicate that enamel matrix protein derivative (EMD) has a key role in periodontal wound healing. Histological results from animal and human studies have shown that treatment with EMD promotes periodontal regeneration. Moreover, clinical studies have indicated that treatment with EMD positively influences periodontal wound healing in humans. This review aims to present an overview of evidence-based clinical indications for regenerative therapy with EMD.

  18. Oleanolic acid and its derivatives: new inhibitor of protein tyrosine phosphatase 1B with cellular activities.

    Science.gov (United States)

    Zhang, Yi-Nan; Zhang, Wei; Hong, Di; Shi, Lei; Shen, Qiang; Li, Jing-Ya; Li, Jia; Hu, Li-Hong

    2008-09-15

    Protein tyrosine phosphatase 1B is a key factor in the negative regulation of insulin pathway and a promising target for treatment of diabetes and obesity. Herein, a series of competitive inhibitors were optimized from oleanolic acid, a natural triterpenoid identified against PTP1B by screening libraries of traditional Chinese medicinal herbs. Modifying at 3 and 28 positions, we obtained compound 13 with a K(i) of 130 nM, which exhibited good selectivity between other phosphatases involved in insulin pathway except T-cell protein tyrosine phosphatase. Further evaluation in cell models illustrated that the derivatives enhanced insulin receptor phosphorylation in CHO/hIR cells and also stimulated glucose uptake in L6 myotubes with or addition of without insulin.

  19. Application of intelligent techniques for classification of bacteria using protein sequence-derived features.

    Science.gov (United States)

    Banerjee, Amit Kumar; Ravi, Vadlamani; Murty, U S N; Sengupta, Neelava; Karuna, Batepatti

    2013-07-01

    Standard molecular experimental methodologies and mathematical procedures often fail to answer many phylogeny and classification related issues. Modern artificial intelligent-based techniques, such as radial basis function, genetic algorithm, artificial neural network, and support vector machines are of ample potential in this regard. Reliance on a large number of essential parameters will aid in enhanced robustness, reliability, and better accuracy as opposed to single molecular parameter. This study was conducted with dataset of computed protein physicochemical properties belonging to 20 different bacterial genera. A total of 57 sequential and structural parameters derived from protein sequences were considered for the initial classification. Feature selection based techniques were employed to find out the most important features influencing the dataset. Various amino acids, hydrophobicity, relative sulfur percentage, and codon number were selected as important parameters during the study. Comparative analyses were performed applying RapidMiner data mining platform. Support vector machine proved to be the best method with maximum accuracy of more than 91%.

  20. SARS Patients-derived Human Recombinant Antibodies to S and M Proteins Efficiently Neutralize SARS-Coronavirus Infectivity

    Institute of Scientific and Technical Information of China (English)

    MI-FANG LIANG; KONG-XING WU; ZHAO-HUI XIONG; QI JIN; DE-XIN LI; RUN-LEI DU; JING-ZHI LIU; CHUAN LI; QUAN-FU ZHANG; LU-LU HAN; JIAN-SHI YU; SHU-MIN DUAN; XIAO-FANG WANG

    2005-01-01

    Objective To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. Methods By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. Coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. Results After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions

  1. Studies on Purification of Methamidophos Monoclonal Antibodies and Comoarative Immunoactivity of Purified Antibodies

    Institute of Scientific and Technical Information of China (English)

    SU-QING ZHAO; YUAN-MING SUN; CHUN-YAN ZHANG; XIAO-YU HUANG; HOU-RUI ZHANG; ZHEN-YU ZHU

    2003-01-01

    Objective To purify Methamidophos (Met) monoclonal antibodies with two methods andcompare immune activity of purified antibodies. Method Caprylic acid ammonium sulphateprecipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method wereused to purify Met monoclonal antibodies, UV spectrum scanning was used to determine proteincontent and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linkedimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mLand 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASPmethod. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was181.26 μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03 μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29 ug/mL and 3.42 μg/mL,respectively. Conclusion Antibodies purified by SPA method are better than those by CAASPmethod, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelledtesting paper for analyzing Met residue in vegetable and drink water.

  2. Unraveling the Roots of Selectivity of Peptide Affinity Reagents for Structurally Similar Ribosomal Inactivating Protein Derivatives

    Directory of Open Access Journals (Sweden)

    Deborah A. Sarkes

    2016-11-01

    Full Text Available Peptide capture agents have become increasingly useful tools for a variety of sensing applications due to their ease of discovery, stability, and robustness. Despite the ability to rapidly discover candidates through biopanning bacterial display libraries and easily mature them to Protein Catalyzed Capture (PCC agents with even higher affinity and selectivity, an ongoing challenge and critical selection criteria is that the peptide candidates and final reagent be selective enough to replace antibodies, the gold-standard across immunoassay platforms. Here, we have discovered peptide affinity reagents against abrax, a derivative of abrin with reduced toxicity. Using on-cell Fluorescence Activated Cell Sorting (FACS assays, we show that the peptides are highly selective for abrax over RiVax, a similar derivative of ricin originally designed as a vaccine, with significant structural homology to abrax. We rank the newly discovered peptides for strongest affinity and analyze three observed consensus sequences with varying affinity and specificity. The strongest (Tier 1 consensus was FWDTWF, which is highly aromatic and hydrophobic. To better understand the observed selectivity, we use the XPairIt peptide–protein docking protocol to analyze binding location predictions of the individual Tier 1 peptides and consensus on abrax and RiVax. The binding location profiles on the two proteins are quite distinct, which we determine is due to differences in pocket size, pocket environment (including hydrophobicity and electronegativity, and steric hindrance. This study provides a model system to show that peptide capture candidates can be quite selective for a structurally similar protein system, even without further maturation, and offers an in silico method of analysis for understanding binding and down-selecting candidates.

  3. Unbiased Quantitative Models of Protein Translation Derived from Ribosome Profiling Data.

    Directory of Open Access Journals (Sweden)

    Alexey A Gritsenko

    2015-08-01

    Full Text Available Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of protein synthesis. Although a number of computational models of the process have been proposed, their application is limited by the assumptions they make. Ribosome profiling (RP, a relatively new sequencing-based technique capable of recording snapshots of the locations of actively translating ribosomes, is a promising source of information for deriving unbiased data-driven translation models. However, quantitative analysis of RP data is challenging due to high measurement variance and the inability to discriminate between the number of ribosomes measured on a gene and their speed of translation. We propose a solution in the form of a novel multi-scale interpretation of RP data that allows for deriving models with translation dynamics extracted from the snapshots. We demonstrate the usefulness of this approach by simultaneously determining for the first time per-codon translation elongation and per-gene translation initiation rates of Saccharomyces cerevisiae from RP data for two versions of the Totally Asymmetric Exclusion Process (TASEP model of translation. We do this in an unbiased fashion, by fitting the models using only RP data with a novel optimization scheme based on Monte Carlo simulation to keep the problem tractable. The fitted models match the data significantly better than existing models and their predictions show better agreement with several independent protein abundance datasets than existing models. Results additionally indicate that the tRNA pool adaptation hypothesis is incomplete, with evidence suggesting that tRNA post-transcriptional modifications and codon context may play a role in determining codon elongation rates.

  4. Unbiased Quantitative Models of Protein Translation Derived from Ribosome Profiling Data.

    Science.gov (United States)

    Gritsenko, Alexey A; Hulsman, Marc; Reinders, Marcel J T; de Ridder, Dick

    2015-08-01

    Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of protein synthesis. Although a number of computational models of the process have been proposed, their application is limited by the assumptions they make. Ribosome profiling (RP), a relatively new sequencing-based technique capable of recording snapshots of the locations of actively translating ribosomes, is a promising source of information for deriving unbiased data-driven translation models. However, quantitative analysis of RP data is challenging due to high measurement variance and the inability to discriminate between the number of ribosomes measured on a gene and their speed of translation. We propose a solution in the form of a novel multi-scale interpretation of RP data that allows for deriving models with translation dynamics extracted from the snapshots. We demonstrate the usefulness of this approach by simultaneously determining for the first time per-codon translation elongation and per-gene translation initiation rates of Saccharomyces cerevisiae from RP data for two versions of the Totally Asymmetric Exclusion Process (TASEP) model of translation. We do this in an unbiased fashion, by fitting the models using only RP data with a novel optimization scheme based on Monte Carlo simulation to keep the problem tractable. The fitted models match the data significantly better than existing models and their predictions show better agreement with several independent protein abundance datasets than existing models. Results additionally indicate that the tRNA pool adaptation hypothesis is incomplete, with evidence suggesting that tRNA post-transcriptional modifications and codon context may play a role in determining codon elongation rates.

  5. MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.

    Directory of Open Access Journals (Sweden)

    Laura Camacho

    Full Text Available Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM versus non-brain metastatic (non-BM cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210 and two down-regulated miRNAs (miR-19a and miR-29c in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis.

  6. MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.

    Science.gov (United States)

    Camacho, Laura; Guerrero, Paola; Marchetti, Dario

    2013-01-01

    Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM) versus non-brain metastatic (non-BM) cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM) variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210) and two down-regulated miRNAs (miR-19a and miR-29c) in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis.

  7. Protein adsorption on the poly(L-lactic acid) surface modified by chitosan and its derivatives

    Institute of Scientific and Technical Information of China (English)

    JIAO YanPeng; ZHOU ChangRen; LI LiHua; DING Shan; LU Lu; LUO BingHong; LI Hong

    2009-01-01

    Surface modification of biomaterials has been adopted over the years to improve their biocompatibility.In this study,aiming to promote hydrophilicity and to introduce natural recognition sites onto poly(L-lactic acid) (PLLA) films,chitosan and its derivatives,carboxymethyl chitosan(CMC) and N-methylene phosphonic chitosan (NPC),were used to modify the surface of PLLA films by an entrapment method. Radiolabeled (12SI) proteins were used to measure the amount of protein adsorbed to PLLA surfaces. Fibronectin (Fn) was used to study the protein adsorption on the modified PLLA surfaces,including isotherm adsorption and adsorption kinetics of single protein,competitive adsorption of binary proteins system and serum multi-proteins and the desorption behavior in serum solution. The results showed that in the isotherm adsorption,Fn had a larger adsorption capacity on the CS-modified surface at lower concentrations,but had a high adsorption capacity at CMC-modified surface at higher concentrations. In the study of absorption kinetics,Fn had a fastest adsorption equilibrium and a highest equilibrium adsorption capacity at the CS-modified surface,while it was opposite at the PCS-modified surface. When BSA and serum were added,it had the greatest effect on the adsorption of Fn on the PCS-modified surface. After 6 hours soaking in the desorption study,Fn had reached desorption equilibrium on all the modified surfaces,which had different effects on the desorption rate and the remaining percentage of Fn.

  8. Expression of domains for protein-protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability.

    Science.gov (United States)

    Jordheim, Lars Petter; Cros-Perrial, Emeline; Matera, Eva-Laure; Bouledrak, Karima; Dumontet, Charles

    2014-10-01

    Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein-protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2-2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4-5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein-protein interactions.

  9. Voluntary exercise protects against stress-induced decreases in brain-derived neurotrophic factor protein expression.

    Science.gov (United States)

    Adlard, P A; Cotman, C W

    2004-01-01

    Exercise is increasingly recognized as an intervention that can reduce CNS dysfunctions such as cognitive decline, depression and stress. Previously we have demonstrated that brain-derived neurotrophic factor (BDNF) is increased in the hippocampus following exercise. In this study we tested the hypothesis that exercise can counteract a reduction in hippocampal BDNF protein caused by acute immobilization stress. Since BDNF expression is suppressed by corticosterone (CORT), circulating CORT levels were also monitored. In animals subjected to 2 h immobilization stress, CORT was elevated immediately following, and at 1 h after the cessation of stress, but remained unchanged from baseline up to 24 h post-stress. The stress protocol resulted in a reduction in BDNF protein at 5 and 10 h post-stress that returned to baseline at 24 h. To determine if exercise could prevent this stress-induced reduction in BDNF protein, animals were given voluntary access to running wheels for 3 weeks prior to the stress. Stressed animals, in the absence of exercise, again demonstrated an initial elevation in CORT (at 0 h) and a subsequent decrease in hippocampal BDNF at the 10 h time point. Exercising animals, both non-stressed and stressed, demonstrated circulating CORT and hippocampal BDNF protein levels that were significantly elevated above control values at both time points examined (0 and 10 h post-stress). Thus, the persistently high CORT levels in exercised animals did not affect the induction of BDNF with exercise, and the effect of immobilization stress on BDNF protein was overcome. To examine the role of CORT in the stress-related regulation of BDNF protein, experiments were carried out in adrenalectomized (ADX) animals. BDNF protein was not downregulated as a result of immobilization stress in ADX animals, while there continued to be an exercise-induced upregulation of BDNF. This study demonstrates that CORT modulates stress-related alterations in BDNF protein. Further, exercise

  10. Short- and long-term antihypertensive effect of egg protein-derived peptide QIGLF.

    Science.gov (United States)

    Yu, Zhipeng; Zhao, Wenzhu; Ding, Long; Wang, Yaqi; Chen, Feng; Liu, Jingbo

    2017-01-01

    The present study aimed to investigate the in vivo antihypertensive effect on spontaneously hypertensive rats (SHRs) induced by egg protein-derived peptide QIGLF, which has been previously characterized in vitro as a potent angiotensin-converting enzyme inhibitor. In vivo antihypertensive effect of QIGLF orally administered was evaluated by the tail-cuff method. The systolic blood pressure and the diastolic blood pressure of rats were measured 0, 5, 10, 15 and 20 h after administration every day. Subsequently, the effect of QIGLF on angiotensin-converting enzyme mRNA expression in the kidney of SHRs was evaluated by a polymerase chain reaction. Systolic blood pressure was found to be reduced markedly in the SHRs after a single oral administration. The results show that the effect of QIGLF (50 mg kg(-1) body weight) was similar to that of captopril (10 mg kg(-1) body weight) with respect to lowering systolic blood pressure in SHRs. Therefore, egg white protein-derived peptide QIGLF may be useful in the prevention or treatment of hypertension. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  11. Protein composition and biomechanical properties of in vivo-derived basement membranes.

    Science.gov (United States)

    Halfter, Willi; Candiello, Joseph; Hu, Haiyu; Zhang, Peng; Schreiber, Emanuel; Balasubramani, Manimalha

    2013-01-01

    Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.

  12. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J. Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H. James; Huband, Michael D.; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y.; Mehrens, Shawn; Mueller, W. Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J.V.N. Vara; Shelly, John A.; Skerlos, Laura; Sulavik, Mark; Thomas, V. Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C. Kendall; (Pfizer)

    2009-06-25

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  13. Effect of different enamel matrix derivative proteins on behavior and differentiation of endothelial cells.

    Science.gov (United States)

    Andrukhov, Oleh; Gemperli, Anja C; Tang, Yan; Howald, Nadia; Dard, Michel; Falkensammer, Frank; Moritz, Andreas; Rausch-Fan, Xiaohui

    2015-07-01

    Enamel matrix derivative (EMD) is an effective biomaterial for periodontal tissue regeneration and might stimulate angiogenesis. In order to clarify mechanisms underlying its biological activity, we separated two EMD fractions with different molecular weight protein components and investigated their effects on human umbilical vein endothelial cells (HUVECs) in vitro. Fraction Low-Molecular Weight (LMW) included proteins with a molecular weight (M.W.)8kDa and lower than approximately 55kDa. The effect of EMD fractions on proliferation/viability, apoptosis, migration and expression of angiopoetin-2 (ang-2), von Willebrand factor (vWF), E-selectin, intracellular adhesion molecules 1 (ICAM-1), vascular endothelial growth factor (VEGF) receptors Flt-1 and KDR was investigated. The proliferation/viability of HUVECs was inhibited by both LMW and LMW-depleted at concentrations 100μg/ml, whereas EMD slightly increased cell proliferation/viability. The expression of all investigated proteins was up-regulated by EMD. However, differences in the effect of EMD fractions on the protein expression were observed. The effect of LMW-depleted on the expression of ICAM-1 and E-selectin was markedly higher compared to LMW. In contrast, the expression of vWF and VEGF receptors Flt-1 and KDR was primarily affected LMW than by LMW depleted. The expression of ang-2 was not influenced by LMW and LMW-depleted. HUVECs migration was stimulated more strongly by LMW than by EMD and LMW-depleted. Our in vitro study shows that the proteins composing EMD have different and specific biological activities and consequently have the ability to cover different aspects of EMD's biological and clinical effects. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  14. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

    Science.gov (United States)

    Miller, J Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H James; Huband, Michael D; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y; Mehrens, Shawn; Mueller, W Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J V N Vara; Shelly, John A; Skerlos, Laura; Sulavik, Mark; Thomas, V Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C Kendall

    2009-02-10

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  15. Screening of soy protein-derived hypotriglyceridemic di-peptides in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Matsui Toshiro

    2011-05-01

    Full Text Available Abstract Background Soy protein and soy peptides have attracted considerable attention because of their potentially beneficial biological properties, including antihypertensive, anticarcinogenic, and hypolipidemic effects. Although soy protein isolate contains several bioactive peptides that have distinct physiological activities in lipid metabolism, it is not clear which peptide sequences are responsible for the triglyceride (TG-lowering effects. In the present study, we investigated the effects of soy protein-derived peptides on lipid metabolism, especially TG metabolism, in HepG2 cells and obese Otsuka Long-Evans Tokushima fatty (OLETF rats. Results In the first experiment, we found that soy crude peptide (SCP-LD3, which was prepared by hydrolyze of soy protein isolate with endo-type protease, showed hypolipidemic effects in HepG2 cells and OLETF rats. In the second experiment, we found that hydrophilic fraction, separated from SCP-LD3 with hydrophobic synthetic absorbent, revealed lipid-lowering effects in HepG2 cells and OLETF rats. In the third experiment, we found that Fraction-C (Frc-C peptides, fractionated from hydrophilic peptides by gel permeation chromatography-high performance liquid chromatography, significantly reduced TG synthesis and apolipoprotein B (apoB secretion in HepG2 cells. In the fourth experiment, we found that the fraction with 0.1% trifluoroacetic acid, isolated from Frc-C peptides by octadecylsilyl column chromatography, showed hypolipidemic effects in HepG2 cells. In the final experiment, we found that 3 di-peptides, Lys-Ala, Val-Lys, and Ser-Tyr, reduced TG synthesis, and Ser-Tyr additionally reduced apoB secretion in HepG2 cells. Conclusion Novel active peptides with TG-lowering effects from soy protein have been isolated.

  16. Milk-derived proteins and peptides of potential therapeutic and nutritive value.

    Science.gov (United States)

    Zimecki, Michal; Kruzel, Marian L

    2007-01-01

    Milk and colostrum are rich in proteins and peptides which play a crucial role in development of the immune system in mammalian offspring. Immunotropic properties of these compounds prompted investigators to search for their utility in prevention and therapy of various disorders in humans. The following constituents of milk are of particular interest: 1) Lactoferrin (LF)--exhibits antibacterial, antifungal, antiviral, antiparasite and antitumor activities. It is protective with regard to intestinal epithelium, promotes bone growth and accelerates recovery of the immune system function in immunocompromised animal; 2) A Proline-Rich Polypeptide (PRP) shows a variety of immunotropic functions, including promotion of T-cell maturation and inhibition'of autoimmune disorders. PRP was recently found to improve or stabilize the Instrumental Activity of Daily Living status in Alzheimer's disease patients. 3) Casein--has been protective in experimental bacteremia by eliciting myelopoiesis. Casein hydrolyzates were also protective in diabetic animals, reduced the tumor growth and diminished colicky symptoms in infants. Casein-derived peptides have been found to have antihypertensive effects. Glycomacropeptide (GMP)--a peptide derived from kappa casein, exhibits antibacterial and antithrombotic activities. 4) Alpha lactalbumin (LA)--demonstrates antiviral, antitumor and anti-stress properties. LA-enriched diets were anxiolytic, lowered blood pressure in rats, prevented diarrhea and led to a better weight gain in malnourished children. 5) Lysozyme--is effective in treatment of periodentitis and prevention of tooth decay. Milk enriched in lysozyme was used in feeding premature infants suffering from concomitant diseases. 6) Lactoperoxidase--shows antibacterial properties. In conclusion, milk-derived proteins and peptides are bio-accessible and safe for the prevention and treatment of numerous disorders in humans.

  17. Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

    Science.gov (United States)

    Li, Hao; Onbe, Keisuke; Liu, Qiushi; Iijima, Masumi; Tatematsu, Kenji; Seno, Masaharu; Tada, Hiroko; Kuroda, Shun' Ichi

    2017-08-19

    Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Leghemoglobin-derived radicals. Evidence for multiple protein-derived radicals and the initiation of peribacteroid membrane damage

    DEFF Research Database (Denmark)

    Moreau, S; Davies, Michael Jonathan; Mathieu, C

    1996-01-01

    Reaction of H2O2 with ferric leghemoglobin (metLb, the monomeric, oxygen-carrying, heme protein from root nodules of nitrogen-fixing plants) has been previously shown to generate an iron(IV)-oxo (ferryl) species and at least one protein radical. The latter has been suggested to be a tyrosine-deri...

  19. Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF1

    Science.gov (United States)

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

    2008-01-01

    Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

  20. Synthetic peptides derived from ribosomal proteins of Leishmania spp. in mucocutaneous leishmaniasis: Diagnostic usefulness.

    Science.gov (United States)

    Florez, Magda Melissa; de Oliveira, Camila Indiani; Puerta, Concepción; Guzmán, Fanny; Ayala, Martha; Montoya, Gladis; Delgado, Gabriela

    2017-07-28

    The serological diagnostic methods currently available for mucocutaneous leishmaniasis (MCL) lack specificity when complete parasites are used; however, such specificity increases when protein fractions are used. Ribosomal proteins have been reported to induce antibodies in animal and humans infected with the parasite, making them a worth candidate to assess its diagnosis potential. This study was thus aimed at evaluating synthetic peptides derived from Leishmania braziliensis ribosomal proteins S25 and S5 as antigen candidates for diagnosing MCL by ELISA; 4 of these 21 peptides (P4, P6, P19 and P21) had the greatest sensitivity (21.7%, 13.04%, 20% and 20%, respectively) as well as having 95%, 100%, 100% and 82.5% specificity, respectively. The study revealed the limited usefulness of the peptides being studied as a diagnostic tool in the conditions used here, because its low sensitivity, but it is worth highlighting that the use of peptides as antigen in the serodiagnosis of MCL may overcome the cross reaction presented with other antigens, thus avoiding false positives. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  1. Potent inhibition of protein tyrosine phosphatases by copper complexes with multi-benzimidazole derivatives.

    Science.gov (United States)

    Li, Ying; Lu, Liping; Zhu, Miaoli; Wang, Qingming; Yuan, Caixia; Xing, Shu; Fu, Xueqi; Mei, Yuhua

    2011-12-01

    A series of copper complexes with multi-benzimidazole derivatives, including mono- and di-nuclear, were synthesized and characterized by Fourier transform IR spectroscopy, UV-Vis spectroscopy, elemental analysis, electrospray ionization mass spectrometry. The speciation of Cu/NTB in aqueous solution was investigated by potentiometric pH titrations. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) were evaluated in vitro. The five copper complexes exhibit potent inhibition against PTP1B, TCPTP and PTP-MEG2 with almost same inhibitory effects with IC(50) at submicro molar level and about tenfold weaker inhibition versus SHP-1, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Fluorescence study on the interaction between PTP1B and complex 2 or 4 suggests that the complexes bind to PTP1B with the formation of a 1:1 complex. The binding constant are about 1.14 × 10(6) and 1.87 × 10(6) M(-1) at 310 K for 2 and 4, respectively.

  2. Spectral study, stability and protein labeling of two Carbazole–Benzothiazole derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Lu; Li, Ling; Fei, Xuening, E-mail: xueningfei888@163.com

    2014-05-01

    3-benzothiazole-9-ethyl-carbazole (BEC) and 3,6-di-benzothiazole-9-ethyl-carbazole (DBEC) were synthesized by using carbazole as base and then characterized by {sup 1}H NMR. The optical properties of them were investigated and compared with carbazole and 9-ethyl-carbazole. The photostability and thermostability of the compounds were studied and the results indicated they are both very stable. Effects of solvent properties including polarity, viscosity and refractive index on the spectra properties of the compounds were investigated. Quantum yield of the compounds in organic solvent was also determined and it is found the most complex compound has the highest quantum yield up to 0.68. Finally the synthesized compounds were used to label bovine serum albumin (BSA) protein and simultaneously stability constant (K{sub s}) between them is calculated and compared with other similar dyes. The results showed that the fluorescent intensity of the compound is increased significantly after labeling with BSA protein. - Highlights: • Synthesis and optical properties of two carbazole-benzothiazole derivatives. • Photostability and thermostability of the compounds and compared the results with each other. • Quantum yields of the compounds and the relationship with the structure. • Protein labeling properties of the compounds and stability constant.

  3. Rational design of a photo-responsive UVR8-derived protein and a self-assembling peptide-protein conjugate for responsive hydrogel formation

    Science.gov (United States)

    Zhang, Xiaoli; Dong, Chunming; Huang, Weiyun; Wang, Huaimin; Wang, Ling; Ding, Dan; Zhou, Hao; Long, Jiafu; Wang, Tingliang; Yang, Zhimou

    2015-10-01

    Responsive hydrogels hold great potential in controllable drug delivery, regenerative medicine, sensing, etc. We introduced in this study the first example of a photo-responsive protein for hydrogel formation. Based on the first example of the crystal structure of a photo-responsive protein, Arabidopsis thaliana protein UVR8, we designed and expressed its derived protein UVR8-1 with a hexa-peptide WRESAI. We also prepared supramolecular nanofibers with a TIP-1 protein at their surface. The simple mixing of these two components resulted in rapid hydrogel formation through the specific interactions between the protein TIP-1 and the peptide WRESAI. Since the protein could show a reversible dimer-monomer transformation, the resulting gels also showed a reversible gel-sol phase transition which was controlled by photo-irradiation. The photo-controllable gel-sol phase transition could be applied for protein delivery and cell separation.Responsive hydrogels hold great potential in controllable drug delivery, regenerative medicine, sensing, etc. We introduced in this study the first example of a photo-responsive protein for hydrogel formation. Based on the first example of the crystal structure of a photo-responsive protein, Arabidopsis thaliana protein UVR8, we designed and expressed its derived protein UVR8-1 with a hexa-peptide WRESAI. We also prepared supramolecular nanofibers with a TIP-1 protein at their surface. The simple mixing of these two components resulted in rapid hydrogel formation through the specific interactions between the protein TIP-1 and the peptide WRESAI. Since the protein could show a reversible dimer-monomer transformation, the resulting gels also showed a reversible gel-sol phase transition which was controlled by photo-irradiation. The photo-controllable gel-sol phase transition could be applied for protein delivery and cell separation. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05213k

  4. Immunotherapy of metastatic breast cancer patients with vitamin D-binding protein-derived macrophage activating factor (GcMAF).

    Science.gov (United States)

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki; Ushijima, Naofumi

    2008-01-15

    Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized beta-galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years.

  5. A New Pyridoxal Derivative for Transamination of N-Terminus of Proteins%A New Pyridoxal Derivative for Transamination of N-Terminus of Proteins

    Institute of Scientific and Technical Information of China (English)

    张美娟; 张学梅; 李娟; 郭庆祥

    2011-01-01

    A new pyridoxal-5-phosphate (PLP) derivative FHMDP was developed for the transamination of different pep- tides with three most hindered amino acid residues (Leu, Ile, Val) as their N-terminus. Compared to the previously reported reactions of PLP derivatives, the N-terminus transamination could be accomplished efficiently with the new compound.

  6. Fetuin-B, a liver-derived plasma protein is essential for fertilization.

    Science.gov (United States)

    Dietzel, Eileen; Wessling, Jennifer; Floehr, Julia; Schäfer, Cora; Ensslen, Silke; Denecke, Bernd; Rösing, Benjamin; Neulen, Joseph; Veitinger, Thomas; Spehr, Marc; Tropartz, Tanja; Tolba, René; Renné, Thomas; Egert, Angela; Schorle, Hubert; Gottenbusch, Yuliya; Hildebrand, André; Yiallouros, Irene; Stöcker, Walter; Weiskirchen, Ralf; Jahnen-Dechent, Willi

    2013-04-15

    The zona pellucida (ZP) is a glycoprotein matrix surrounding mammalian oocytes. Upon fertilization, ZP hardening prevents sperm from binding to and penetrating the ZP. Here, we report that targeted gene deletion of the liver-derived plasma protein fetuin-B causes premature ZP hardening and, consequently, female infertility. Transplanting fetuin-B-deficient ovaries into wild-type recipients restores fertility, indicating that plasma fetuin-B is necessary and sufficient for fertilization. In vitro fertilization of oocytes from fetuin-B-deficient mice only worked after rendering the ZP penetrable by laser perforation. Mechanistically, fetuin-B sustains fertility by inhibiting ovastacin, a cortical granula protease known to trigger ZP hardening. Thus, plasma fetuin-B is necessary to restrain protease activity and thereby maintain ZP permeability until after gamete fusion. These results also show that premature ZP hardening can cause infertility in mice.

  7. Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Kati Juuti-Uusitalo

    Full Text Available Retinal pigment epithelial (RPE cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP, the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC and RPE derived from the hESC (hESC-RPE. Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.

  8. Stromal cell-derived factor-1 potentiates bone morphogenetic protein-2 induced bone formation.

    Science.gov (United States)

    Higashino, Kosaku; Viggeswarapu, Manjula; Bargouti, Maggie; Liu, Hui; Titus, Louisa; Boden, Scott D

    2011-02-01

    The mechanisms driving bone marrow stem cell mobilization are poorly understood. A recent murine study found that circulating bone marrow-derived osteoprogenitor cells (MOPCs) were recruited to the site of recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone formation. Stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4 have been shown to mediate the homing of stem cells to injured tissues. We hypothesized that chemokines, such as SDF-1, are also involved with mobilization of bone marrow cells. The CD45(-) fraction is a major source of MOPCs. In this report we determined that the addition of BMP-2 or SDF-1 to collagen implants increased the number of MOPCs in the peripheral blood. BMP-2-induced mobilization was blocked by CXCR4 antibody, confirming the role of SDF-1 in mobilization. We determined for the first time that addition of SDF-1 to implants containing BMP-2 enhances mobilization, homing of MOPCs to the implant, and ectopic bone formation induced by suboptimal BMP-2 doses. These results suggest that SDF-1 increases the number of osteoprogenitor cells that are mobilized from the bone marrow and then home to the implant. Thus, addition of SDF-1 to BMP-2 may improve the efficiency of BMPs in vivo, making their routine use for orthopaedic applications more affordable and available to more patients.

  9. Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

    Directory of Open Access Journals (Sweden)

    Mariana Conceição Souza

    2015-06-01

    Full Text Available A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP-expressing Plasmodium berghei strain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3 inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

  10. Quantification of protein thiols using ThioGlo 1 fluorescent derivatives and HPLC separation.

    Science.gov (United States)

    Hoff, Signe; Larsen, Flemming H; Andersen, Mogens L; Lund, Marianne N

    2013-04-07

    A method for quantification of total soluble protein-derived thiols in beer was developed based on the formation of fluorescent adducts with the maleimide compound ThioGlo 1. The problem of interference from fluorescent adducts of sulfite and ThioGlo 1 was solved by HPLC separation of the adducts followed by fluorescence detection. Using standard addition of GSH, a detection limit of 0.028 μM thiols was achieved. The application and validation of the method was demonstrated for beers with different color intensities, and the application range is in principle for any biological system containing thiols. However, the quantification of cysteine was complicated by a lower fluorescence response of its ThioGlo 1 adducts. Based on the studies of the responses of a series of cysteine-derived thiols and (1)H NMR studies of the structures of ThioGlo 1 adducts with GSH and cysteine, it was concluded that thiols with a neighboring free amino group yield ThioGlo 1 adducts with a reduced fluorescence intensity.

  11. Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies.

    Science.gov (United States)

    Calpe, Silvia; Wagner, Koen; El Khattabi, Mohamed; Rutten, Lucy; Zimberlin, Cheryl; Dolk, Edward; Verrips, C Theo; Medema, Jan Paul; Spits, Hergen; Krishnadath, Kausilia K

    2015-11-01

    Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4(+) malignancies.

  12. Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF).

    Science.gov (United States)

    Yamamoto, Nobuto; Ushijima, Naofumi; Koga, Yoshihiko

    2009-01-01

    Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.

  13. Construction of a New Fusion Protein Vector Associated to Fibronectin Binding Protein A and Clumping Factor A derived from Staphylococcus aureus NCTC8325

    Directory of Open Access Journals (Sweden)

    Jamshid Faghri

    2009-03-01

    Full Text Available Objective(s Staphylococcus aureus is a leading cause of many nosocomial and community acquired infections. According to many reports, antibiotic therapy can not guarantee the eradication of S. aureus infections. Thus designing an adhesin based vaccine could restrain the S. aureus infections. This study designed for construction of a new fusion protein vaccine against S. aureus infections based on adhesin molecules fibronectin binding protein A (FnBPA and clumping factor A (ClfA. Materials and Methods Bioinformatic experiments were performed using Oligo analyzer and DNAMAN softwares. The fragments corresponding to fnbA binding domain and a C-terminal fragment from clfA were amplified from S. aureus NCTC8325 genomic DNA. Purified PCR products and the vector, pET15b, were digested with NcoI and BamHI. The digested PCR products were hybridized together and then ligated to digested vector. Finally incomplete construct was assembled by Taq DNA polymerase. To quick confirmation of cloning procedure the new construct designated pfnbA-clfA was digested with NcoI and BamHI. To further verification, the product was sent for sequencing. Results The data based on bioinformatic analysis showed no homology between fusion protein and human proteins. Digestion of new vector with NcoI and BamHI confirmed the ligation of fusion protein sequence into pET15b. Sequencing results verified the integrity of target sequences. Conclusion This study is the first effort to construct a new fusion protein vector based on S. aureus adhesins using a new design. This project is being continued to study the expression and biological activity of the fusion protein in a cell culture model.

  14. Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

    Indian Academy of Sciences (India)

    Chandrasekaran Sivakamasundari; Ramakrishnan Nagaraj

    2009-06-01

    We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from –1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide–membrane interactions.

  15. Interaction of Whey Proteins with Phenolic Derivatives Under Neutral and Acidic pH Conditions.

    Science.gov (United States)

    Cao, Yanyun; Xiong, Youling L

    2017-02-01

    Integration of gallic acid (GA) and its derivative epigallocatechin gallate (EGCG; 20, 120, and 240 μmol/g, protein basis) into whey protein isolate (WPI) at room temperature and pH 3.0 and pH 7.0 was investigated. At pH 7.0, both phenolics caused significant structural changes and EGCG induced greater digestibility of WPI. Total sulfhydryl in WPI decreased from 28.6 to 7.6 μmol/g and surface hydrophobicity declined by nearly 50% with 240 μmol/g EGCG at pH 7.0. Similar but less appreciable changes were produced by GA and at pH 3.0. Isothermal titration and fluorescence quenching tests showed moderately weak binding of WPI with GA but strong binding with EGCG at pH 7.0. Both phenolics at high concentrations lowered the thermal transition temperature of β-lactoglobulin by 0.5 °C to 1.4 °C and promoted its digestion. The phenolics also displayed a remarkable synergism with peptides in the WPI digest exerting radical scavenging activity. © 2017 Institute of Food Technologists®.

  16. Reduced expression of brain-derived neurotrophic factor protein in Parkinson's disease substantia nigra.

    Science.gov (United States)

    Parain, K; Murer, M G; Yan, Q; Faucheux, B; Agid, Y; Hirsch, E; Raisman-Vozari, R

    1999-02-25

    Several in vitro and in vivo studies have shown that brain-derived neurotrophic factor (BDNF) promotes survival of damaged mesencephalic dopaminergic neurons. Using a specific antibody directed against human recombinant BDNF, we studied the expression of the protein at the cellular level in the post-mortem mesencephalon of control subjects and patients with Parkinson's disease (PD). In control subjects, BDNF was expressed in all mesencephalic regions containing dopaminergic neurons, and in the substantia nigra pars compacta (SNpc) 65% of the melanized neurons expressed BDNF. In the PD SNpc, the total number of pigmented neurons containing BDNF was reduced to 9.6% of the corresponding control value. In contrast, the number of pigmented neurons non-immunoreactive for BDNF was reduced to 23.9% of the corresponding control value. This result appears to indicate that SNpc melanized neurons not expressing BDNF have a 2.5-fold greater probability of surviving than BDNF-positive melanized neurons. Furthermore, we found that in parkinsonian mesencephalon almost all dopaminergic neurons containing Lewy bodies were immunoreactive for BDNF. These findings demonstrate a reduced expression of BDNF in PD and suggest that BDNF protein expression does not protect melanized SNpc neurons from the degenerative process in this disease.

  17. Bacteria that purify sludge; Des bacteries epuratrices

    Energy Technology Data Exchange (ETDEWEB)

    Peignen-Seraline, P.; Manem, J. [Cirsee, Lyonnaise des Eaux, 92 - Nanterre (France)

    1997-03-01

    Inherent in water purification processes, the formation of sludges is intensively studied. Recently, original bacteria have been observed by searchers: some of them purify water making ``tassels``, others separate them and some of them even participate in the elimination of the first. This research study is described into details and will probably be used in the future at the industrial scale. (O.M.)

  18. An improved classification of G-protein-coupled receptors using sequence-derived features

    Directory of Open Access Journals (Sweden)

    Peng Zhen-Ling

    2010-08-01

    Full Text Available Abstract Background G-protein-coupled receptors (GPCRs play a key role in diverse physiological processes and are the targets of almost two-thirds of the marketed drugs. The 3 D structures of GPCRs are largely unavailable; however, a large number of GPCR primary sequences are known. To facilitate the identification and characterization of novel receptors, it is therefore very valuable to develop a computational method to accurately predict GPCRs from the protein primary sequences. Results We propose a new method called PCA-GPCR, to predict GPCRs using a comprehensive set of 1497 sequence-derived features. The principal component analysis is first employed to reduce the dimension of the feature space to 32. Then, the resulting 32-dimensional feature vectors are fed into a simple yet powerful classification algorithm, called intimate sorting, to predict GPCRs at five levels. The prediction at the first level determines whether a protein is a GPCR or a non-GPCR. If it is predicted to be a GPCR, then it will be further predicted into certain family, subfamily, sub-subfamily and subtype by the classifiers at the second, third, fourth, and fifth levels, respectively. To train the classifiers applied at five levels, a non-redundant dataset is carefully constructed, which contains 3178, 1589, 4772, 4924, and 2741 protein sequences at the respective levels. Jackknife tests on this training dataset show that the overall accuracies of PCA-GPCR at five levels (from the first to the fifth can achieve up to 99.5%, 88.8%, 80.47%, 80.3%, and 92.34%, respectively. We further perform predictions on a dataset of 1238 GPCRs at the second level, and on another two datasets of 167 and 566 GPCRs respectively at the fourth level. The overall prediction accuracies of our method are consistently higher than those of the existing methods to be compared. Conclusions The comprehensive set of 1497 features is believed to be capable of capturing information about amino acid

  19. Proteome profiling of neuroblastoma-derived exosomes reveal the expression of proteins potentially involved in tumor progression.

    Directory of Open Access Journals (Sweden)

    Danilo Marimpietri

    Full Text Available Neuroblastoma (NB is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133, basigin (CD147 and B7-H3 (CD276. Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.

  20. Application of a Low-Level Laser Therapy and the Purified Protein from Natural Latex (Hevea brasiliensis in the Controlled Crush Injury of the Sciatic Nerve of Rats: A Morphological, Quantitative, and Ultrastructural Study

    Directory of Open Access Journals (Sweden)

    Fernando José Dias

    2013-01-01

    Full Text Available This study analyzed the effects of a low-level laser therapy (LLLT, 15 J/cm2, 780 nm wavelength and the natural latex protein (P1, 0.1% in sciatic nerve after crush injury (15 Kgf, axonotmesis in rats. Sixty rats (male, 250 g were allocated into the 6 groups (n=10: CG—control group; EG—nerve exposed; IG—injured nerve without treatment; LG—crushed nerve treated with LLLT; PG—injured nerve treated with P1; and LPG—injured nerve treated with LLLT and P1. After 4 or 8 weeks, the nerve samples were processed for morphological, histological quantification and ultrastructural analysis. After 4 weeks, the myelin density and morphological characteristics improved in groups LG, PG, and LPG compared to IG. After 8 weeks, PG, and LPG were similar to CG and the capillary density was higher in the LG, PG, and LPG. In the ultrastructural analysis the PG and LPG had characteristics that were similar to the CG. The application of LLLT and/or P1 improved the recovery from the nerve crush injury, and in the long term, the P1 protein was the better treatment used, since only the application of LLLT has not reached the same results, and these treatments applied together did not potentiate the recovery.

  1. Encapsulation of bone morphogenic protein-2 with Cbfa1-overexpressing osteogenic cells derived from human embryonic stem cells in hydrogel accelerates bone tissue regeneration.

    Science.gov (United States)

    Kim, Min Jung; Park, Ji Sun; Kim, Sinae; Moon, Sung-Hwan; Yang, Han Na; Park, Keun-Hong; Chung, Hyung-Min

    2011-08-01

    Bone tissue defects caused by trauma and disease are significant problems in orthopedic surgery. Human embryonic stem cells (hESCs) hold great promise for the treatment of bone tissue disease in regenerative medicine. In this study, we have established an effective method for the differentiation of osteogenic cells derived from hESCs using a lentiviral vector containing the transcription factor Cbfa1. Differentiation was initiated in embryoid body formation of Cbfa1-expressing hESCs, resulting in a highly purified population of osteogenic cells based on flow cytometric analysis. These cells also showed characteristics of osteogenic cells in vitro, as determined by reverse-transcription (RT)-polymerase chain reaction and immunocytochemistry using osteoblast-specific markers. We also evaluated the regenerative potential of Cbfa1-expressing cells derived from hESCs (hESC-CECs) compared with hESCs and the osteogenic effects of bone morphogenic protein-2 (BMP2) encapsulated in thermoreversible hydrogel in vivo. hESC-CECs were embedded in hydrogel constructs enriched with BMP2 to promote bone regeneration. We observed prominent mineralization and the formation of nodule-like structures using von Kossa and alizarin red S staining. In addition, the expression patterns of osteoblast-specific genes were verified by RT-polymerase chain reaction, and immunohistochemical analysis revealed that collagen type 1 and Cbfa1 were highly expressed in hESC-CECs compared with other cell types. Taken together, our results suggest that encapsulation of hESC-CECs with BMP2 in hydrogel constructs appears to be a promising method to enhance the in vitro osteoblastic differentiation and in vivo osteogenic activity of hESC-CECs.

  2. Cellufine sulfate column chromatography as a simple, rapid, and effective method to purify dengue virus.

    Science.gov (United States)

    Kanlaya, Rattiyaporn; Thongboonkerd, Visith

    2016-08-01

    Conventional method to purify/concentrate dengue virus (DENV) is time-consuming with low virus recovery yield. Herein, we applied cellufine sulfate column chromatography to purify/concentrate DENV based on the mimicry between heparan sulfate and DENV envelope protein. Comparative analysis demonstrated that this new method offered higher purity (as determined by less contamination of bovine serum albumin) and recovery yield (as determined by greater infectivity). Moreover, overall duration used for cellufine sulfate column chromatography to purify/concentrate DENV was approximately 1/20 of that of conventional method. Therefore, cellufine sulfate column chromatography serves as a simple, rapid, and effective alternative method for DENV purification/concentration.

  3. Human cord blood derived immature basophils show dual characteristics, expressing both basophil and eosinophil associated proteins

    OpenAIRE

    Jeanette Grundström; Jenny M Reimer; Sofia E Magnusson; Gunnar Nilsson; Sara Wernersson; Lars Hellman

    2012-01-01

    Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transc...

  4. Leukocyte-Reduced Platelet-Rich Plasma Alters Protein Expression of Adipose Tissue-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Loibl, Markus; Lang, Siegmund; Hanke, Alexander; Herrmann, Marietta; Huber, Michaela; Brockhoff, Gero; Klein, Silvan; Nerlich, Michael; Angele, Peter; Prantl, Lukas; Gehmert, Sebastian

    2016-08-01

    Application of platelet-rich plasma and stem cells has become important in regenerative medicine. Recent literature supports the use of platelet-rich plasma as a cell culture media supplement to stimulate proliferation of adipose tissue-derived mesenchymal stem cells. The underlying mechanism of proliferation stimulation by platelet-rich plasma has not been investigated so far. Adipose tissue-derived mesenchymal stem cells were cultured in α-minimal essential medium supplemented with platelet-rich plasma or fetal calf serum. Cell proliferation was assessed with cell cycle kinetics using flow cytometric analyses after 48 hours. Differences in proteome expression of the adipose tissue-derived mesenchymal stem cells were analyzed using a reverse-phase protein array to quantify 214 proteins. Complementary Ingenuity Pathways Analysis and gene set enrichment analysis were performed using protein data, and confirmed by Western blot analysis. A higher percentage of adipose tissue-derived mesenchymal stem cells in the S phase in the presence of platelet-rich plasma advocates the proliferation stimulation. Ingenuity Pathways Analysis and gene set enrichment analysis confirm the involvement of the selected proteins in the process of cell growth and proliferation. Ingenuity Pathways Analysis revealed a participation in the top-ranked canonical pathways PI3K/AKT, PTEN, ILK, and IGF-1. Gene set enrichment analysis identified the authors' protein set as being part of significantly regulated protein sets with the focus on cell cycle, metabolism, and the Kyoto Encyclopedia of Genes and Genomes transforming growth factor-β signaling pathway. The present study provides evidence that platelet-rich plasma stimulates proliferation and induces a unique change in the proteomic profile of adipose tissue-derived mesenchymal stem cells. The interpretation of altered expression of regulatory proteins represents a step forward toward achieving good manufacturing practice-compliant criteria

  5. Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants.

    Science.gov (United States)

    Woodard, Susan L; Mayor, Jocelyne M; Bailey, Michele R; Barker, Donna K; Love, Robert T; Lane, Jeffrey R; Delaney, Donna E; McComas-Wagner, Janet M; Mallubhotla, Hanuman D; Hood, Elizabeth E; Dangott, Lawrence J; Tichy, Shane E; Howard, John A

    2003-10-01

    Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins.

  6. In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

    Directory of Open Access Journals (Sweden)

    Intekhab Islam

    2016-01-01

    Full Text Available Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs are potentially an unlimited source of healthy and functional osteoprogenitors (OPs that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs expressing green fluorescent protein (GFP and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

  7. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  8. Why CLEAN when you can PURIFY?

    CERN Document Server

    Carrillo, Rafael E; Wiaux, Yves

    2013-01-01

    We extend previously proposed radio-interferometric imaging approaches based on convex optimization to handle continuous visibilities and large-scale optimization problems. We propose a general algorithmic framework based on the simultaneous-direction method of multipliers to solve sparse imaging problems. The algorithm offers a parallel implementation structure, thus providing a significant gain in terms of speed and scalability to very high dimensions. We implement various state-of-the-art sparsity regularization priors, including our recent average sparsity approach SARA, in a new imaging software dubbed PURIFY. We evaluate through realistic simulations the performance of the software in terms of reconstruction quality and computational speed. Simulation results confirm both the superiority of SARA for continuous Fourier measurements and the fact that the new algorithmic structure offers a promising path to handle large-scale problems. Code is available at https://github.com/basp-group/purify

  9. Immunotherapy of metastatic colorectal cancer with vitamin D-binding protein-derived macrophage-activating factor, GcMAF.

    Science.gov (United States)

    Yamamoto, Nobuto; Suyama, Hirofumi; Nakazato, Hiroaki; Yamamoto, Nobuyuki; Koga, Yoshihiko

    2008-07-01

    Serum vitamin D binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of colorectal cancer patients was lost or reduced because Gc protein is deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Deglycosylated Gc protein cannot be converted to MAF, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage-activating factor (GcMAF) ever discovered, but it produces no side effect in humans. Macrophages treated with GcMAF (100 microg/ml) develop an enormous variation of receptors and are highly tumoricidal to a variety of cancers indiscriminately. Administration of 100 nanogram (ng)/ human maximally activates systemic macrophages that can kill cancerous cells. Since the half-life of the activated macrophages is approximately 6 days, 100 ng GcMAF was administered weekly to eight nonanemic colorectal cancer patients who had previously received tumor-resection but still carried significant amounts of metastatic tumor cells. As GcMAF therapy progressed, the MAF precursor activities of all patients increased and conversely their serum Nagalase activities decreased. Since serum Nagalase is proportional to tumor burden, serum Nagalase activity was used as a prognostic index for time course analysis of GcMAF therapy. After 32-50 weekly administrations of 100 ng GcMAF, all colorectal cancer patients exhibited healthy control levels of the serum Nagalase activity, indicating eradication of metastatic tumor cells. During 7 years after the completion of GcMAF therapy, their serum Nagalase activity did not increase, indicating no recurrence of cancer, which was also supported by the annual CT scans of these patients.

  10. Structural basis for the site-specific incorporation of lysine derivatives into proteins.

    Directory of Open Access Journals (Sweden)

    Veronika Flügel

    Full Text Available Posttranslational modifications (PTMs of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS:tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.

  11. Effect of enzyme hydrolysis on solubility and antioxidative properties of potato protein isolates derived from heat coagulation

    OpenAIRE

    MIKOVÁ, Klára

    2014-01-01

    The bachelor thesis is focused on effect of enzyme hydrolysis on solubility and antioxidative properties of potato protein. It was used industrial potato fruit juice and potato fruit juice from tubers two varieties Sibu and Ornella. Isolates derived from heat coagulation are generally poorly soluble so their use is limited (only feeding purporses). This work describes positive effect of enzyme hydrolysis on solubility and antioxidative properties of potato protein isolates. For example, antio...

  12. Utilization of purified cellulose in fiber studies.

    Science.gov (United States)

    Penner, M H; Liaw, E T

    1990-01-01

    Purified cellulose-type fiber products are widely used in experimental nutrition. Their use in a broad spectrum of studies may potentially lead to the acceptance of the misconception that the various commercially available cellulose products are equivalent. In this paper we have attempted to show that this is not the case. The comparative structural data of Table 2 and the compositional data of Olsen et al provide examples which indicate that purified cellulose preparations should not necessarily be considered equivalent. Unfortunately, our current lack of understanding of how fibers are metabolized and how they may affect specific physiological parameters makes it difficult to determine which, if any, of the measurable structural and chemical properties will be of relevance for a given in vivo study. At present, it appears that researchers utilizing/evaluating the consequences of consuming a purified cellulose-type fiber would be prudent to provide at least a limited amount of data on the properties of the cellulose preparation used in their studies. The characterization of the cellulose product may be done by a variety of methods depending on the expertise of the laboratory. The methods and results discussed in this paper provide an example of the type of information which may be obtained from an in vitro characterization of cellulose products.

  13. Some Properties of Purified and Non-purified Rumen Tissue Arginase in Cattle

    OpenAIRE

    ERİŞİR, Mine; OZAN, Sema Temizer

    1998-01-01

    Some biochemical properties of purified and non-purified rumen tissue arginase were compared. Homogenization, heating, treatment with aceton, precipitation with ammonium sulfate, dialysis, several centrifugations, gel filtration on sephadex G-200 processes were utilized in the purification procedure of the enzyme. It was found that pre-incubation temperature (60 °C) of arginase and Km (4mM) to its substrate, L-arginine, did not change before and after purification. While pre-incubation peri...

  14. Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis.

    Science.gov (United States)

    Stec, Jozef; Vilchèze, Catherine; Lun, Shichun; Perryman, Alexander L; Wang, Xin; Freundlich, Joel S; Bishai, William; Jacobs, William R; Kozikowski, Alan P

    2014-11-01

    New triclosan (TRC) analogues were evaluated for their activity against the enoyl-acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of InhA, and specific modifications to its positions 5 and 4' afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 4-(n-butyl)-1,2,3-triazolyl TRC derivative 3, had an MIC value of 0.6 μg mL(-1) (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC50 value of 90 nM. Compound 3 and the 5-methylisoxazole-modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc(2) 4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14, supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Tumour-derived PTH-related protein triggers adipose tissue browning and cancer cachexia.

    Science.gov (United States)

    Kir, Serkan; White, James P; Kleiner, Sandra; Kazak, Lawrence; Cohen, Paul; Baracos, Vickie E; Spiegelman, Bruce M

    2014-09-04

    Cachexia is a wasting disorder of adipose and skeletal muscle tissues that leads to profound weight loss and frailty. About half of all cancer patients suffer from cachexia, which impairs quality of life, limits cancer therapy and decreases survival. One key characteristic of cachexia is higher resting energy expenditure levels than in healthy individuals, which has been linked to greater thermogenesis by brown fat. How tumours induce brown fat activity is unknown. Here, using a Lewis lung carcinoma model of cancer cachexia, we show that tumour-derived parathyroid-hormone-related protein (PTHrP) has an important role in wasting, through driving the expression of genes involved in thermogenesis in adipose tissues. Neutralization of PTHrP in tumour-bearing mice blocked adipose tissue browning and the loss of muscle mass and strength. Our results demonstrate that PTHrP mediates energy wasting in fat tissues and contributes to the broader aspects of cancer cachexia. Thus, neutralization of PTHrP might hold promise for ameliorating cancer cachexia and improving patient survival.

  16. A mathematical model of T lymphocyte calcium dynamics derived from single transmembrane protein properties

    Directory of Open Access Journals (Sweden)

    Christine Dorothee Schmeitz

    2013-09-01

    Full Text Available Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

  17. Assay of flippase activity in proteoliposomes using fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Marek, Magdalena; Günther-Pomorski, Thomas

    2016-01-01

    Specific membrane proteins, termed lipid flippases, play a central role in facilitating the movement of lipids across cellular membranes. In this protocol, we describe the reconstitution of ATP-driven lipid flippases in liposomes and the analysis of their in vitro flippase activity based on the use...... of fluorescent lipid derivatives. Working with purified and reconstituted systems provides a well-defined experimental setup and allows to directly characterize these membrane proteins at the molecular level....

  18. Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

    Science.gov (United States)

    Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

    2017-01-01

    Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

  19. Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria.

    Directory of Open Access Journals (Sweden)

    Ingo L Brand

    Full Text Available Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins may underestimate their activity.

  20. Derivation of factors to estimate daily fat, protein, and somatic cell score from one milking of cows milked twice daily

    Science.gov (United States)

    The objective was to derive factors to predict daily fat (F) and protein (P) yield or somatic cell score (SCS) when milk is sampled once for cows milked twice per d. Milk samples were collected for each milking on test-day by Dairy Herd Improvement personnel from herds recording milking times and m...

  1. Acute differential effects of milk-derived dietary proteins on postprandial lipaemia in obese non-diabetic subjects

    DEFF Research Database (Denmark)

    Holmer-Jensen, J; Hartvigsen, M L; Mortensen, L S

    2012-01-01

    Postprandial lipaemia is an established risk factor for atherosclerosis. To investigate the acute effect of four milk-derived dietary proteins (alpha-lactalbumin, whey isolate, caseinoglycomacropeptide and whey hydrolysate) on postprandial lipaemia, we have conducted a randomized, acute, single-b......-blinded clinical intervention study with crossover design....

  2. The roles of bone morphogenetic proteins and their signaling in the osteogenesis of adipose-derived stem cells

    NARCIS (Netherlands)

    Zhang, X.; Guo, J.; Zhou, Y.; Wu, G.

    2014-01-01

    Large-size bone defects can severely compromise both aesthetics and musculoskeletal functions. Adipose-derived stem cells (ASCs)-based bone tissue engineering has recently become a promising treatment strategy for the above situation. As robust osteoinductive cytokines, bone morphogenetic proteins (

  3. Effects of heterodimeric bone morphogenetic protein-2/7 on osteogenesis of human adipose-derived stem cells

    NARCIS (Netherlands)

    Zhang, X.; Guo, J.; Wu, G.; Zhou, Y.

    2015-01-01

    Objective Roles of bone morphogenetic proteins (BMPs) on osteogenesis of human adipose-derived stem cells (hASCs) remain ambiguous. In this study, we evaluated in vitro and in vivo functional characteristics of BMPs of different dimerization types, with the aim of determining osteoinductive efficien

  4. Identification of MHC class I H-2 Kb/Db-restricted immunogenic peptides derived from retinal proteins

    DEFF Research Database (Denmark)

    Wang, Mingjun; Bai, Fang; Pries, Mette

    2006-01-01

    . The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. CONCLUSIONS: Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes...... in the development of inflammatory eye diseases, such as uveitis....

  5. Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions

    Directory of Open Access Journals (Sweden)

    Munguia Agustin

    2009-07-01

    Full Text Available Abstract Background Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts. Results Two types of procedures were used for the preparation of biocatalysts from purified wild type LS (WT LS B. subtilis and the R360K and Y429N LS mutants: purified enzymes aggregated with glutaraldehyde (cross-linked enzyme aggregates: CLEAs, and covalently immobilized enzymes in Eupergit C®. The biocatalysts were characterized and used for fructoside synthesis using xylose as an acceptor model. CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes. The specific activity of CLEAs prepared from wild type LS (44.9 U/mg of CLEA, R360K (56.5 U/mg of CLEA and Y429N (1.2 U/mg of CLEA mutants were approximately 70, 40 and 200-fold higher, respectively, than equivalent Eupergit C® immobilized enzyme preparations (U/mg of Eupergit, where units refer to global LS activity. In contrast, the specific activity of the free enzymes was 160, 171.2 and 1.5 U/mg of protein, respectively. Moreover, all CLEAs had higher thermal stability than corresponding soluble enzymes. In the long term, the operational stability was affected by levan synthesis. Conclusion This is the first report of cross-linked transglycosidases aggregates. CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized

  6. Design and modular parallel synthesis of a MCR derived α-helix mimetic protein-protein interaction inhibitor scaffold

    NARCIS (Netherlands)

    Antuch, Walfrido; Menon, Sanjay; Chen, Quin-Zene; Lu, Yingchun; Sakamuri, Sukumar; Beck, Barbara; Schauer-Vukašinović, Vesna; Agarwal, Seema; Hess, Sibylle; Dömling, Alexander

    2006-01-01

    A terphenyl α-helix mimetic scaffold recognized to be capable of disrupting protein-protein interactions was structurally morphed into an easily amenable and versatile multicomponent reaction (MCR) backbone. The design, modular in-parallel library synthesis, initial cell based biological data, and p

  7. Protein conjugated with aldehydes derived from lipid peroxidation as an independent parameter of the carbonyl stress in the kidney damage

    Directory of Open Access Journals (Sweden)

    Medina-Navarro Rafael

    2011-11-01

    Full Text Available Abstract Background One of the well-defined and characterized protein modifications usually produced by oxidation is carbonylation, an irreversible non-enzymatic modification of proteins. However, carbonyl groups can be introduced into proteins by non-oxidative mechanisms. Reactive carbonyl compounds have been observed to have increased in patients with renal failure. In the present work we have described a procedure designed as aldehyde capture to calculate the protein carbonyl stress derived solely from lipid peroxidation. Methods Acrolein-albumin adduct was prepared as standard at alkaline pH. Rat liver microsomal membranes and serum samples from patients with diabetic nephropathy were subjected to the aldehyde capture procedure and aldol-protein formation. Before alkalinization and incubation, samples were precipitated and redisolved in 6M guanidine. The absorbances of the samples were read with a spectrophotometer at 266 nm against a blank of guanidine. Results Evidence showed abundance of unsaturated aldehydes derived from lipid peroxidation in rat liver microsomal membranes and in the serum of diabetic patients with advanced chronic kidney disease. Carbonyl protein and aldol-proteins resulted higher in the diabetic nephropathy patients (p Conclusion The aldehyde-protein adduct represents a non oxidative component of carbonyl stress, independent of the direct amino acid oxidation and could constitute a practical and novelty strategy to measure the carbonyl stress derived solely from lipid peroxidation and particularly in diabetic nephropathy patients. In addition, we are in a position to propose an alternative explanation of why alkalinization of urine attenuates rhabdomyolysis-induced renal dysfunction.

  8. Soybean meal substitution with a yeast-derived microbial protein source in dairy cow diets.

    Science.gov (United States)

    Sabbia, J A; Kalscheur, K F; Garcia, A D; Gehman, A M; Tricarico, J M

    2012-10-01

    The objective of this study was to examine the effects substituting soybean meal with a yeast-derived microbial protein (YMP) on rumen and blood metabolites, dry matter intake, and milk production of high-producing dairy cows. Sixteen Holstein cows (12 multiparous and 4 primiparous), 93 ± 37 DIM (mean ± SD) at the beginning of the experiment, were used in a 4×4 Latin square design with four 28-d periods. Cows were blocked by parity and production, with 1 square consisting of 4 animals fitted with rumen cannulas. Basal diets, formulated for 16.1% crude protein and 1.56 Mcal/kg of net energy for lactation, contained 40% corn silage, 20% alfalfa hay, and 40% concentrate mix. During each period, cows were fed 1 of 4 treatment diets corresponding to YMP (DEMP; Alltech Inc., Nicholasville, KY) concentrations of 0, 1.14, 2.28, and 3.41% DM. Soybean meal (44% CP) was replaced by YMP to attain isonitrogenous and isoenergetic diets. Dietary treatments had no effect on pH and on most ruminal volatile fatty acid concentrations, with the exception of isovalerate, which decreased linearly with the addition of YMP. Rumen ammonia concentration decreased linearly, whereas free amino acids, total amino acid nitrogen, and soluble proteins weighing more than 10 kDa showed a cubic response on rumen N fractionation. A quadratic response was observed in oligopeptides that weighed between 3 and 10 kDa and peptides under 3kDa when expressed as percentages of total amino acids and total nitrogen. Although nonesterified fatty acid concentration in blood did not differ between treatments, β-hydroxybutyrate and plasma glucose increased linearly as YMP increased. Dry matter intake showed a cubic effect, where cows fed 1.14, and 3.41% YMP had the highest intake. Milk production was not affected by YMP, whereas a trend was observed for a quadratic increase for 4% fat-corrected milk and energy-corrected milk. Medium- and long-chain fatty acid concentrations in milk increased quadratically

  9. Human-derived physiological heat shock protein 27 complex protects brain after focal cerebral ischemia in mice.

    Directory of Open Access Journals (Sweden)

    Shinichiro Teramoto

    Full Text Available Although challenging, neuroprotective therapies for ischemic stroke remain an interesting strategy for countering ischemic injury and suppressing brain tissue damage. Among potential neuroprotective molecules, heat shock protein 27 (HSP27 is a strong cell death suppressor. To assess the neuroprotective effects of HSP27 in a mouse model of transient middle cerebral artery occlusion, we purified a "physiological" HSP27 (hHSP27 from normal human lymphocytes. hHSP27 differed from recombinant HSP27 in that it formed dimeric, tetrameric, and multimeric complexes, was phosphorylated, and contained small amounts of αβ-crystallin and HSP20. Mice received intravenous injections of hHSP27 following focal cerebral ischemia. Infarct volume, neurological deficit scores, physiological parameters, and immunohistochemical analyses were evaluated 24 h after reperfusion. Intravenous injections of hHSP27 1 h after reperfusion significantly reduced infarct size and improved neurological deficits. Injected hHSP27 was localized in neurons on the ischemic side of the brain. hHSP27 suppressed neuronal cell death resulting from cytochrome c-mediated caspase activation, oxidative stress, and inflammatory responses. Recombinant HSP27 (rHSP27, which was artificially expressed and purified from Escherichia coli, and dephosphorylated hHSP27 did not have brain protective effects, suggesting that the phosphorylation of hHSP27 may be important for neuroprotection after ischemic insults. The present study suggests that hHSP27 with posttranslational modifications provided neuroprotection against ischemia/reperfusion injury and that the protection was mediated through the inhibition of apoptosis, oxidative stress, and inflammation. Intravenously injected human HSP27 should be explored for the treatment of acute ischemic strokes.

  10. Gc-protein-derived macrophage activating factor counteracts the neuronal damage induced by oxaliplatin.

    Science.gov (United States)

    Morucci, Gabriele; Branca, Jacopo J V; Gulisano, Massimo; Ruggiero, Marco; Paternostro, Ferdinando; Pacini, Alessandra; Di Cesare Mannelli, Lorenzo; Pacini, Stefania

    2015-02-01

    Oxaliplatin-based regimens are effective in metastasized advanced cancers. However, a major limitation to their widespread use is represented by neurotoxicity that leads to peripheral neuropathy. In this study we evaluated the roles of a proven immunotherapeutic agent [Gc-protein-derived macrophage activating factor (GcMAF)] in preventing or decreasing oxaliplatin-induced neuronal damage and in modulating microglia activation following oxaliplatin-induced damage. The effects of oxaliplatin and of a commercially available formula of GcMAF [oleic acid-GcMAF (OA-GcMAF)] were studied in human neurons (SH-SY5Y cells) and in human microglial cells (C13NJ). Cell density, morphology and viability, as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration [neuromodulin or growth associated protein-43 (Gap-43)] and markers of microglia activation [ionized calcium binding adaptor molecule 1 (Iba1) and B7-2], were determined. OA-GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability. The neuroprotective effect was accompanied by increased intracellular cAMP production, as well as by increased expression of VEGF and neuromodulin. OA-GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it increased microglial activation following oxaliplatin-induced damage, resulting in an increased expression of the markers Iba1 and B7-2 without any concomitant increase in cell number. When neurons and microglial cells were co-cultured, the presence of OA-GcMAF significantly counteracted the toxic effects of oxaliplatin. Our results demonstrate that OA-GcMAF, already used in the immunotherapy of advanced cancers, may significantly contribute to neutralizing the neurotoxicity induced by oxaliplatin, at the same time possibly concurring to an integrated anticancer effect. The association between these two powerful anticancer molecules would probably produce

  11. Air Purifiers Eliminate Pathogens, Preserve Food

    Science.gov (United States)

    2009-01-01

    NASA-funded researchers produced an ethylene reduction device for a plant growth unit. KES Science & Technology Inc., a Kennesaw, Georgia-based company specializing in sustaining perishable foods, licensed the ethylene scrubbing technology. KES partnered with Akida Holdings, of Jacksonville, Florida, which now markets the NASA-developed technology as AiroCide. According to the company, it is the only air purifier that completely destroys airborne bacteria, mold, fungi, mycotoxins, viruses, volatile organic compounds (like ethylene), and odors. What?s more, the devices have no filters that need changing and produce no harmful byproducts, such as the ozone created by some filtration systems.

  12. Dimerization capacities of FGF2 purified with or without heparin-affinity chromatography.

    Directory of Open Access Journals (Sweden)

    Natalia Platonova

    Full Text Available Fibroblast growth factor-2 (FGF2 is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.

  13. Altered regulation of brain-derived neurotrophic factor protein in hippocampus following slice preparation.

    Science.gov (United States)

    Danzer, S C; Pan, E; Nef, S; Parada, L F; McNamara, J O

    2004-01-01

    Brain-derived neurotrophic factor (BDNF) and its cognate receptor tyrosine kinase B (TrkB) play important roles in regulating survival, structure, and function of CNS neurons. One method of studying the functions of these molecules has utilized in vitro hippocampal slice preparations. An important caveat to using slices, however, is that slice preparation itself might alter the expression of BDNF, thereby confounding experimental results. To address this concern, BDNF immunoreactivity was examined in rodent slices using two different methods of slice preparation. Rapid and anatomically selective regulation of BDNF content followed slice preparation using both methodologies; however, different patterns of altered BDNF immunoreactivity were observed. First, in cultured slices, BDNF content decreased in the dentate molecular layer and increased in the CA3 pyramidal cell layer and the mossy fiber pathway of the hippocampus after 30 min. Furthermore, an initially "punctate" pattern of BDNF labeling observed in the mossy fiber pathway of control sections changed to homogenous labeling of the pathway in vitro. In contrast to these findings, slices prepared as for acute slice physiology exhibited no change in BDNF content in the molecular layer and mossy fiber pathway 30 min after slicing, but exhibited significant increases in the dentate granule and CA3 pyramidal cell layers. These findings demonstrate that BDNF protein content is altered following slice preparation, that different methods of slice preparation produce different patterns of BDNF regulation, and raise the possibility that BDNF release and TrkB activation may also be regulated. These consequences of hippocampal slice preparation may confound analyses of exogenous or endogenous BDNF on hippocampal neuronal structure or function.

  14. Identification of a myeloid-derived suppressor cell cystatin-like protein that inhibits metastasis.

    Science.gov (United States)

    Boutté, Angela M; Friedman, David B; Bogyo, Matthew; Min, Yongfen; Yang, Li; Lin, P Charles

    2011-08-01

    Myeloid-derived suppressor cells (MDSCs) are significantly increased in cancer patients and tumor bearing-animals. MDSCs infiltrate into tumors and promote tumor invasion and metastasis. To identify the mediator responsible for the prometastatic property of MDSCs, we used proteomics. We found neutrophilic granule protein (NGP) was decreased >2-fold in MDSCs from metastatic 4T1 tumor-bearing mice compared to nonmetastatic 67NR controls. NGP mRNA levels were decreased in bone marrow and in tumor-infiltrating MDSCs by 45 and 66%, respectively, in 4T1 tumor-bearing mice compared to 67NR controls. Interestingly, 4T1-conditioned medium reduced myeloid cell NGP expression by ∼ 40%, suggesting that a secreted factor mediates gene reduction. Sequence analysis shows a putative cystatin domain in NGP, and biochemical analysis confirms NGP a novel cathepsin inhibitor. It inhibited cathepsin B activity by nearly 40% in vitro. NGP expression in 4T1 tumor cells suppressed cell invasion, delayed primary tumor growth, and greatly reduced lung metastasis in vivo. A 2.8-fold reduction of cathepsin activity was found in tumors expressing NGP compared to controls. NGP significantly reduced tumor angiogenesis to 12.6 from 19.6 and lymphangiogenesis to 4.6 from 9.1 vessels/field. Necrosis was detectable only in NGP-expressing tumors, and the number of apoptotic cells increased to 22.4 from 8.3 in controls. Taken together, this study identifies a negative regulator of tumor metastasis in MDSCs, NGP, which is down-regulated in metastatic conditions. The finding suggests that malignant tumors promote invasion/metastasis not only through up-regulation of proteases but also down-regulation of protease inhibitors.

  15. Immunotoxicity of nucleic acid reduced BioProtein - a bacterial derived single cell protein - in Wistar rats

    DEFF Research Database (Denmark)

    Mølck, Anne-marie; Poulsen, Morten; Christensen, Hanne Risager;

    2002-01-01

    BioProtein is a single cell protein produced by a mixed methanotrophic and heterotrophic bacteria culture using natural gas as energy source, which has been approved for animal feed. BioProtein contains a large amount of nucleic acids making the product less suitable for human consumption...... the same tendency, although, not statistically significant (P = 0.09). The subsets of cells identified as neutrophils and eosinophils were increased and lymphocytes decreased. The histopathological examination revealed histiocytosis and accumulation of foamy macrophages in the mesenteric lymph nodes...

  16. High-mobility group protein HMGA2-derived fragments stimulate the proliferation of chondrocytes and adipose tissue-derived stem cells.

    Science.gov (United States)

    Richter, A; Lübbing, M; Frank, H G; Nolte, I; Bullerdiek, J C; von Ahsen, I

    2011-04-11

    In previous research, it was shown that recombinant HMGA2 protein enhances the proliferation of porcine chondrocytes grown in vitro, opening up promising applications of this embryonic architectural transcription factor for tissue engineering, such as in cartilage repair. In this paper, we describe the development and analyses of two synthetic fragments comprising the functional AT-hook motifs of the HMGA2 protein, as well as the nuclear transport domain. They can be synthesised up to large scales, while eliminating some of the problems of recombinant protein production, including unwanted modification or contamination by the expression hosts, or of gene therapy approaches such as uncontrolled viral integration and transgene expression even after therapy. Application of one of these peptides onto porcine hyaline cartilage chondrocytes, grown in in vitro monolayer cell culture, showed a growth-promoting effect similar to that of the wild type HMGA2 protein. Furthermore, it also promoted cell growth of adult adipose tissue derived stem cells. Due to its proliferation inducing function and vast availability, this peptide is thus suitable for further application and investigation in various fields such as tissue engineering and stem cell research.

  17. Study on soot purifying of molding shop in coking factory

    Institute of Scientific and Technical Information of China (English)

    LI Duo-song; ZHANG Hui; BAI Xiang-yu

    2006-01-01

    Exhaust gas in molding shop was complicated in component and characteristic in Iow thickness asphalt smoke, mass steam-gas and dust. It was difficult to purify the soot with common purifier. So we must consider them roundly and develop new multifunction purifier. PFP multifunction soot purifier was made on the base of design optimization and was installed at Shenhuo Coking Factory in 2004. The combined effects of multi- mechanism in purifier make purifying ratio keep in high level. The remove ratio of smut reaches at 92.8%, and asphalt smoke at 83.7%.

  18. Analysis the Sources of Soy Protein Derivative%酱油中蛋白衍生物来源分析

    Institute of Scientific and Technical Information of China (English)

    刘艳民

    2011-01-01

    During fermentation of soy sauce, microbial protein decomposition of raw materials was not complete. Hie soy sauce and protein hydrolysates of protein derivatives showed that the current soy sauce brewing process to the hydrolysis of proteins was incomplete, only about 60% . Protein degradation in protein hydrolysate was not entirely, its protein degradation ability at about 80%. Because of the testing technology was not very peptide mature ill qualitative aspects, there were some difficulties. It could be only as a difference method between soy sauce and a of protein hydrolysates.%酱油在发酵过程中,微生物对原料中蛋白质分解是不完全的.通过对酿造酱油和蛋白水解液中蛋白衍生物的分析表明,目前的酱油酿造工艺对蛋白质的水解是不完全的,仅仅在60%左右,蛋白水解液中蛋白质降解也不完全,其蛋白质降解能力在80%左右.由于肽的检测技术不是很成熟,在定性方面存在一定的难度,因此可以仅作为区别酿造酱油和蛋白水解液的一种方法.

  19. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    Science.gov (United States)

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  20. Protein structure validation and refinement using amide proton chemical shifts derived from quantum mechanics

    DEFF Research Database (Denmark)

    Christensen, Anders Steen; Linnet, Troels Emtekær; Borg, Mikael;

    2013-01-01

    We present the ProCS method for the rapid and accurate prediction of protein backbone amide proton chemical shifts - sensitive probes of the geometry of key hydrogen bonds that determine protein structure. ProCS is parameterized against quantum mechanical (QM) calculations and reproduces high level...... QM results obtained for a small protein with an RMSD of 0.25 ppm (r = 0.94). ProCS is interfaced with the PHAISTOS protein simulation program and is used to infer statistical protein ensembles that reflect experimentally measured amide proton chemical shift values. Such chemical shift...

  1. Synthesis of gold and silver nanoparticles using purified URAK.

    Science.gov (United States)

    Deepak, Venkataraman; Umamaheshwaran, Paneer Selvam; Guhan, Kandasamy; Nanthini, Raja Amrisa; Krithiga, Bhaskar; Jaithoon, Nagoor Meeran Hasika; Gurunathan, Sangiliyandi

    2011-09-01

    This study aims at developing a new eco-friendly process for the synthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) using purified URAK. URAK is a fibrinolytic enzyme produced by Bacillus cereus NK1. The enzyme was purified and used for the synthesis of AuNPs and AgNPs. The enzyme produced AgNPs when incubated with 1 mM AgNO3 for 24 h and AuNPs when incubated with 1 mM HAuCl4 for 60 h. But when NaOH was added, the synthesis was rapid and occurred within 5 min for AgNPs and 12 h for AuNPs. The synthesized nanoparticles were characterized by a peak at 440 nm and 550 nm in the UV-visible spectrum. TEM analysis showed that AgNPs of the size 60 nm and AuNPs of size 20 nm were synthesized. XRD confirmed the crystalline nature of the nanoparticles and AFM showed the morphology of the nanoparticle to be spherical. FT-IR showed that protein was responsible for the synthesis of the nanoparticles. This process is highly simple, versatile and produces AgNPs and AuNPs in environmental friendly manner. Moreover, the synthesized nanoparticles were found to contain immobilized enzyme. Also, URAK was tested on RAW 264.7 macrophage cell line and was found to be non-cytotoxic until 100 μg/ml.

  2. Translationally-controlled tumor protein activates the transcription of Oct-4 in kidney-derived stem cells.

    Science.gov (United States)

    Jing, Ying; He, Liang-Liang; Mei, Chang-Lin

    2017-01-01

    The molecular mechanisms underlying translationally-controlled tumor protein (TCTP) in the activation of octamer-binding transcription factor 4 (Oct-4) in kidney-derived stem cells have not been characterized. The aim of the present study was to identify the transcriptional activation of Oct-4 by TCTP in kidney-derived stem cells. Homology-directed repair cDNA inserted into Fisher 344 transgenic (Tg) rats and the mouse strain 129/Svj were used for the experiments. Diphtheria toxin (DT; 10 ng/kg) injected into the Tg rats created the kidney injury, which was rapidly restored by the activation of kidney-derived stem cells. Kidney-derived stem cells were isolated from the DT-injured Tg rats using cell culture techniques. The co-expression of Oct-4 and TCTP were observed in the isolated kidney-derived stem cells. Immunoblotting and reverse transcription-polymerase chain reaction analysis of TCTP null mutant (TCTP(-)/(-)) embryos at day 9.5 (E9.5) demonstrated the absence of co-expression of Oct-4 and TCTP, but expression of paired box-2 was detected. This was in contrast with the E9.5 control embryos, which expressed all three proteins. In conclusion, the results of the present study demonstrated that TCTP activates the transcription of Oct-4 in kidney-derived stem cells, as TCTP(-)/(-) embryos exhibited knock down of TCTP and Oct-4 without disturbing the expression of Pax-2 The characteristics and functional nature of TCTP in association with Oct-4 in kidney-derived stem cells was identified.

  3. A model study of artificial linker system using self-assembled calix[4]arene derivative monolayers for protein immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Minsu; An, Won Gun; Kim, Jae-Ho; Choi, Heung-Jin; Kim, Sung-Hoon; Han, Moon-Hi; Koh, Kwangnak

    2004-01-05

    The attachment of biomolecules, in particular proteins, onto solid supports is fundamental in the development of advanced biosensors, biochips, bioreactors, and many diagnostic techniques. In addition, the effective investigation of biomolecular structure and function with chip-based modern instruments often requires effective attachment of the biomolecule to a substrate. For this reason, it is very important to construct well-characterized linker system that can immobilize protein efficiently. Here, we investigate the formation of self-assembled monolayers (SAMs) with calix[4]arene ethylester and carboxylic acid derivatives that can serve as a model system for protein immobilization at solid surfaces. The calix[4]arene derivative monolayers were formed on Au surface and carefully characterized by atomic force microscopy (AFM), Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS) and surface plasmon resonance (SPR). Immobilization process of protein using bovine serum albumin (BSA) on the artificial linker layer was measured by SPR. The surface concentration of BSA was calculated by simulation of experimental SPR data. The surface concentration of BSA on the carboxylic acid form was higher than that of the ethylester. These results can help in modeling and understanding of protein immobilization on the linker layer.

  4. Ozone Air Purifiers: Can They Improve Asthma Symptoms?

    Science.gov (United States)

    ... daughter has asthma. Would she benefit from an ozone air purifier in her room? Answers from James ... Li, M.D., Ph.D. Despite manufacturers' claims, ozone air purifiers don't remove asthma triggers from ...

  5. Prediction of Spontaneous Protein Deamidation from Sequence-Derived Secondary Structure and Intrinsic Disorder.

    Directory of Open Access Journals (Sweden)

    J Ramiro Lorenzo

    Full Text Available Asparagine residues in proteins undergo spontaneous deamidation, a post-translational modification that may act as a molecular clock for the regulation of protein function and turnover. Asparagine deamidation is modulated by protein local sequence, secondary structure and hydrogen bonding. We present NGOME, an algorithm able to predict non-enzymatic deamidation of internal asparagine residues in proteins in the absence of structural data, using sequence-based predictions of secondary structure and intrinsic disorder. Compared to previous algorithms, NGOME does not require three-dimensional structures yet yields better predictions than available sequence-only methods. Four case studies of specific proteins show how NGOME may help the user identify deamidation-prone asparagine residues, often related to protein gain of function, protein degradation or protein misfolding in pathological processes. A fifth case study applies NGOME at a proteomic scale and unveils a correlation between asparagine deamidation and protein degradation in yeast. NGOME is freely available as a webserver at the National EMBnet node Argentina, URL: http://www.embnet.qb.fcen.uba.ar/ in the subpage "Protein and nucleic acid structure and sequence analysis".

  6. Isolating and Purifying Clostridium difficile Spores

    Science.gov (United States)

    Edwards, Adrianne N.; McBride, Shonna M.

    2016-01-01

    Summary The ability for the obligate anaerobe, Clostridium difficile, to form a metabolically dormant spore is critical for the survival of this organism outside of the host. This spore form is resistant to a myriad of environmental stresses, including heat, desiccation and exposure to disinfectants and antimicrobials. These intrinsic properties of spores allow C. difficile to survive long-term in an oxygenated environment, to be easily transmitted from host-to-host and to persist within the host following antibiotic treatment. Because of the importance of the spore form to the C. difficile lifecycle and treatment and prevention of C. difficile infection (CDI), the isolation and purification of spores are necessary to study the mechanisms of sporulation and germination, investigate spore properties and resistances, and for use in animal models of CDI. This chapter provides basic protocols, in vitro growth conditions and additional considerations for purifying C. difficile spores for a variety of downstream applications. PMID:27507337

  7. Host-derived smooth muscle cells accumulate in cardiac allografts: role of inflammation and monocyte chemoattractant protein 1.

    Directory of Open Access Journals (Sweden)

    Piotr Religa

    Full Text Available Transplant arteriosclerosis is characterized by inflammation and intimal thickening caused by accumulation of smooth muscle cells (SMCs both from donor and recipient. We assessed the relationship between clinical factors and the presence of host-derived SMCs in 124 myocardial biopsies from 26 consecutive patients who received hearts from opposite-sex donors. Clinical and demographic information was obtained from the patients' medical records. Host-derived SMCs accounted for 3.35+/-2.3% of cells in arterioles (range, 0.08-12.51%. As shown by linear regression analysis, an increased number of SMCs was associated with rejection grade (mean, 1.41+/-1.03, p = 0.034 and the number of leukocytes (19.1+/-12.7 per 20 high-power fields, p = 0.01. The accumulation of host-derived SMCs was associated with an increased number of leukocytes in the allografts. In vitro, monocyte chemoattractant protein 1 (MCP-1 released from leukocytes was crucial for SMC migration. After heart allotransplantation, mice treated with MCP-1-specific antibodies had significantly fewer host-derived SMCs in the grafts than mice treated with isotypic antibody controls. We conclude that the number of host-derived SMCs in human cardiac allografts is associated with the rejection grade and that MCP-1 may play pivotal role in recruiting host-derived SMCs into cardiac allografts.

  8. Host-Derived Smooth Muscle Cells Accumulate in Cardiac Allografts: Role of Inflammation and Monocyte Chemoattractant Protein 1

    Science.gov (United States)

    Bojakowski, Krzysztof; Soin, Joanna; Nozynski, Jerzy; Zakliczynski, Michal; Gaciong, Zbigniew; Zembala, Marian; Söderberg-Nauclér, Cecilia

    2009-01-01

    Transplant arteriosclerosis is characterized by inflammation and intimal thickening caused by accumulation of smooth muscle cells (SMCs) both from donor and recipient. We assessed the relationship between clinical factors and the presence of host-derived SMCs in 124 myocardial biopsies from 26 consecutive patients who received hearts from opposite-sex donors. Clinical and demographic information was obtained from the patients' medical records. Host-derived SMCs accounted for 3.35±2.3% of cells in arterioles (range, 0.08–12.51%). As shown by linear regression analysis, an increased number of SMCs was associated with rejection grade (mean, 1.41±1.03, p = 0.034) and the number of leukocytes (19.1±12.7 per 20 high-power fields, p = 0.01). The accumulation of host-derived SMCs was associated with an increased number of leukocytes in the allografts. In vitro, monocyte chemoattractant protein 1 (MCP-1) released from leukocytes was crucial for SMC migration. After heart allotransplantion, mice treated with MCP-1-specific antibodies had significantly fewer host-derived SMCs in the grafts than mice treated with isotypic antibody controls. We conclude that the number of host-derived SMCs in human cardiac allografts is associated with the rejection grade and that MCP-1 may play pivotal role in recruiting host-derived SMCs into cardiac allografts. PMID:19142231

  9. Adipocyte protein modification by Krebs cycle intermediates and fumarate ester-derived succination.

    Science.gov (United States)

    Manuel, Allison M; Frizzell, Norma

    2013-11-01

    Protein succination, the non-enzymatic modification of cysteine residues by fumarate, is distinguishable from succinylation, an enzymatic reaction forming an amide bond between lysine residues and succinyl-CoA. Treatment of adipocytes with 30 mM glucose significantly increases protein succination with only a small change in succinylation. Protein succination may be significantly increased intracellularly after treatment with fumaric acid esters, however, the ester must be removed by saponification to permit 2SC-antibody detection of the fumarate adduct.

  10. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  11. Lymphocyte activation by purified HLA-DR molecules fused into autochthonous "stimulating cells".

    Science.gov (United States)

    Diu, A; Abikar, K; Rode, H N; Gordon, J

    1985-08-01

    Affinity-purified Ia molecules derived from the Daudi cell line were reconstituted into vesicles with Sendai virus envelope glycoproteins. These vesicles inserted into human peripheral leukocytes could induce stimulation of autologous lymphocytes, as measured by thymidine uptake, 6 days later. It is suggested that this method could provide a means to study allostimulation at the molecular level.

  12. Protein Structure Validation and Refinement Using Chemical Shifts Derived from Quantum Mechanics

    DEFF Research Database (Denmark)

    Bratholm, Lars Andersen

    In this thesis, my work involving dierent aspects of protein structure determination by computer modeling is presented. Determination of several protein's native fold were carried out with Markov chain Monte Carlo simulations in the PHAISTOS protein structure simulation framework, utilizing...... to within 3 A. Furthermore, a fast quantum mechanics based chemical shift predictor was developed together with methodology for using chemical shifts in structure simulations. The developed predictor was used for renement of several protein structures and for reducing the computational cost of quantum...

  13. Host cell proteins in biotechnology-derived products: A risk assessment framework.

    Science.gov (United States)

    de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

    2015-11-01

    To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins.

  14. Envelope glycoproteins of HIV-1, HIV-2, and SIV purified with Galanthus nivalis agglutinin induce strong immune responses.

    Science.gov (United States)

    Gilljam, G

    1993-05-01

    Lectin affinity chromatography was used to purify in a single step the envelope glycoproteins of HIV-1, HIV-2, and SIV. Envelope glycoproteins carry the major determinants essential for protection by the humoral immune response. The purification of these proteins has previously been a laborious procedure. The glycoproteins were purified by a one-step procedure to a high level of purity by using Galanthus nivalis agglutinin (GNA). The purified glycoprotein had CD4-binding and antigenic reactivities. Strong immune responses to envelope proteins and peptides were seen in mice and primates after immunization with these preparations.

  15. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons

    Directory of Open Access Journals (Sweden)

    Jessica Lenzi

    2015-07-01

    Full Text Available Patient-derived induced pluripotent stem cells (iPSCs provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUSR514S and FUSR521C patient fibroblasts, whereas in the case of the severe FUSP525L mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.

  16. Schiff Base Metal Derivatives Enhance the Expression of HSP70 and Suppress BAX Proteins in Prevention of Acute Gastric Lesion

    Directory of Open Access Journals (Sweden)

    Shahram Golbabapour

    2013-01-01

    Full Text Available Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg, the positive control (Tween 20 5% v/v, 5 mL/kg, and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg. After 1 h, all of the groups received ethanol 95% (5 mL/kg but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg. The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E, immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

  17. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons

    Science.gov (United States)

    Lenzi, Jessica; De Santis, Riccardo; de Turris, Valeria; Morlando, Mariangela; Laneve, Pietro; Calvo, Andrea; Caliendo, Virginia; Chiò, Adriano; Rosa, Alessandro; Bozzoni, Irene

    2015-01-01

    ABSTRACT Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUSR514S and FUSR521C patient fibroblasts, whereas in the case of the severe FUSP525L mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis. PMID:26035390

  18. Purification and characterisation of antibacterial peptide-containing compound derived from palm kernel cake.

    Science.gov (United States)

    Tan, Yen Nee; Ayob, Mohd Khan; Wan Yaacob, Wan Ahmad

    2013-01-01

    Palm kernel cake (PKC), the most useful by-product resulted from palm kernel oil production. In this study, PKC-derived protein product was found suitable for use as an antimicrobial agent with potent antibacterial activity, particularly against Bacillus species, after enzymatic hydrolysis with alcalase. The hydrolysate was further purified by gel filtration chromatography. The purified fraction was found to have 14.63±0.70% (w/w) protein, a molecular mass of 2.4kDa and low hemolytic activity (antibacterial effect of purified PKC fraction. This study suggests that the antibacterial PKC compound may be not a pure peptide but instead a peptide-containing compound high in lauric acid derivative.

  19. Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.; Belair, David G.; Rettko, Nicholas J.; Murphy, William L.; Forest, Katrina T.; Gellman, Samuel H. (UW)

    2015-04-14

    Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF₁₆₅-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

  20. Unbiased quantitative models of protein translation derived from ribosome profiling data

    NARCIS (Netherlands)

    Gritsenko, A.A.; Hulsman, M.; Reinders, M.J.T.; Ridder, de D.

    2015-01-01

    Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of prot

  1. Unbiased Quantitative Models of Protein Translation Derived from Ribosome Profiling Data

    NARCIS (Netherlands)

    Gritsenko, A.A.; Hulsman, M.; Reinders, M.J.T.; De Ridder, D.

    2015-01-01

    Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of prot

  2. Protein structure validation and refinement using amide proton chemical shifts derived from quantum mechanics

    DEFF Research Database (Denmark)

    Christensen, Anders Steen; Linnet, Troels Emtekær; Borg, Mikael;

    2013-01-01

    We present the ProCS method for the rapid and accurate prediction of protein backbone amide proton chemical shifts - sensitive probes of the geometry of key hydrogen bonds that determine protein structure. ProCS is parameterized against quantum mechanical (QM) calculations and reproduces high level...

  3. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    Science.gov (United States)

    Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-08-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.

  4. Detergent activation of the binding protein in the folate radioassay

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with ..beta..-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to ..beta..-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants (lipids/detergents).

  5. Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant.

    Science.gov (United States)

    Hoffmann, Toni; Stadler, Lukas Kurt Josef; Busby, Michael; Song, Qifeng; Buxton, Anthony T; Wagner, Simon D; Davis, Jason J; Ko Ferrigno, Paul

    2010-05-01

    Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein-protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in 'loop 2' allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.

  6. Effects of different routes of administration on the immunogenicity of the Tat protein and a Tat-derived peptide.

    Science.gov (United States)

    Finessi, Valentina; Nicoli, Francesco; Gallerani, Eleonora; Sforza, Fabio; Sicurella, Mariaconcetta; Cafaro, Aurelio; Caputo, Antonella; Ensoli, Barbara; Gavioli, Riccardo

    2015-01-01

    The use of the Tat protein of HIV in vaccines against AIDS showed promising results in primate and human studies. To characterize the impact of the administration route on the induction of humoral responses at systemic and mucosal levels, we compared intradermal, intramuscular and mucosal immunizations with Tat and a Tat-derived peptide. Mice were immunized with the Tat protein by different routes and the titer and isotype of anti-Tat antibodies were assessed in serum and mucosal lavages. Intramuscular and intradermal administrations showed comparable immunogenicity, while the mucosal administration was unable to induce IgM in serum and IgG at mucosal sites but showed superior immunogenicity in terms of IgA induction. Anti-Tat antibodies were also obtained upon vaccination with the immunodominant Tat 1-20 peptide which was, however, less immunogenic than the whole Tat protein.

  7. Purified oleosins at air-water interfaces

    NARCIS (Netherlands)

    Nikiforidis, C.V.; Ampatzidis, C.; Lalou, S.; Scholten, E.; Karapantsios, T.D.; Kiosseoglou, V.

    2013-01-01

    Oleosins are low molecular mass proteins that are distinguished from other proteins for their extended central hydrophobic domain which covers almost half of its entity. For this work, they were extracted from isolated maize germ oil bodies. The purification steps included washing with diethylether

  8. Purified oleosins at air-water interfaces

    NARCIS (Netherlands)

    Nikiforidis, C.V.; Ampatzidis, C.; Lalou, S.; Scholten, E.; Karapantsios, T.D.; Kiosseoglou, V.

    2013-01-01

    Oleosins are low molecular mass proteins that are distinguished from other proteins for their extended central hydrophobic domain which covers almost half of its entity. For this work, they were extracted from isolated maize germ oil bodies. The purification steps included washing with diethylether

  9. Protein structure validation and refinement using amide proton chemical shifts derived from quantum mechanics

    CERN Document Server

    Christensen, Anders S; Borg, Mikael; Boomsma, Wouter; Lindorff-Larsen, Kresten; Hamelryck, Thomas; Jensen, Jan H

    2013-01-01

    We present the ProCS method for the rapid and accurate prediction of protein backbone amide proton chemical shifts - sensitive probes of the geometry of key hydrogen bonds that determine protein structure. ProCS is parameterized against quantum mechanical (QM) calculations and reproduces high level QM results obtained for a small protein with an RMSD of 0.25 ppm (r = 0.94). ProCS is interfaced with the PHAISTOS protein simulation program and is used to infer statistical protein ensembles that reflect experimentally measured amide proton chemical shift values. Such chemical shift-based structural refinements, starting from high-resolution X-ray structures of Protein G, ubiquitin, and SMN Tudor Domain, result in average chemical shifts, hydrogen bond geometries, and trans-hydrogen bond (h3JNC') spin-spin coupling constants that are in excellent agreement with experiment. We show that the structural sensitivity of the QM-based amide proton chemical shift predictions is needed to refine protein structures to this...

  10. Milk proteins-derived bioactive peptides in dairy products: molecular, biological and methodological aspects

    Directory of Open Access Journals (Sweden)

    Bartłomiej Dziuba

    2014-03-01

    Full Text Available Proteins are one of the primary components of the food, both in terms of nutrition and function. They are main source of amino acids, essential for synthesis of proteins, and also source of energy. Additionally, many proteins exhibit specifi c biological activities, which may have effect on functional or pro-health properties of food products. These proteins and their hydrolysis products, peptides, may infl uence the properties of food and human organism. The number of commercially available food products containing bioactive peptides is very low, apart from that milk proteins are their rich source. It could be supposed that number of available products with declared activity will rise in near future because of observed strong uptrend on interest in such products. Molecular and biological properties of milk proteins, as precursors of bioactive peptides was characterised in the work. Therefore, the strategy of research and obtaining of such peptides both in laboratory and industrial scale, as well as the range of their commercial application, was presented. Several examples of research efforts presenting high potential to develop new products containing bioactive peptides from milk proteins and predetermined as nutraceuticals was described.

  11. Milk proteins-derived bioactive peptides in dairy products: molecular, biological and methodological aspects.

    Science.gov (United States)

    Dziuba, Bartłomiej; Dziuba, Marta

    2014-01-01

    Proteins are one of the primary components of the food, both in terms of nutrition and function. They are main source of amino acids, essential for synthesis of proteins, and also source of energy. Additionally, many proteins exhibit specific biological activities, which may have effect on functional or pro-health properties of food products. These proteins and their hydrolysis products, peptides, may influence the properties of food and human organism. The number of commercially available food products containing bioactive peptides is very low, apart from that milk proteins are their rich source. It could be supposed that number of available products with declared activity will rise in near future because of observed strong uptrend on interest in such products. Molecular and biological properties of milk proteins, as precursors of bioactive peptides was characterised in the work. Therefore, the strategy of research and obtaining of such peptides both in laboratory and industrial scale, as well as the range of their commercial application, was presented. Several examples of research efforts presenting high potential to develop new products containing bioactive peptides from milk proteins and predetermined as nutraceuticals was described.

  12. The challenging measurement of protein in complex biomass-derived samples

    DEFF Research Database (Denmark)

    Haven, M.O.; Jørgensen, H.

    2014-01-01

    and fast protein measurement on this type of samples was the ninhydrin assay. This method has also been used widely for this purpose, but with two different methods for protein hydrolysis prior to the assay - alkaline or acidic hydrolysis. In samples containing glucose or ethanol, there was significant......Measurement of the protein content in samples from production of lignocellulosic bioethanol is an important tool when studying the adsorption of cellulases. Several methods have been used for this, and after reviewing the literature, we concluded that one of the most promising assays for simple...

  13. Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Jing Liu; Li-Xin Zhu; Yu-Ying Kong; Guang-Di Li; Yuan Wang

    2005-01-01

    AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E.coli)and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally trunczated E1 fragments were expressed in E. coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chromatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot.RESULTS: Full-length E1 protein proved difficult to express in E. coli. C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization.CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.

  14. Assessment of interactions between four proteins and benzothiazole derivatives by DSC and CD

    Energy Technology Data Exchange (ETDEWEB)

    Hassan, Natalia [Soft Matter and Molecular Biophysics Group, Department of Applied Physics, Faculty of Physics, Campus Vida, University of Santiago de Compostela, E-15782 Santiago de Compostela (Spain); Verdes, Pedro V., E-mail: pedro.vazquez@usc.e [Soft Matter and Molecular Biophysics Group, Department of Applied Physics, Faculty of Physics, Campus Vida, University of Santiago de Compostela, E-15782 Santiago de Compostela (Spain); Ruso, Juan M. [Soft Matter and Molecular Biophysics Group, Department of Applied Physics, Faculty of Physics, Campus Vida, University of Santiago de Compostela, E-15782 Santiago de Compostela (Spain)

    2011-03-15

    The thermal denaturation of ovalbumin, lysozyme, myoglobin and fibrinogen at different BTS concentrations have been investigated using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Thermodynamic parameters: melting temperatures (T{sub m}), calorimetric enthalpy ({Delta}H), van't Hoff enthalpy ({Delta}H{sub v}) were obtained for all the systems under study. Thermal denaturation of the four proteins was completely irreversible. Changes in the protein conformation due to the adsorption of BTS molecules have been monitored by using UV-CD spectra. Greater changes in {alpha}-helical contents correspond with the BTS higher concentrations. The lysozyme denaturation temperature increases at low concentrations BTS indicating that BTS acts as a structure stabilizer; meanwhile it acts as a destabilizer at higher concentrations in all the proteins studied. The major effect is observed in the case of myoglobin, the protein with the highest {alpha}-helical secondary structure (75%).

  15. Relationship of Soluble Grape-Derived Proteins to Condensed Tannin Extractability during Red Wine Fermentation.

    Science.gov (United States)

    Springer, Lindsay F; Chen, Lei-An; Stahlecker, Avery C; Cousins, Peter; Sacks, Gavin L

    2016-11-02

    In red winemaking, the extractability of condensed tannins (CT) can vary considerably even under identical fermentation conditions, and several explanations for this phenomenon have been proposed. Recent work has demonstrated that grape pathogenesis-related proteins (PRPs) may limit retention of CT added to finished wines, but their relevance to CT extractability has not been evaluated. In this work, Vitis vinifera and interspecific hybrids (Vitis ssp.) from both hot and cool climates were vinified under small-scale, controlled conditions. The final CT concentration in wine was well modeled from initial grape tannin and juice protein concentrations using the Freundlich equation (r(2)= 0.686). In follow-up experiments, separation and pretreatment of juice by bentonite, heating, freezing, or exogenous tannin addition reduced protein concentrations in juices from two grape varieties. The bentonite treatment also led to greater wine CT for one of the varieties, indicating that prefermentation removal of grape protein may be a viable approach to increasing wine CT.

  16. Catalysis of Strand Annealing by Replication Protein A Derives from Its Strand Melting Properties*

    OpenAIRE

    Bartos, Jeremy D.; Willmott, Lyndsay J.; Binz, Sara K.; Wold, Marc S.; Bambara, Robert A.

    2008-01-01

    Eukaryotic DNA-binding protein replication protein A (RPA) has a strand melting property that assists polymerases and helicases in resolving DNA secondary structures. Curiously, previous results suggested that human RPA (hRPA) promotes undesirable recombination by facilitating annealing of flaps produced transiently during DNA replication; however, the mechanism was not understood. We designed a series of substrates, representing displaced DNA flaps generated during ma...

  17. Carbonyl J Acid Derivatives Block Protein Priming of Hepadnaviral P Protein and DNA-Dependent DNA Synthesis Activity of Hepadnaviral Nucleocapsids

    Science.gov (United States)

    Wang, Yong-Xiang; Wen, Yu-Mei

    2012-01-01

    Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 μM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives. PMID:22787212

  18. Pyrrolopyrimidine derivatives and purine analogs as novel activators of Multidrug Resistance-associated Protein 1 (MRP1, ABCC1).

    Science.gov (United States)

    Schmitt, Sven Marcel; Stefan, Katja; Wiese, Michael

    2017-01-01

    Multidrug resistance (MDR) is the main cause of diminished success in cancer chemotherapy. ABC transport proteins are considered to be one important factor of MDR. Besides P-glycoprotein (P-gp, ABCB1) and Breast Cancer Resistance Protein (BCRP, ABCG2), Multidrug Resistance-associated Protein 1 (MRP1, ABCC1) is associated with non-response to chemotherapy in different cancers. While considerable effort was spent in overcoming MDR during the last two decades, almost nothing is known with respect to activators of transport proteins. In this work we present certain pyrrolo[3,2-d]pyrimidine derivatives with variations at positions 4 and 5 and purine analogs with variations at position 6 as novel activators of MRP1-mediated transport of the MRP1 substrate calcein AM and the anticancer drug daunorubicin in low nanomolar concentration range. Two different MRP1 overexpressing cell lines were used, the doxorubicin-selected human lung cancer cell line H69 AR and the transfected Madin-Darby Canine Kidney cell line MDCK II MRP1. No effect was observed in the sensitive counterparts H69 and MDCK II wild type (wt). Derivatives with higher molecular weight possessed also inhibitory properties at low micromolar concentrations, although most compounds were rather poor MRP1 inhibitors. Purine analogs derived from potent MRP1 inhibitors of the pyrrolopyrimidine class showed equal activating, but no inhibiting effects at all. All tested compounds were non-toxic and had only minor impact on P-gp or BCRP, showing no inhibition or activation. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Synthesis of fluorinated maltose derivatives for monitoring protein interaction by 19F NMR

    Directory of Open Access Journals (Sweden)

    Michaela Braitsch

    2012-03-01

    Full Text Available A novel reporter system, which is applicable to the 19F NMR investigation of protein interactions, is presented. This approach uses 2-F-labeled maltose as a spy ligand to indirectly probe protein–ligand or protein–protein interactions of proteins fused or tagged to the maltose-binding protein (MBP. The key feature is the simultaneous NMR observation of both 19F NMR signals of gluco/manno-type-2-F-maltose-isomers; one isomer (α-gluco-type binds to MBP and senses the protein interaction, and the nonbinding isomers (β-gluco- and/or α/β-manno-type are utilized as internal references. Moreover, this reporter system was used for relative affinity studies of fluorinated and nonfluorinated carbohydrates to the maltose-binding protein, which were found to be in perfect agreement with published X-ray data. The results of the NMR competition experiments together with the established correlation between 19F chemical shift data and molecular interaction patterns, suggest valuable applications for studies of protein–ligand interaction interfaces.

  20. Emerging trends in nutraceutical applications of whey protein and its derivatives.

    Science.gov (United States)

    Patel, Seema

    2015-11-01

    The looming food insecurity demands the utilization of nutrient-rich residues from food industries as value-added products. Whey, a dairy industry waste has been characterized to be excellent nourishment with an array of bioactive components. Whey protein comprises 20 % of total milk protein and it is rich in branched and essential amino acids, functional peptides, antioxidants and immunoglobulins. It confers benefits against a wide range of metabolic diseases such as cardiovascular complications, hypertension, obesity, diabetes, cancer and phenylketonuria. The protein has been validated to boost recovery from resistance exercise-injuries, stimulate gut physiology and protect skin against detrimental radiations. Apart from health invigoration, whey protein has proved its suitability as fat replacer and emulsifier. Further, its edible and antimicrobial packaging potential renders its highly desirable in food as well as pharmaceutical sectors. Considering the enormous nutraceutical worth of whey protein, this review emphasizes on its established and emerging biological roles. Present and future scopes in food processing and dietary supplement formulation are discussed. Associated hurdles are identified and how technical advancement might augment its applications are explored. This review is expected to provide valuable insight on whey protein-fortified functional foods, associated technical hurdles and scopes of improvement.

  1. Selective translational repression of truncated proteins from frameshift mutation-derived mRNAs in tumors.

    Directory of Open Access Journals (Sweden)

    Kwon Tae You

    2007-05-01

    Full Text Available Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs. Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD system. However, PTCs located within 50-55 nucleotides of the last exon-exon junction are not recognized by NMD (NMD-irrelevant, and some PTC-containing mRNAs can escape from the NMD system (NMD-escape. We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.

  2. The Effect of Plant Proteins Derived from Cereals and Legumes on Heme Iron Absorption.

    Science.gov (United States)

    Weinborn, Valerie; Pizarro, Fernando; Olivares, Manuel; Brito, Alex; Arredondo, Miguel; Flores, Sebastián; Valenzuela, Carolina

    2015-10-30

    The aim of this study is to determine the effect of proteins from cereals and legumes on heme iron (Fe) absorption. The absorption of heme Fe without its native globin was measured. Thirty adult females participated in two experimental studies (15 per study). Study I focused on the effects of cereal proteins (zein, gliadin and glutelin) and study II on the effects of legume proteins (soy, pea and lentil) on heme Fe absorption. When heme was given alone (as a control), study I and II yielded 6.2% and 11.0% heme absorption (p > 0.05). In study I, heme Fe absorption was 7.2%, 7.5% and 5.9% when zein, gliadin and glutelin were added, respectively. From this, it was concluded that cereal proteins did not affect heme Fe absorption. In study II, heme Fe absorption was 7.3%, 8.1% and 9.1% with the addition of soy, pea and lentil proteins, respectively. Only soy proteins decreased heme Fe absorption (p legumes do not affect heme Fe absorption.

  3. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    Directory of Open Access Journals (Sweden)

    Michał Śmiga

    Full Text Available Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins and T. forsythia (Tfo protein and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

  4. Single molecule DNA compaction by purified histones

    Institute of Scientific and Technical Information of China (English)

    RAN ShiYong; WANG XiaoLing; FU WenBo; WANG WeiChi; LI Ming

    2008-01-01

    The compaction of single DNA molecules by purified histones is studied using magnetic tweezers, The compaction rate increases rapidly when the histone concentration is increased from 0.002 to 0.2 mmol/L, and saturates when the concentration is beyond 0.2 mmol/L, The time course of compaction is exponential at low histone concentrations. It becomes sigmoidal at high concentrations. Cooperativity between the histones bound to DNA is proposed to be responsible for the transition. The histones are loaded onto DNA randomly at low concentrations. They tend to bind DNA cooperatively at high con-centrations because the structural torsions of DNA induced by the bound histones become overlapping so that the binding of one histone facilitates the binding of others. Under very large forces, the com-pacted histone-DNA complex can be disrupted in a discrete manner with a step size of ~60 nm. But the histones cannot be completely stripped off DNA, as is revealed by the lowered B-S transition plateau of the histone-bound DNA.

  5. Involvement of dynamin-related protein 1 in free fatty acid-induced INS-1-derived cell apoptosis.

    Science.gov (United States)

    Peng, Liang; Men, Xiuli; Zhang, Wenjian; Wang, Haiyan; Xu, Shiqing; Fang, Qing; Liu, Honglin; Yang, Wenying; Lou, Jinning

    2012-01-01

    Elevated extracellular free fatty acids (FFAs) can induce pancreatic beta cell apoptosis, thereby contributing to the pathogenesis of type 2 diabetes mellitus (T2D). Mitochondrial dysfunction has been implicated in FFA-induced beta cell apoptosis. However, molecular mechanisms linking mitochondrial dysfunction and FFA-induced beta cell apoptosis are not clear. Dynamin-related protein 1 (DRP-1) is a mitochondrial fission modulator. In this study, we investigated its role in FFA-induced INS-1 beta cell apoptosis. DRP-1 protein was promptly induced in INS-1 cells and rat islets after stimulation by FFAs, and this DRP-1 upregulation was accompanied by increased INS-1 cell apoptosis. Induction of DRP-1 expression significantly promoted FFA-induced apoptosis in DRP-1 WT (DRP-1 wild type) inducible INS-1-derived cell line, but not in DRP-1K38A (a dominant negative mutant of DRP-1) inducible INS-1-derived cell line. To validate these in vitro results, we transplanted DRP-1 WT or DRP-1 K38A cells into renal capsules of streptozotocin (STZ)-treated diabetic mice to study the apoptosis in xenografts. Consistent with the in vitro results, the over-expression of DRP-1 led to aggravated INS-1-derived cell apoptosis triggered by FFAs. In contrast, dominant-negative suppression of DRP-1 function as represented by DRP-1 K38A significantly prevented FFA-induced apoptosis in xenografts. It was further demonstrated that mitochondrial membrane potential decreased, while cytochrome c release, caspase-3 activation, and generation of reactive oxygen species (ROS) were enhanced by the induction of DRP-1WT, but prevented by DRP-1 K38A in INS-1-derived cells under FFA stimulation. These results indicated that DRP-1 mediates FFA-induced INS-1-derived cell apoptosis, suggesting that suppression of DRP-1 is a potentially useful therapeutic strategy for protecting against beta cell loss that leads to type 2 diabetes.

  6. Purified and synthetic Alzheimer's amyloid beta (Aβ) prions.

    Science.gov (United States)

    Stöhr, Jan; Watts, Joel C; Mensinger, Zachary L; Oehler, Abby; Grillo, Sunny K; DeArmond, Stephen J; Prusiner, Stanley B; Giles, Kurt

    2012-07-03

    The aggregation and deposition of amyloid-β (Aβ) peptides are believed to be central events in the pathogenesis of Alzheimer's disease (AD). Inoculation of brain homogenates containing Aβ aggregates into susceptible transgenic mice accelerated Aβ deposition, suggesting that Aβ aggregates are capable of self-propagation and hence might be prions. Recently, we demonstrated that Aβ deposition can be monitored in live mice using bioluminescence imaging (BLI). Here, we use BLI to probe the ability of Aβ aggregates to self-propagate following inoculation into bigenic mice. We report compelling evidence that Aβ aggregates are prions by demonstrating widespread cerebral β-amyloidosis induced by inoculation of either purified Aβ aggregates derived from brain or aggregates composed of synthetic Aβ. Although synthetic Aβ aggregates were sufficient to induce Aβ deposition in vivo, they exhibited lower specific biological activity compared with brain-derived Aβ aggregates. Our results create an experimental paradigm that should lead to identification of self-propagating Aβ conformations, which could represent novel targets for interrupting the spread of Aβ deposition in AD patients.

  7. Structural conversion of the transformer protein RfaH: new insights derived from protein structure prediction and molecular dynamics simulations.

    Science.gov (United States)

    Balasco, Nicole; Barone, Daniela; Vitagliano, Luigi

    2015-01-01

    Recent structural investigations have shown that the C-terminal domain (CTD) of the transcription factor RfaH undergoes unique structural modifications that have a profound impact into its functional properties. These modifications cause a complete change in RfaH(CTD) topology that converts from an α-hairpin to a β-barrel fold. To gain insights into the determinants of this major structural conversion, we here performed computational studies (protein structure prediction and molecular dynamics simulations) on RfaH(CTD). Although these analyses, in line with literature data, suggest that the isolated RfaH(CTD) has a strong preference for the β-barrel fold, they also highlight that a specific region of the protein is endowed with a chameleon conformational behavior. In particular, the Leu-rich region (residues 141-145) has a good propensity to adopt both α-helical and β-structured states. Intriguingly, in the RfaH homolog NusG, whose CTD uniquely adopts the β-barrel fold, the corresponding region is rich in residues as Val or Ile that present a strong preference for the β-structure. On this basis, we suggest that the presence of this Leu-rich element in RfaH(CTD) may be responsible for the peculiar structural behavior of the domain. The analysis of the sequences of RfaH family (PfamA code PF02357) unraveled that other members potentially share the structural properties of RfaH(CTD). These observations suggest that the unusual conformational behavior of RfaH(CTD) may be rare but not unique.

  8. A knowledge-based scoring function for protein-RNA interactions derived from a statistical mechanics-based iterative method.

    Science.gov (United States)

    Huang, Sheng-You; Zou, Xiaoqin

    2014-04-01

    Protein-RNA interactions play important roles in many biological processes. Given the high cost and technique difficulties in experimental methods, computationally predicting the binding complexes from individual protein and RNA structures is pressingly needed, in which a reliable scoring function is one of the critical components. Here, we have developed a knowledge-based scoring function, referred to as ITScore-PR, for protein-RNA binding mode prediction by using a statistical mechanics-based iterative method. The pairwise distance-dependent atomic interaction potentials of ITScore-PR were derived from experimentally determined protein-RNA complex structures. For validation, we have compared ITScore-PR with 10 other scoring methods on four diverse test sets. For bound docking, ITScore-PR achieved a success rate of up to 86% if the top prediction was considered and up to 94% if the top 10 predictions were considered, respectively. For truly unbound docking, the respective success rates of ITScore-PR were up to 24 and 46%. ITScore-PR can be used stand-alone or easily implemented in other docking programs for protein-RNA recognition.

  9. The neuroendocrine-derived peptide parathyroid hormone-related protein promotes prostate cancer cell growth by stabilizing the androgen receptor.

    Science.gov (United States)

    DaSilva, John; Gioeli, Daniel; Weber, Michael J; Parsons, Sarah J

    2009-09-15

    During progression to an androgen-independent state following androgen ablation therapy, prostate cancer cells continue to express the androgen receptor (AR) and androgen-regulated genes, indicating that AR is critical for the proliferation of hormone-refractory prostate cancer cells. Multiple mechanisms have been proposed for the development of AR-dependent hormone-refractory disease, including changes in expression of AR coregulatory proteins, AR mutation, growth factor-mediated activation of AR, and AR protein up-regulation. The most prominent of these progressive changes is the up-regulation of AR that occurs in >90% of prostate cancers. A common feature of the most aggressive hormone-refractory prostate cancers is the accumulation of cells with neuroendocrine characteristics that produce paracrine factors and may provide a novel mechanism for the regulation of AR during advanced stages of the disease. In this study, we show that neuroendocrine-derived parathyroid hormone-related protein (PTHrP)-mediated signaling through the epidermal growth factor receptor (EGFR) and Src pathways contributes to the phenotype of advanced prostate cancer by reducing AR protein turnover. PTHrP-induced accumulation of AR depended on the activity of Src and EGFR and consequent phosphorylation of the AR on Tyr(534). PTHrP-induced tyrosine phosphorylation of AR resulted in reduced AR ubiquitination and interaction with the ubiquitin ligase COOH terminus of Hsp70-interacting protein. These events result in increased accumulation of AR and thus enhanced growth of prostate cancer cells at low levels of androgen.

  10. Hepatitis C virus core proteins derived from different quasispecies of genotype 1b inhibit the growth of Chang liver cells

    Institute of Scientific and Technical Information of China (English)

    Xue-Bing Yan; Lei Mei; Xia Feng; Mei-Rong Wan; Zhi Chen; Nicole Pavia; Christian Brechot

    2008-01-01

    AIM: To investigate the influence of different quasispecies of hepatitis C virus (HCV) genotype 1b core protein on growth of Chang liver cells.METHODS: Three eukaryotic expression plasmids (pEGFP-N1/core) that contained different quasispecies truncated core proteins of HCV genotype 1b were constructed. These were derived from tumor (T) and nontumor (NT) tissues of a patient infected with HCV and C191 (HCV-J6). The core protein expression plasmids were transiently transfected into Chang liver cells. At different times, the cell cycle and apoptosis was assayed by flow cytometry, and cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) assay.RESULTS: The proportion of S-phase Chang liver cells transfected with pEGFP-N1/core was significantly lower than that of cells transfected with blank plasmid at three different times after transfection (all P NT > C191), and apoptosis was increased in cells transfected with pEGFP-N1/core as the transfection time increased (72 h > 48 h > 24 h).CONCLUSION: These results suggest that HCV genotype 1b core protein induces apoptosis, and inhibits cellcycle progression and proliferation of Chang liver cells.Different quasispecies core proteins of HCV genotype 1b might have some differences in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.

  11. Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP).

    Science.gov (United States)

    Schubert, Thomas; Schlegel, Jacqueline; Schmid, Rainer; Opolka, Alfred; Grassel, Susanne; Humphries, Martin; Bosserhoff, Anja-Katrin

    2010-03-31

    Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.

  12. Marinopyrrole derivatives with sulfide spacers as selective disruptors of Mcl-1 binding to pro-apoptotic protein Bim.

    Science.gov (United States)

    Cheng, Chunwei; Liu, Yan; Balasis, Maria E; Garner, Thomas P; Li, Jerry; Simmons, Nicholas L; Berndt, Norbert; Song, Hao; Pan, Lili; Qin, Yong; Nicolaou, K C; Gavathiotis, Evripidis; Sebti, Said M; Li, Rongshi

    2014-07-29

    A series of novel marinopyrroles with sulfide and sulphone spacers were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-xL, was evaluated using ELISA assays. Fluorescence-quenching (FQ) assays confirmed the direct binding of marinopyrroles to Mcl-1. Benzyl- and benzyl methoxy-containing sulfide derivatives 4 and 5 were highly potent dual Mcl-1/Bim and Bcl-xL/Bim disruptors (IC50 values of 600 and 700 nM), whereas carboxylate-containing sulfide derivative 9 exhibited 16.4-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding. In addition, a nonsymmetrical marinopyrrole 12 is as equally potent as the parent marinopyrrole A (1) for disrupting both Mcl-1/Bim and Bcl-xL/Bim binding. Some of the derivatives were also active in intact human breast cancer cells where they reduced the levels of Mcl-1, induced programd cell death (apoptosis) and inhibited cell proliferation.

  13. Marinopyrrole Derivatives with Sulfide Spacers as Selective Disruptors of Mcl-1 Binding to Pro-Apoptotic Protein Bim

    Directory of Open Access Journals (Sweden)

    Chunwei Cheng

    2014-07-01

    Full Text Available A series of novel marinopyrroles with sulfide and sulphone spacers were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-xL, was evaluated using ELISA assays. Fluorescence-quenching (FQ assays confirmed the direct binding of marinopyrroles to Mcl-1. Benzyl- and benzyl methoxy-containing sulfide derivatives 4 and 5 were highly potent dual Mcl-1/Bim and Bcl-xL/Bim disruptors (IC50 values of 600 and 700 nM, whereas carboxylate-containing sulfide derivative 9 exhibited 16.4-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding. In addition, a nonsymmetrical marinopyrrole 12 is as equally potent as the parent marinopyrrole A (1 for disrupting both Mcl-1/Bim and Bcl-xL/Bim binding. Some of the derivatives were also active in intact human breast cancer cells where they reduced the levels of Mcl-1, induced programd cell death (apoptosis and inhibited cell proliferation.

  14. Liposomal leakage induced by virus-derived peptides, viral proteins, and entire virions: rapid analysis by chip electrophoresis.

    Science.gov (United States)

    Weiss, Victor U; Bilek, Gerhard; Pickl-Herk, Angela; Subirats, Xavier; Niespodziana, Katarzyna; Valenta, Rudolf; Blaas, Dieter; Kenndler, Ernst

    2010-10-01

    Permeabilization of model lipid membranes by virus-derived peptides, viral proteins, and entire virions of human rhinovirus was assessed by quantifying the release of a fluorescent dye from liposomes via a novel chip electrophoretic assay. Liposomal leakage readily occurred upon incubation with the pH-sensitive synthetic fusogenic peptide GALA and, less efficiently, with a 24mer peptide (P1-N) derived from the N-terminus of the capsid protein VP1 of human rhinovirus 2 (HRV2) at acidic pH. Negative stain transmission electron microscopy showed that liposomes incubated with the rhinovirus-derived peptide remained largely intact. At similar concentrations, the GALA peptide caused gross morphological changes of the liposomes. On a molar basis, the leakage-inducing efficiency of the P1 peptide was by about 2 orders of magnitude inferior to that of recombinant VP1 (from HRV89) and entire HRV2. This underscores the role in membrane destabilization of VP1 domains remote from the N-terminus and the arrangement of the peptide in the context of the icosahedral virion. Our method is rapid, requires tiny amounts of sample, and allows for the parallel determination of released and retained liposomal cargo.

  15. Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs.

    Science.gov (United States)

    He, Mian; Qin, Hao; Poon, Terence C W; Sze, Siu-Ching; Ding, Xiaofan; Co, Ngai Na; Ngai, Sai-Ming; Chan, Ting-Fung; Wong, Nathalie

    2015-09-01

    Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic RNAs and proteins, such as MET protooncogene, S100 family members and the caveolins. Of interest, we found that exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis.

  16. New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

    Science.gov (United States)

    Dziuba, Bartłomiej; Dziuba, Marta

    2014-01-01

    New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins. PMID:25141106

  17. New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

    Directory of Open Access Journals (Sweden)

    Bartłomiej Dziuba

    2014-08-01

    Full Text Available New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM, random forest (RF, artificial neural networks (ANN and discriminant analysis (DA available in the Collection of Anti-Microbial Peptides (CAMP database. Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.

  18. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  19. Purifying selection, drift, and reversible mutation with arbitrarily high mutation rates.

    Science.gov (United States)

    Charlesworth, Brian; Jain, Kavita

    2014-12-01

    Some species exhibit very high levels of DNA sequence variability; there is also evidence for the existence of heritable epigenetic variants that experience state changes at a much higher rate than sequence variants. In both cases, the resulting high diversity levels within a population (hyperdiversity) mean that standard population genetics methods are not trustworthy. We analyze a population genetics model that incorporates purifying selection, reversible mutations, and genetic drift, assuming a stationary population size. We derive analytical results for both population parameters and sample statistics and discuss their implications for studies of natural genetic and epigenetic variation. In particular, we find that (1) many more intermediate-frequency variants are expected than under standard models, even with moderately strong purifying selection, and (2) rates of evolution under purifying selection may be close to, or even exceed, neutral rates. These findings are related to empirical studies of sequence and epigenetic variation.

  20. Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein.

    Science.gov (United States)

    Sung, Min Sun; Mun, Ji-Young; Kwon, Ohsuk; Kwon, Ki-Sun; Oh, Doo-Byoung

    2013-07-19

    Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Exploring the unbinding of Leishmania (L.) amazonensis CPB derived-epitopes from H2 MHC class I proteins.

    Science.gov (United States)

    Brandt, Artur M L; Batista, Paulo Ricardo; Souza-Silva, Franklin; Alves, Carlos Roberto; Caffarena, Ernesto Raul

    2016-04-01

    New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C-terminus extension (cyspep) can play a decisive role in the host-parasite interaction. The interaction between cyspep-derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep-derived peptides, previously identified as capable of interacting with H-2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H-2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔGd ) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔGd values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H-2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H-2 proteins occurring at ∼ 25 Å.

  2. INHIBITION OF THE DNA-BINDING ACTIVITY OF DROSOPHILA SUPPRESSOR OF HAIRLESS AND OF ITS HUMAN HOMOLOG, KBF2/RBP-J-KAPPA, BY DIRECT PROTEIN-PROTEIN INTERACTION WITH DROSOPHILA HAIRLESS

    NARCIS (Netherlands)

    BROU, C; LOGEAT, F; LECOURTOIS, M; VANDEKERCKHOVE, Joël; KOURILSKY, P; SCHWEISGUTH, F; ISRAEL, A

    1994-01-01

    We have purified the sequence-specific DNA-binding protein KBF2 and cloned the corresponding cDNA, which is derived from the previously described RBP-J kappa gene, the human homolog of the Drosophila Suppressor of Hairless [Su(H)] gene. Deletion studies of the RBP-J kappa and Su(H) proteins allowed

  3. Peptide vaccination against multiple myeloma using peptides derived from anti-apoptotic proteins

    DEFF Research Database (Denmark)

    Jørgensen, Nicolai Grønne; Ahmad, Shamaila Munir; Abildgaard, Niels

    2016-01-01

    The B-cell lymphoma-2 (Bcl-2) family of proteins play a crucial role in multiple myeloma (MM), contributing to lacking apoptosis which is a hallmark of the disease. This makes the Bcl-2 proteins interesting targets for therapeutic peptide vaccination. We report a phase I trial of therapeutic...... vaccination with peptides from the proteins Bcl-2, Bcl-XL and Mcl-1 in patients with relapsed MM. Vaccines were given concomitant with bortezomib. Out of 7 enrolled patients, 4 received the full course of 8 vaccinations. The remaining 3 patients received fewer vaccinations due to progression, clinical...... decision of lacking effect and development of hypercalcemia, respectively. There were no signs of toxicity other than what was to be expected from bortezomib. Immune responses to the peptides were seen in all 6 patients receiving more than 2 vaccinations. Three patients had increased immune responses after...

  4. Binding properties of HABA-type azo derivatives to avidin and avidin-related protein 4.

    Science.gov (United States)

    Repo, Susanna; Paldanius, Tiina A; Hytönen, Vesa P; Nyholm, Thomas K M; Halling, Katrin K; Huuskonen, Juhani; Pentikäinen, Olli T; Rissanen, Kari; Slotte, J Peter; Airenne, Tomi T; Salminen, Tiina A; Kulomaa, Markku S; Johnson, Mark S

    2006-10-01

    The chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to D-biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands beta3 and beta4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 A) X-ray structure for avidin reported here. These results may be valuable in designing improved tools for avidin-based life science and nanobiotechnology applications.

  5. Dual host-defence functions of SPLUNC2/PSP and synthetic peptides derived from the protein.

    Science.gov (United States)

    Gorr, Sven-Ulrik; Abdolhosseini, Mahsa; Shelar, Anuradha; Sotsky, Julie

    2011-08-01

    PSP (parotid secretory protein)/SPLUNC2 (short palate, lung and nasal epithelium clone 2) is expressed in human salivary glands and saliva. The protein exists as an N-glycosylated and non-glycosylated form and both appear to induce agglutination of bacteria, a major antibacterial function for salivary proteins. Both forms of PSP/SPLUNC2 bind LPS (lipopolysaccharide), suggesting that the protein may also play an anti-inflammatory role. Based on the predicted structure of PSP/SPLUNC2 and the location of known antibacterial and anti-inflammatory peptides in BPI (bactericidal/permeability-increasing protein) and LBP (LPS-binding protein), we designed GL13NH2 and GL13K, synthetic peptides that capture these proposed functions of PSP/SPLUNC2. GL13NH3 agglutinates bacteria, leading to increased clearance by macrophages and reduced spread of infection in a plant model. GL13K kills bacteria with a minimal inhibitory concentration of 5-10 μg/ml, kills bacteria in biofilm and retains activity in 150 mM NaCl and 50% saliva. Both peptides block endotoxin action, but only GL13K appears to bind endotoxin. The peptides do not cause haemolysis, haemagglutination in serum, inhibit mammalian cell proliferation or induce an inflammatory response in macrophages. These results suggest that the GL13NH2 and the modified peptide GL13K capture the biological activity of PSP/SPLUNC2 and can serve as lead compounds for the development of novel antimicrobial and anti-inflammatory peptides.

  6. High resolution preparation of monocyte-derived macrophages (MDM protein fractions for clinical proteomics

    Directory of Open Access Journals (Sweden)

    Olivieri Oliviero

    2009-02-01

    Full Text Available Abstract Background Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels. Results Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis indicated no fraction cross contamination. On 2D-PAGE mini gels (7 × 8 cm we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS. Conclusion This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.

  7. Quantification of protein-derived thiols during atmosphere-controlled brewing in laboratory scale

    DEFF Research Database (Denmark)

    Murmann, Anne Nordmark; Andersen, Preben; Mauch, Alexander

    2016-01-01

    An atmosphere-controlled brewing system was built to study thiol oxidation during brewing in laboratory scale under conditions with limited oxygen exposure. Quantification of free and total thiols and protein showed that thiols were lost during wort boiling possibly owing to protein precipitation...... was more pronounced at longer incubation times. However, the reduction of the pool of oxidized thiols by sulfite was inefficient for sulfite concentrations typically found in beer, and the reaction was found to be relatively slow compared with reduction by tris(carboxyethyl)phosphine....

  8. Functionality of lipids and lipid-protein interactions in cereal-derived food products

    Directory of Open Access Journals (Sweden)

    Marion Didier

    2003-01-01

    Full Text Available Lipids and especially cereal lipids play a significant role in the processing and quality of cereals and baked cereal foods (bread, biscuits and beverages (beer. Most of the physico-chemical mechanisms responsible for the lipid functionality has been investigated and recently the specific role of lipid-binding proteins, e.g. lipid transfer proteins and puroindolines, has been highlighted. The state of the researches performed in this field are briefly presented in this review and the data obtained until now show that new perspectives are opened in cereal breeding and processing for improving the quality of cereals and cereal products.

  9. Evolution of a derived protein-protein interaction between HoxA11 and Foxo1a in mammals caused by changes in intramolecular regulation.

    Science.gov (United States)

    Brayer, Kathryn J; Lynch, Vincent J; Wagner, Günter P

    2011-08-09

    Current models of developmental evolution suggest changes in gene regulation underlie the evolution of morphology. Despite the fact that protein complexes regulate gene expression, the evolution of regulatory protein complexes is rarely studied. Here, we investigate the evolution of a protein-protein interaction (PPI) between Homeobox A11 (HoxA11) and Forkhead box 01A (Foxo1a). Using extant and "resurrected" ancestral proteins, we show that the physical interaction between HoxA11 and Foxo1a originated in the mammalian stem lineage. Functional divergence tests and coimmunoprecipitation with heterologous protein pairs indicate that the evolution of interaction was attributable to changes in HoxA11, and deletion studies demonstrate that the interaction interface is located in the homeodomain region of HoxA11. However, there are no changes in amino acid sequence in the homeodomain region during this time period, indicating that the origin of the derived PPI was attributable to changes outside the binding interface. We infer that the amino acid substitutions in HoxA11 altered Foxo1a's access to the conserved binding interface at the HoxA11 homeodomain. We also found an expansion in the number of paired Hox/Fox binding sites in the genomes of mammalian lineage species suggesting the complex has a biological function. Our data indicate that the physical interaction between HoxA11 and Foxo1a evolved through noninterface changes that facilitate the PPI, which prevents inappropriate interactions, rather than through the evolution of a novel binding interface. We speculate that evolutionary changes of intramolecular regulation have limited pleiotropic effects compared with changes to interaction domains themselves.

  10. Verruculides A and B, two new protein tyrosine phosphatase 1B inhibitors from an Indonesian ascidian-derived Penicillium verruculosum.

    Science.gov (United States)

    Yamazaki, Hiroyuki; Nakayama, Wataru; Takahashi, Ohgi; Kirikoshi, Ryota; Izumikawa, Yuta; Iwasaki, Kohei; Toraiwa, Kengo; Ukai, Kazuyo; Rotinsulu, Henki; Wewengkang, Defny S; Sumilat, Deiske A; Mangindaan, Remy E P; Namikoshi, Michio

    2015-08-15

    Two new merosesquiterpenes, verruculides A (1) and B (2), were isolated from a culture broth of the Indonesian ascidian-derived Penicillium verruculosum TPU1311, together with three known congeners, chrodrimanins A (3), B (4), and H (5). The structures of 1 and 2 were assigned on the basis of their spectroscopic data (1D and 2D NMR, HRMS, UV, CD, and IR). Compound 2 had a linear sesquiterpene moiety and was considered to be the derivative of the biosynthetic precursor for 1 and 3-5. Compounds 1, 3, and 5 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 8.4, 8.5, and 14.9 μM, respectively. Compound 2 showed 40% inhibition at 23.1 μM, while 4 was not active at 20.7 μM.

  11. Protein hairy enhancer of split-1 expression during differentiation of muscle-derived stem cells into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Mina Huang; Zhanpeng Guo; Kun Liu; Xifan Mei; Shiqiang Fang; Jinhao Zeng; Yansong Wang; Yajiang Yuan

    2012-01-01

    Muscle-derived stem cells were isolated from the skeletal muscle of Sprague-Dawley neonatal rats aged 3 days old.Cells at passage 5 were incubated in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum,20 μg/L nerve growth factor,20 μg/L basic fibroblast growth factor and 1% (v/v) penicillin for 6 days.Cells presented with long processes, similar to nerve cells.Connections were formed between cell processes.Immunocytochemical staining with neuron specific enolase verified that cells differentiated into neuron-like cells. Immunofluorescence cytochemistry and western blot results revealed that the expression of protein hairy enhancer of split-1 was significantly reduced.These results indicate that low expression of protein hairy enhancer of split-1 participates in the differentiation of muscle-derived stem cells into neuron-like cells.

  12. CoMFA, CoMSIA and Eigenvalue Analysis on Dibenzodioxepinone and Dibenzodioxocinone Derivatives as Cholesteryl Ester Transfer Protein Inhibitors

    Directory of Open Access Journals (Sweden)

    Mao-sheng Cheng

    2008-08-01

    Full Text Available Abstract: CoMFA, CoMSIA and eigenvalue analysis (EVA were performed to study the structural features of 61 diverse dibenzodioxepinone and dibenzodioxocinone analogues to probe cholesteryl ester transfer protein (CETP inhibitory activity. Three methods yielded statistically significant models upon assessment of cross-validation, bootstrapping, and progressive scrambling. This was further validated by an external set of 13 derivatives. Our results demonstrate that three models have a good interpolation as well as extrapolation. The hydrophobic features were confirmed to contribute significantly to inhibitor potencies, while a pre-oriented hydrogen bond provided by the hydroxyl group at the 3-position indicated a good correlation with previous SAR, and a hydrogen bond acceptor may play a crucial role in CETP inhibition. These derived models may help us to gain a deeper understanding of the binding interaction of these lactone-based compounds and aid in the design of new potent compounds against CETP.

  13. Evaluating purifying selection in the mitochondrial DNA of various mammalian species.

    Directory of Open Access Journals (Sweden)

    Pedro Soares

    Full Text Available Mitochondrial DNA (mtDNA, the circular DNA molecule inside the mitochondria of all eukaryotic cells, has been shown to be under the effect of purifying selection in several species. Traditional testing of purifying selection has been based simply on ratios of nonsynonymous to synonymous mutations, without considering the relative age of each mutation, which can be determined by phylogenetic analysis of this non-recombining molecule. The incorporation of a mutation time-ordering from phylogeny and of predicted pathogenicity scores for nonsynonymous mutations allow a quantitative evaluation of the effects of purifying selection in human mtDNA. Here, by using this additional information, we show that purifying selection undoubtedly acts upon the mtDNA of other mammalian species/genera, namely Bos sp., Canis lupus, Mus musculus, Orcinus orca, Pan sp. and Sus scrofa. The effects of purifying selection were comparable in all species, leading to a significant major proportion of nonsynonymous variants with higher pathogenicity scores in the younger branches of the tree. We also derive recalibrated mutation rates for age estimates of ancestors of these various species and proposed a correction curve in order to take into account the effects of selection. Understanding this selection is fundamental to evolutionary studies and to the identification of deleterious mutations.

  14. Characterisation of the 1st SSI purified MBL standard

    DEFF Research Database (Denmark)

    Laursen, Inga; Højrup, Peter; Houen, Gunnar

    2008-01-01

    BACKGROUND: Mannan-binding lectin (MBL) is of importance in innate immunity. MBL-deficiency, the most common immune defect, is significant in several clinical contexts. The request for MBL diagnostic is increasing, hence we developed a high-purity MBL standard assigned with a traceable value. MET...... purified MBL standard has been produced, and assigned the value 192.6 microg MBL/ml, traceable to an accurate realisation of the unit.......BACKGROUND: Mannan-binding lectin (MBL) is of importance in innate immunity. MBL-deficiency, the most common immune defect, is significant in several clinical contexts. The request for MBL diagnostic is increasing, hence we developed a high-purity MBL standard assigned with a traceable value....... METHODS AND RESULTS: The standard material was produced from human plasma; and the protein concentration determined by amino acid analysis after a preceding desalting. The standard value was assessed by two series of sub-sample analyses from nine vials by the grand mean: 235.7 microg protein/ml (range 191...

  15. Targeted mass spectrometry analysis of neutrophil-derived proteins released during sepsis progression

    DEFF Research Database (Denmark)

    Malmström, E; Davidova, A; Mörgelin, M

    2014-01-01

    Early diagnosis of severe infectious diseases is essential for timely implementation of lifesaving therapies. In a search for novel biomarkers in sepsis diagnosis we focused on polymorphonuclear neutrophils (PMNs). Notably, PMNs have their protein cargo readily stored in granules and following sy...

  16. Effect of high contents of dietary animal-derived protein or carbohydrates on canine fecal microbiota

    NARCIS (Netherlands)

    Hang, I.; Rinttila, T.; Zentek, J.; Kettunen, A.; Alaja, S.; Apajalahti, J.A.; Harmoinen, J.; Vos, de W.M.; Spillmann, T.

    2012-01-01

    BACKGROUND: Considerable evidence suggests that food impacts both the gastro-intestinal (GI) function and the microbial ecology of the canine GI tract. The aim of this study was to evaluate the influence of high-carbohydrate (HC), high-protein (HP) and dry commercial (DC) diets on the canine colonic

  17. Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants.

    Science.gov (United States)

    Chuah, Jo-Ann; Horii, Yoko; Numata, Keiji

    2016-12-16

    The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

  18. A simple pressure cell and delivery system for the preparation of Xe derivatives for protein crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Stowell, M.H.; Soltis, S.M.; Kisker, C.; Peters, J.W.; Schindelin, H.; Rees, D.C.; Cascio, D.; Beamer, L.; Hart, J.; Whitby, F.G. [Carl F. and Winifred H. Braun Laboratories, Mail Stop 147-75CH, California Institute of Technology, Pasadena, CA 91125 (United States)]|[Stanford Synchrotron Radiation Laboratory, SLAC, P. O. Box 4349, Bin 69, Stanford University, CA 94309 (United States)]|[University of California Los Angeles, Institute of Molecular Biology, Department of Chemistry and Biochemistry, Los Angeles, CA 90024 (United States)]|[University of Utah Medical Center, Department of Biochemistry, 50 North Medical Drive, Salt Lake City, UT 84132 (United States)

    1996-09-01

    We have developed a simple device for preparing Xe derivatives under moderate gaseous pressure (1{endash}100 atm). The device employs a Cajon ultra-torr fitting to ensure a gas-tight seal around a standard x-ray capillary. As such, the cell can accommodate standard x-ray capillaries up to 1.5 mm in diameter without any modification. The device is straightforward to utilize, and samples can be mounted and pressurized in a matter of seconds. In addition, a simple and safe purging and pressurization system has been designed and constructed for the use at beamline 7-1 at the Stanford Synchrotron Radiation Laboratory (SSRL). We describe the construction of both the pressure cell and the delivery system and present results on the cells use in the preparation of xenon derivatives. {copyright} {ital 1996 American Institute of Physics.}

  19. Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme.

    Science.gov (United States)

    Avila, Frank W; Cohen, Allie B; Ameerudeen, Fatima S; Duneau, David; Suresh, Shruthi; Mattei, Alexandra L; Wolfner, Mariana F

    2015-08-01

    Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females. Copyright © 2015 by the Genetics Society of America.

  20. Inhibitory effects of gallic acid ester derivatives on Saccharomyces cerevisiae multidrug resistance protein Pdr5p.

    Science.gov (United States)

    Pereira Rangel, Luciana; Fritzen, Márcio; Yunes, Rosendo Augusto; Leal, Paulo César; Creczynski-Pasa, Tânia Beatriz; Ferreira-Pereira, Antônio

    2010-05-01

    Overexpression of the Saccharomyces cerevisiae ABC transporter Pdr5p confers resistance to a range of structurally unrelated xenobiotics. This property allows Pdr5p to be used as a target for novel multidrug resistance reversal reagents or chemosensitizers. Herein, we report the effects of gallic acid derivatives with substitutions either on the ester moiety or in the benzene ring on the activity of Pdr5p. Compounds with a longer side chain (8-16 carbons) resulted in greater inhibition of Pdr5p ATPase. Derivatives with side chains of 8-12 carbons that retained hydroxyl groups on the benzene ring extensively inhibited Pdr5p ATPase activity. These compounds almost completely inhibited the efflux of the Pdr5p fluorescent substrate Rhodamine 6G and at 25 muM chemosensitized the Pdr5p-overexpressing strain AD124567 to fluconazole (0.4 mg mL(-1)). Gallic acid derivatives may be a new class of Pdr5p inhibitors.

  1. In vivo incisional wound healing augmented by platelet-derived growth factor and recombinant c-sis gene homodimeric proteins

    OpenAIRE

    1988-01-01

    Human platelet-derived growth factor (hPDGF) is likely to be important in stimulating tissue repair, based upon its in vivo chemotactic and stimulatory activities for inflammatory cells and fibroblasts and upon the presence of PDGF and related proteins in platelets, macrophages, and activated fibroblasts, cell types that make up the milieu of the healing wound. Recombinant human c-sis (rPDGF-B), homodimers of the B chain of PDGF, were compared with hPDGF in vitro. rPDGF-B was immunologically ...

  2. Data on the identity and myristoylation state of recombinant, purified hippocalcin

    Directory of Open Access Journals (Sweden)

    Anuradha Krishnan

    2016-09-01

    Full Text Available In this data article we report on the purity and post translation modification of bacterially expressed and purified recombinant hippocalcin (HPCA: a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. MALDI-TOF in source decay (ISD analysis was used to identify both the myristoylated or non-myristoylated forms of the protein. MALDI-TOF ISD data on the identity of the protein, amino acid sequence and myristoylation efficiency are provided. This data relates to the article “Single-Column Purification of the Tag-free, Recombinant Form of the Neuronal Calcium Sensor Protein, Hippocalcin Expressed in Eschericia coli” [1].

  3. Zellbiologie des Chondrozyten im Hinblick auf die Arthroseentstehung: Rolle der knorpelspezifischen Wachstumsfaktoren Cartilage-Derived Morphogenetic Proteins

    Directory of Open Access Journals (Sweden)

    Erlacher L

    1999-01-01

    Full Text Available Cartilage-derived morphogenetic proteins-1 and -2 (CDMP-1 and -2 sind Wachstumsfaktoren der Bone morphogenetic protein-Gruppe, die bei der embryonalen Skelettentwicklung maßgeblich beteiligt ist. In vivo führten CDMP-1 und -2 zur Neubildung von Knorpel- und Knochengewebe und mittels in vitro Untersuchungen an Knorpelzellen wurde eine Stimulation der Proteoglykansynthese nachgewiesen. Rezeptorbindungsstudien zeigten eine spezifische Bindung an den BMPR-IB/BMPR-II Rezeptorkomplex. Weiters konnte anhand immunhistochemischer Untersuchungen die Expression von CDMP-1 und -2 in gesundem als auch osteoarthrotischem Gelenksknorpel erbracht werden. In einem in vitro Modell für Osteoarthrose führten beide CDMPs nachhaltig zu einer Steigerung der Proteoglykansynthese und somit zu einer Förderung der Reparaturmechanismen des Knorpelgewebes.

  4. Utilization of glycerin byproduct derived from soybean oil biodiesel as a carbon source for heterologous protein production in Pichia pastoris.

    Science.gov (United States)

    Anastácio, G S; Santos, K O; Suarez, P A Z; Torres, F A G; De Marco, J L; Parachin, N S

    2014-01-01

    Crude glycerol, also known as glycerin, is the main byproduct of the biodiesel industry. It has been estimated that up to 40,000 tons of glycerin will be produced each year by 2020. This study evaluated the value-added use of crude glycerol derived from soybean biodiesel preparation as a carbon source for heterologous protein production using the yeast Pichia pastoris. Eleven glycerin samples were obtained by methanolysis of soybean oil using different acids or bases as catalysts. Cell growth experiments showed that crude glycerol containing either potassium or sodium hydroxide resulted in 1.5-2 times higher final cell densities when compared to glycerol P.A. Finally, crude glycerol containing sodium hydroxide was successfully utilized for constitutive heterologous α-amylase production in P. pastoris. This study demonstrated that crude glycerol without any purification steps may be directly used as carbon source for protein production in P. pastoris.

  5. STUDY ON THE ANTI-TUMOR EFFICACY INDUCED BY HEAT SHOCK PROTEIN 70-PEPTIDE COMPLEXES DERIVED FROM TUMOR CELLS

    Institute of Scientific and Technical Information of China (English)

    傅庆国; 张玮; 孟凡东; 郭仁宣; 姚振宇

    2002-01-01

    Objective. To study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells. Methods. Cells culture, flow cytometric analysis, affinity chromatography for protein purification, SDS-PAGE, Western-blotting and animal experiment were used. Results. HSP70-PC immunization rendered protective effect to both naive and tumorl-bearing mice. All of the naive mice obtained complete resistance to Hcaf cell attack; 40% of the tumor-bearing mice survived for over 90 days, whereas the mice of control group died within 2 weeks (P<0.01). CD8+ subset of T lymphocytes in the peripheral blood of immunized mice increased by 12% . Conclusion. HSP70-PC induces anti-tumor immunity via activation of cytotoxic T lymphocytes (CTLs), and it possesses strong tumor vaccine effect. Our research adds more evidence to support the clinical use of HSP70-PC to fight human cancers.

  6. Emerging roles for the amyloid precursor protein and derived peptides in the regulation of cellular and systemic metabolism.

    Science.gov (United States)

    Czeczor, Juliane K; McGee, Sean L

    2017-03-28

    The amyloid precursor protein (APP) is a transmembrane protein that can be cleaved by proteases through two different pathways to yield a number of small peptides, each with distinct physiological properties and functions. It has been extensively studied in the context of Alzheimer's disease, with the APP-derived amyloid beta (Aβ) peptide being a major constituent of the amyloid plaques observed in this disease. It has been known for some time that APP can regulate neuronal metabolism, however this review will examine evidence that APP and its peptides can also regulate key metabolic processes such as insulin action, lipid synthesis and storage and mitochondrial function in peripheral tissues. This review will present a hypothesis that amyloidogenic processing of APP in peripheral tissues plays a key role in the response to nutrient excess and that this could contribute to the pathogenesis of metabolic diseases such as obesity and type 2 diabetes (T2D). This article is protected by copyright. All rights reserved.

  7. Molecular characterization and chromosomal assignment of equine cartilage derived retinoic acid sensitive protein (CD-RAP)/melanoma inhibitory activity (MIA)

    DEFF Research Database (Denmark)

    Berg, Lise Charlotte; Mata, Xavier; Thomsen, Preben Dybdahl

    2008-01-01

    Cartilage-derived retinoic acid sensitive protein (CD-RAP) also known as melanoma inhibitory activity (MIA) has already been established as a marker for chondrocyte differentiation and a number of cancerous condition sin humans. Studies have also shown that CD-RAP/MIA is a potential marker of joint...... to the human gene is 90% for the translated region. The upstream sequence includes regulatory elements and putative transcription factor binding sites previously described in the human and murine promoter regions. The deduced amino acid sequence consists of 130 aa including a signal peptide of 23 aa, and has...... a 91% identity to the human protein. Using radiation hybrid mapping, the CD-RAP/MIA gene was localized to the p arm of equine chromosome 10 (ECA10p), which is in accordance with prediction based on the current human-equine comparative map. Gene expression studies showed expression of CD-RAP/MIA m...

  8. Activation and localization of cartilage-derived morphogenetic protein-1 at the site of ossification of the ligamentum flavum.

    Science.gov (United States)

    Nakase, T; Ariga, K; Yonenobu, K; Tsumaki, N; Luyten, F P; Mukai, Y; Sato, I; Yoshikawa, H

    2001-08-01

    Localization and expression of cartilage-derived morphogenetic protein (CDMP)-1 in tissues at the site of ossification of the ligamentum flavum (OLF) were examined by immunohistochemistry and in situ hybridization. The CDMP-1 protein and messenger ribonucleic acid (mRNA) were localized in spindle-shaped cells and chondrocytes in the OLF tissues. CDMP-1 was not detected in cells in non-ossified sites. These data indicate that CDMP-1 is locally activated and localized in spindle-shaped cells and chondrocytes at the site of OLE. Given the previously reported promoting action of CDMP-1 for chondrogenesis, the current results suggest that CDMP-1 may be involved in the progression of OLF, leading to the narrowing of spinal canal and thus causing severe clinical manifestations.

  9. Estrogen and exercise interact to regulate brain-derived neurotrophic factor mRNA and protein expression in the hippocampus.

    Science.gov (United States)

    Berchtold, N C; Kesslak, J P; Pike, C J; Adlard, P A; Cotman, C W

    2001-12-01

    We investigated the possibility that estrogen and exercise interact in the hippocampus and regulate brain-derived neurotrophic factor (BDNF), a molecule increasingly recognized for its role in plasticity and neuron function. An important aspect of this study is to examine the effect of different time intervals between estrogen loss and estrogen replacement intervention. We demonstrate that in the intact female rat, physical activity increases hippocampal BDNF mRNA and protein levels. However, the exercise effect on BDNF up-regulation is reduced in the absence of estrogen, in a time-dependent manner. In addition, voluntary activity itself is stimulated by the presence of estrogen. In exercising animals, estrogen deprivation reduced voluntary activity levels, while estrogen replacement restored activity to normal levels. In sedentary animals, estrogen deprivation (ovariectomy) decreased baseline BDNF mRNA and protein, which were restored by estrogen replacement. Despite reduced activity levels in the ovariectomized condition, exercise increased BDNF mRNA levels in the hippocampus after short-term (3 weeks) estrogen deprivation. However, long-term estrogen-deprivation blunted the exercise effect. After 7 weeks of estrogen deprivation, exercise alone no longer affected either BDNF mRNA or protein levels. However, exercise in combination with long-term estrogen replacement increased BDNF protein above the effects of estrogen replacement alone. Interestingly, protein levels across all conditions correlated most closely with mRNA levels in the dentate gyrus, suggesting that expression of mRNA in this hippocampal region may be the major contributor to the hippocampal BDNF protein pool. The interaction of estrogen, physical activity and hippocampal BDNF is likely to be an important issue for maintenance of brain health, plasticity and general well-being, particularly in women.

  10. Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives.

    Science.gov (United States)

    Schopfer, F J; Batthyany, C; Baker, P R S; Bonacci, G; Cole, M P; Rudolph, V; Groeger, A L; Rudolph, T K; Nadtochiy, S; Brookes, P S; Freeman, B A

    2009-05-01

    Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with beta-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial beta-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.

  11. An inhibitor-driven study for enhancing the selectivity of indirubin derivatives towards leishmanial Glycogen Synthase Kinase-3 over leishmanial cdc2-related protein kinase 3.

    Science.gov (United States)

    Efstathiou, Antonia; Gaboriaud-Kolar, Nicolas; Smirlis, Despina; Myrianthopoulos, Vassilios; Vougogiannopoulou, Konstantina; Alexandratos, Alexandros; Kritsanida, Marina; Mikros, Emmanuel; Soteriadou, Ketty; Skaltsounis, Alexios-Leandros

    2014-05-20

    In search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery. A new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm. We have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 μΜ. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial

  12. Extraction and characterization of highly purified collagen from bovine pericardium for potential bioengineering applications

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Maria Helena, E-mail: mariahelena.santos@gmail.com [Department of Dentistry, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Center for Assessment and Development of Biomaterials-BioMat, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Silva, Rafael M.; Dumont, Vitor C. [Department of Dentistry, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Center for Assessment and Development of Biomaterials-BioMat, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Neves, Juliana S. [Center for Assessment and Development of Biomaterials-BioMat, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Mansur, Herman S. [Department of Metallurgical and Materials Engineering, Federal University of Minas Gerais-UFMG, Belo Horizonte/MG 31270-901 (Brazil); Heneine, Luiz Guilherme D. [Department of Health Science, Ezequiel Dias Foundation-FUNED, Belo Horizonte/MG 30510-010 (Brazil)

    2013-03-01

    Bovine pericardium is widely used as a raw material in bioengineering as a source of collagen, a fundamental structural molecule. The physical, chemical, and biocompatibility characteristics of these natural fibers enable their broad use in several areas of the health sciences. For these applications, it is important to obtain collagen of the highest possible purity. The lack of a method to produce these pure biocompatible materials using simple and economically feasible techniques presents a major challenge to their production on an industrial scale. This study aimed to extract, purify, and characterize the type I collagen protein originating from bovine pericardium, considered to be an abundant tissue resource. The pericardium tissue was collected from male animals at slaughter age. Pieces of bovine pericardium were enzymatically digested, followed by a novel protocol developed for protein purification using ion-exchange chromatography. The material was extensively characterized by electrophoresis, scanning electron microscopy, energy dispersive X-ray spectroscopy, and infrared spectroscopy. The results showed a purified material with morphological properties and chemical functionalities compatible with type I collagen and similar to a highly purified commercial collagen. Thus, an innovative and relatively simple processing method was developed to extract and purify type I collagen from bovine tissue with potential applications as a biomaterial for regenerative tissue engineering. - Highlights: Black-Right-Pointing-Pointer Type I collagen was obtained from bovine pericardium, an abundant tissue resource. Black-Right-Pointing-Pointer A simple and feasible processing technique was developed to purify bovine collagen. Black-Right-Pointing-Pointer The appropriate process may be performed on industrial scale. Black-Right-Pointing-Pointer The pure collagen presented appropriate morphological and molecular characteristics. Black-Right-Pointing-Pointer The purify

  13. Characterization of Protein and Peptide Binding to Nanogels Formed by Differently Charged Chitosan Derivatives

    Directory of Open Access Journals (Sweden)

    Anastasia Zubareva

    2013-07-01

    Full Text Available Chitosan (Chi is a natural biodegradable cationic polymer with remarkable potency as a vehicle for drug or vaccine delivery. Chi possesses multiple groups, which can be used both for Chi derivatization and for particle formation. The aim of this work was to produce stable nanosized range Chi gels (nanogels, NGs with different charge and to study the driving forces of complex formation between Chi NGs and proteins or peptides. Positively charged NGs of 150 nm in diameter were prepared from hexanoyl chitosan (HC by the ionotropic gelation method while negatively charged NGs of 190 nm were obtained from succinoyl Chi (SC by a Ca2+ coacervation approach. NGs were loaded with a panel of proteins or peptides with different weights and charges. We show that NGs preferentially formed complexes with oppositely charged molecules, especially peptides, as was demonstrated by gel-electrophoresis, confocal microscopy and HPLC. Complex formation was accompanied by a change in zeta-potential and decrease in size. We concluded that complex formation between Chi NGs and peptide/proteins is mediated mostly by electrostatic interactions.

  14. Effects of extracellular matrix proteins in chondrocyte-derived matrices on chondrocyte functions.

    Science.gov (United States)

    Hoshiba, Takashi; Lu, Hongxu; Kawazoe, Naoki; Yamada, Tomoe; Chen, Guoping

    2013-01-01

    Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0-ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein-coated substrata and P0-ECM. Low chondrocyte attachment was observed on aggrecan-coated substratum and P0-ECM. Cell proliferation on aggrecan- and type II collagen/aggrecan-coated substrata and P0-ECM was lower than that on the other ECM protein (type I collagen and type II collagen)-coated substrata. When chondrocytes were subcultured on aggrecan-coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0-ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0-ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM.

  15. Protein structural information derived from NMR chemical shift with the neural network program TALOS-N.

    Science.gov (United States)

    Shen, Yang; Bax, Ad

    2015-01-01

    Chemical shifts are obtained at the first stage of any protein structural study by NMR spectroscopy. Chemical shifts are known to be impacted by a wide range of structural factors, and the artificial neural network based TALOS-N program has been trained to extract backbone and side-chain torsion angles from (1)H, (15)N, and (13)C shifts. The program is quite robust and typically yields backbone torsion angles for more than 90 % of the residues and side-chain χ 1 rotamer information for about half of these, in addition to reliably predicting secondary structure. The use of TALOS-N is illustrated for the protein DinI, and torsion angles obtained by TALOS-N analysis from the measured chemical shifts of its backbone and (13)C(β) nuclei are compared to those seen in a prior, experimentally determined structure. The program is also particularly useful for generating torsion angle restraints, which then can be used during standard NMR protein structure calculations.

  16. Whey protein-derived biomaterials and their use as bioencapsulation and delivery systems

    Directory of Open Access Journals (Sweden)

    Subirade Muriel

    2003-01-01

    Full Text Available The emergence of bioactive food compounds (nutraceutical compounds with health benefits provides an excellent opportunity for improving public health. The incorporation of bioactive compounds into food systems is therefore of great interest to researchers in their efforts to develop innovative functional foods that may have physiological benefits or reduce the risk of disease beyond basic nutritional functions. However, the effectiveness of these products in preventing diseases relies on preserving the bioavailability of their active ingredients. This represents undoubtedly a great challenge since these molecules are generally sensitive to environmental conditions encountered in food processes (i.e., temperature oxygen, and light or in the gastrointestinal tract (i.e., pH, enzymes presence of other nutrients, which limit their activity and potential health benefits. However, bio- and microencapsulation can be used to overcome these limitations. Whey proteins, also known as the serum proteins of milk, are widely used in food products, because of their high nutritional value and their ability to form gels, emulsions, or foams. The aim of this article is to provide information on the different types of materials obtained from whey proteins and to examine their use as bioencapsulation and delivery systems.

  17. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Jinghuan Li

    2015-05-01

    Full Text Available Extracellular vesicles (EVs play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors, some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

  18. Evidence that beta-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin.

    Science.gov (United States)

    Cook, L; Nagi, M N; Suneja, S K; Hand, A R; Cinti, D L

    1992-01-01

    The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase. Images Fig. 4. Fig. 7. Fig. 8. PMID:1417796

  19. Use of a Monoclonal Antibody to Purify the Tetrodotoxin Binding Component from the Electroplax of Electrophorus electricus

    OpenAIRE

    Nakayama, H; Withy, R M; Raftery, M A

    1982-01-01

    The tetrodotoxin binding component of the voltage-sensitive sodium channel from Electrophorus electricus electroplax was purified by using a monoclonal antibody. An impure preparation of tetrodotoxin binding component was mixed with the pure monoclonal antibody, and the immune complex so formed was isolated by affinity chromatography on a protein A-Sepharose column. Excess antibody was removed by ion-exchange chromatography. The purified material has a specific activity of over 1,800 pmol of ...

  20. T cell-mediated cytotoxicity against p53-protein derived peptides in bulk and limiting dilution cultures of healthy donors

    DEFF Research Database (Denmark)

    Röpke, M; Regner, M; Claesson, M H

    1995-01-01

    The p53 tumour suppressor gene product plays an important role in the development of most human cancers. Point mutations in the p53 gene are common in malignant states and results in over-expression of wild type and mutant determinants of the p53 protein. This process might generate MHC-I restric......The p53 tumour suppressor gene product plays an important role in the development of most human cancers. Point mutations in the p53 gene are common in malignant states and results in over-expression of wild type and mutant determinants of the p53 protein. This process might generate MHC......-I restricted epitopes for T cell recognition and p53-derived peptides have been suggested as targets for tumour-specific cytotoxic T lymphocytes (CTL). Our primary aim was to estimate the frequencies of p53-peptide reactive CTL precursors (CTLp) in peripheral blood from healthy young individuals. We selected...... wild type and mutated peptides derived from the p53 sequence with a binding motif for HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from healthy HLA-A2 donors were stimulated in vitro in bulk cultures as well as in limiting dilution cultures using autologous cells pulsed with p53...

  1. The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability

    Science.gov (United States)

    Myers, Katie N.; Barone, Giancarlo; Ganesh, Anil; Staples, Christopher J.; Howard, Anna E.; Beveridge, Ryan D.; Maslen, Sarah; Skehel, J. Mark; Collis, Spencer J.

    2016-01-01

    It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions. PMID:27739501

  2. Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

    Science.gov (United States)

    Myint, Khine; Li, Yan; Paxton, James; McKeage, Mark

    2015-01-01

    The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In

  3. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    Directory of Open Access Journals (Sweden)

    Gay Bernard

    2011-09-01

    Full Text Available Abstract Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.

  4. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells.

    Science.gov (United States)

    Clerc, Isabelle; Laverdure, Sylvain; Torresilla, Cynthia; Landry, Sébastien; Borel, Sophie; Vargas, Amandine; Arpin-André, Charlotte; Gay, Bernard; Briant, Laurence; Gross, Antoine; Barbeau, Benoît; Mesnard, Jean-Michel

    2011-09-19

    Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.

  5. Radioiododestannylation. Convenient synthesis of a stable penicillin derivative for rapid penicillin binding protein (PBP) assay

    Energy Technology Data Exchange (ETDEWEB)

    Blaszczak, L.C.; Halligan, N.G. (Lilly (Eli) and Co., Indianapolis, IN (USA). Lilly Research Labs.); Seitz, D.E. (Indiana Univ.-Purdue Univ., Indianapolis, IN (USA). School of Medicine)

    1989-04-01

    Radioiodination of p-(trimethylstannyl)penicillin V with ({sup 125}I)Na using a modification of the chloramine-T method is simple, high yielding, and site-specific. The structure and penicillin binding protein (PBP) affinity of p-({sup 125}I)-penicillin V (IPV) are similar to penicillin G and the product can be used directly without purification in the PBP assay. Because of the high degree of stability toward autoradiolysis and equivalent PBP binding affinity, IPV can be used in place of ({sup 3}H)-penicillin G or ({sup 14}C)-penicillin G for these experiments. (author).

  6. RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages.

    Science.gov (United States)

    Chen, J J; Brot, N; Weissbach, H

    1980-07-01

    RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.

  7. Suppression of zinc finger protein 467 alleviates osteoporosis through promoting differentiation of adipose derived stem cells to osteoblasts

    Directory of Open Access Journals (Sweden)

    You Li

    2012-01-01

    Full Text Available Abstract Osteoblast and adipocyte are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. In this study, a comparative analysis of gene expression profiling using cDNA microarray and realtime-PCR indicated that Zinc finger protein 467 (Zfp467 involved in adipocyte and osteoblast differentiation of cultured adipose derived stem cells (ADSCs. Our results showed that RNA interference for Zfp467 in ADSCs inhibited adipocyte formation and stimulated osteoblast commitment. The mRNA levels of osteogenic and adipogenic markers in ADSCs were regulated by si-Zfp467. Zfp467 RNAi in ADSCs could restore bone function and structure in an ovariectomized (OVX-induced osteoporotic mouse model. Thus Zfp467 play an important role in ADSCs differentiation to adipocyte and osteoblast. This has relevance to therapeutic interventions in osteoporosis, including si-Zfp467-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering.

  8. Nematode-derived proteins suppress proliferation and cytokine production of antigen-specific T cells via induction of cell death.

    Directory of Open Access Journals (Sweden)

    Wiebke Hartmann

    Full Text Available In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host's immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2 and novel larval transcript-1 (OvNLT-1 to cell cultures of T cell receptor transgenic CD4(+ and CD8(+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4(+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by (3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8(+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103, did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro.

  9. Nematode-Derived Proteins Suppress Proliferation and Cytokine Production of Antigen-Specific T Cells via Induction of Cell Death

    Science.gov (United States)

    Hartmann, Wiebke; Brenz, Yannick; Kingsley, Manchang Tanyi; Ajonina-Ekoti, Irene; Brattig, Norbert W.; Liebau, Eva; Breloer, Minka

    2013-01-01

    In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2) and novel larval transcript-1 (OvNLT-1) to cell cultures of T cell receptor transgenic CD4+ and CD8+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by 3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro. PMID:23861729

  10. Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

    Science.gov (United States)

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng'an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-05-06

    Aerolysins are virulence factors belonging to the bacterial β-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

  11. Recent Research in Antihypertensive Activity of Food Protein-derived Hydrolyzates and Peptides.

    Science.gov (United States)

    Saleh, Ahmed S M; Zhang, Qing; Shen, Qun

    2016-01-01

    Year to year obesity prevalence, reduced physical activities, bad habits/or stressful lifestyle, and other environmental and physiological impacts lead to increase in diseases such as coronary heart disease, stroke, cancer, diabetes, and hypertension worldwide. Hypertension is considered as one of the most common serious chronic diseases; however, discovery of medications with high efficacy and without side effects for treatment of patients remains a challenge for scientists. Recent trends in functional foods have evidenced that food bioactive proteins play a major role in the concepts of illness and curing; therefore, nutritionists, biomedical scientists, and food scientists are working together to develop improved systems for the discovery of peptides with increased potency and therapeutic benefits. This review presents a recent research carried out to date for the purpose of isolation and identification of bioactive hydrolyzates and peptides with angiotensin I converting enzyme inhibitory activity and antihypertensive effect from animal, marine, microbial, and plant food proteins. Effects of food processing and hydrolyzation conditions as well as some other impacts on formation, activity, and stability of these hydrolyzates and peptides are also presented.

  12. SuperLigands – a database of ligand structures derived from the Protein Data Bank

    Directory of Open Access Journals (Sweden)

    Preissner Robert

    2005-05-01

    Full Text Available Abstract Background Currently, the PDB contains approximately 29,000 protein structures comprising over 70,000 experimentally determined three-dimensional structures of over 5,000 different low molecular weight compounds. Information about these PDB ligands can be very helpful in the field of molecular modelling and prediction, particularly for the prediction of protein binding sites and function. Description Here we present an Internet accessible database delivering PDB ligands in the MDL Mol file format which, in contrast to the PDB format, includes information about bond types. Structural similarity of the compounds can be detected by calculation of Tanimoto coefficients and by three-dimensional superposition. Topological similarity of PDB ligands to known drugs can be assessed via Tanimoto coefficients. Conclusion SuperLigands supplements the set of existing resources of information about small molecules bound to PDB structures. Allowing for three-dimensional comparison of the compounds as a novel feature, this database represents a valuable means of analysis and prediction in the field of biological and medical research.

  13. SuperLigands – a database of ligand structures derived from the Protein Data Bank

    Science.gov (United States)

    Michalsky, Elke; Dunkel, Mathias; Goede, Andrean; Preissner, Robert

    2005-01-01

    Background Currently, the PDB contains approximately 29,000 protein structures comprising over 70,000 experimentally determined three-dimensional structures of over 5,000 different low molecular weight compounds. Information about these PDB ligands can be very helpful in the field of molecular modelling and prediction, particularly for the prediction of protein binding sites and function. Description Here we present an Internet accessible database delivering PDB ligands in the MDL Mol file format which, in contrast to the PDB format, includes information about bond types. Structural similarity of the compounds can be detected by calculation of Tanimoto coefficients and by three-dimensional superposition. Topological similarity of PDB ligands to known drugs can be assessed via Tanimoto coefficients. Conclusion SuperLigands supplements the set of existing resources of information about small molecules bound to PDB structures. Allowing for three-dimensional comparison of the compounds as a novel feature, this database represents a valuable means of analysis and prediction in the field of biological and medical research. PMID:15943884

  14. A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia

    Science.gov (United States)

    Saraiya, Ashesh A.; Li, Wei; Wang, Ching C.

    2011-01-01

    We have previously shown that a snoRNA-derived microRNA, miR2, in Giardia lamblia potentially regulates the expression of 22 variant surface protein (VSP) genes. Here, we identified another miRNA, miR4, also capable of regulating the expression of several VSPs but derived from an unannotated open reading frame (ORF) rather than a snoRNA, suggesting a canonical miRNA biogenesis pathway in Giardia. miR4 represses expression of a reporter containing two miR4 antisense sequences at the 3′ UTR without causing a corresponding decrease in the mRNA level. This repression requires the presence of the Giardia Argonaute protein (GlAgo) and is reversed by 2′ O–methylated antisense oligo to miR4, suggesting an RNA-induced silencing complex (RISC)–mediated mechanism. Furthermore, in vivo and in vitro evidence suggested that the Giardia Dicer protein (GlDcr) is required for miR4 biogenesis. Coimmunoprecipitation of miR4 with GlAgo further verified miR4 as a miRNA. A total of 361 potential target sites for miR4 were bioinformatically identified in Giardia, out of which 69 (32.7%) were associated with VSP genes. miR4 reduces the expression of a reporter containing two copies of the target site from VSP (GL50803_36493) at the 3′ UTR. Sixteen of the 69 VSP genes were further found to contain partially overlapping miR2 and miR4 targeting sites. Expression of a reporter carrying the two overlapping sites was inhibited by either miR2 or miR4, but the inhibition was neither synergistic nor additive, suggesting a complex mechanism of miRNA regulation of VSP expression and the presence of a rich miRNAome in Giardia. PMID:22033329

  15. Overexpression of the endothelial protein C receptor is detrimental during pneumonia-derived gram-negative sepsis (Melioidosis.

    Directory of Open Access Journals (Sweden)

    Liesbeth M Kager

    Full Text Available The endothelial protein C receptor (EPCR enhances anticoagulation by accelerating activation of protein C to activated protein C (APC and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B. pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumosepsis in South-East Asia.Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT mice and mice with either EPCR-overexpression (Tie2-EPCR or EPCR-deficiency (EPCR(-/-. Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR(-/- mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma.Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumosepsis.

  16. Extraction and characterization of highly purified collagen from bovine pericardium for potential bioengineering applications.

    Science.gov (United States)

    Santos, Maria Helena; Silva, Rafael M; Dumont, Vitor C; Neves, Juliana S; Mansur, Herman S; Heneine, Luiz Guilherme D

    2013-03-01

    Bovine pericardium is widely used as a raw material in bioengineering as a source of collagen, a fundamental structural molecule. The physical, chemical, and biocompatibility characteristics of these natural fibers enable their broad use in several areas of the health sciences. For these applications, it is important to obtain collagen of the highest possible purity. The lack of a method to produce these pure biocompatible materials using simple and economically feasible techniques presents a major challenge to their production on an industrial scale. This study aimed to extract, purify, and characterize the type I collagen protein originating from bovine pericardium, considered to be an abundant tissue resource. The pericardium tissue was collected from male animals at slaughter age. Pieces of bovine pericardium were enzymatically digested, followed by a novel protocol developed for protein purification using ion-exchange chromatography. The material was extensively characterized by electrophoresis, scanning electron microscopy, energy dispersive X-ray spectroscopy, and infrared spectroscopy. The results showed a purified material with morphological properties and chemical functionalities compatible with type I collagen and similar to a highly purified commercial collagen. Thus, an innovative and relatively simple processing method was developed to extract and purify type I collagen from bovine tissue with potential applications as a biomaterial for regenerative tissue engineering. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Biological properties of purified recombinant HCV particles with an epitope-tagged envelope